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textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/01%3A_In_The_Beginning/1.01%3A_Introduction_-_Basic_Biology.txt
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• 1.1: Introduction - Basic Biology
The most obvious thing about living organisms is their astounding diversity. Estimates put the number of eukaryotic species at about 8.7 million, while bacteria account for anywhere between 107 and 109 different species. The number of species of archaea is still uncertain, but is expected to be very large. These organisms, representing the three great domains of life, together occupy every environmental niche imaginable.
• 1.2: Introduction - Basic Chemistry
To understand biochemistry, one must possess at least a basic understanding of organic and general chemistry. In this brief section, we will provide a rapid review of the simple concepts necessary to understand cellular chemistry. Chemistry is chemistry, whether in a cell or outside it, but biological chemistry is a particular subset of organic chemistry that often involves enormous macromolecules, and that happens in the aqueous environment of the cell.
• 1.3: Introduction - Water and Buffers
When it comes to water, we’re literally drowning in it, as water is by far the most abundant component of every cell. To understand life, we begin the discussion with the basics of water, because everything that happens in cells, even reactions buried deep inside enzymes, away from water, is influenced by water’s chemistry.
01: In The Beginning
Figure 1.2 Slices of cork as seen by Hooke
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1.02: Introduction - Basic Chemistry
Source: BiochemFFA_1_2.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
“Organic chemistry is the chemistry of carbon compounds. Biochemistry is the chemistry of carbon compounds that crawl” -Michael Adams.
To understand biochemistry, one must possess at least a basic understanding of organic and general chemistry. In this brief section, we will provide a rapid review of the simple concepts necessary to understand cellular chemistry. Chemistry is chemistry, whether in a cell or outside it, but biological chemistry is a particular subset of organic chemistry that often involves enormous macromolecules, and that happens in the aqueous environment of the cell.
Covalent bonds, as you know, are the result of sharing of electrons between two atoms. Ionic bonds, by contrast, are formed when one atom donates an electron to another, such as in the formation of sodium chloride. Single covalent bonds can rotate freely, but double bonds cannot. Single bonds around a carbon atom are arranged in a tetrahedron with bond angles of 109.5° relative to each other, with the carbon at the center (Figure 1.19). Double bonded carbons create a planar structure with bond angles typically of about 120°.
Electronegativity
Electronegativity is a measure of the affinity a nucleus has for outer shell electrons (Table 1.2). High electronegativity corresponds to high affinity. Electrons in a covalent bond are held closer to the nucleus with a greater electronegativity compared to a nucleus with lower electronegativity.
Table 1.2 Image by Aleia Kim
For example, in a molecule of water, with hydrogen covalently bonded to oxygen, the electrons are “pulled” toward the oxygen, which is more electronegative. Because of this, there is a slightly greater negative charge near the oxygen atom of water, compared to the hydrogen (which, correspondingly has a slightly higher positive charge). This unequal charge distribution sets up a dipole, with one side being somewhat negative and the other somewhat positive. Because of this, the molecule is described as polar.
Hydrogen bonds between water molecules are the result of the attraction of the partial positive and partial negative charges on different water molecules (Figure 1.20). Hydrogen bonds can also form between hydrogens with a partial positive charge and other strongly electronegative atoms, like nitrogen, with a partial negative charge. It is important to remember that hydrogen bonds are interactions between molecules (or parts of molecules) and are not bonds between atoms, like covalent or ionic bonds. Bonds between hydrogen and carbon do not form significant partial charges because the electronegativities of the two atoms are similar. Consequently, molecules containing many carbon-hydrogen bonds will not form hydrogen bonds and therefore, do not mix well with water. Such molecules are called hydrophobic. Other compounds with the ability to make hydrogen bonds are polar and can dissolve in water. They are called hydrophilic. Molecules possessing both characteristics are called amphiphilic.
Weak interactions
Hydrogen bonds are one kind of electrostatic (i.e., based on charge) interaction between dipoles. Other forms of electrostatic interactions that are important in biochemistry include weak interactions between a polar molecule and a transient dipole, or between two temporary dipoles. These temporary dipoles result from the movement of electrons in a molecule. As electrons move around, the place where they are, at a given time, becomes temporarily more negatively charged and could now attract a temporary positive charge on another molecule. Since electrons don’t stay put, these dipoles are very short-lived. Thus, the attraction that depends on these dipoles fluctuates and is very weak. Weak interactions like these are sometimes called van der Waals forces. Many molecular interactions in cells depend on weak interactions. Although the individual hydrogen bonds or other dipole-dipole interactions are weak, because of their large numbers, they can result in quite strong interactions between molecules.
Oxidation/reduction
Oxidation involves loss of electrons and reduction results in gain of electrons. For every biological oxidation, there is a corresponding reduction - one molecule loses electrons to another molecule. Oxidation reactions tend to release energy and are a source of bioenergy for chemotrophic cells.
Ionization
Ionization of biomolecules, by contrast does not involve oxidation/reduction. In ionization, a hydrogen ion (H+) leaves behind its electron as it exits (leaving behind a negative charge) or joins a group (adding a positive charge). Biological ionizations typically involve carboxyl groups or amines, though phosphates or sulfates can also be ionized. A carboxyl group can have two ionization states - a charge of -1 corresponds to the carboxyl without its proton and a charge of zero corresponds to the charge of the carboxyl with its proton on. An amine also has two ionization states. A charge of zero corresponds to a nitrogen with three covalent bonds (usually in the form of C-NH2) and a charge of +1 corresponds to a nitrogen making four covalent bonds (usually X-NH3 +).
Stereochemistry
A carbon has the ability to make four single bonds (forming a tetrahedral structure) and if it bonds to four different chemical groups, their atoms can be arranged around the carbon in two different ways, giving rise to stereochemical “handedness” (Figure 1.21). Each carbon with such a property is referred to as an asymmetric center. The property of handedness only occurs when a carbon has four different groups bonded to it. Enzymes have very specific 3-D structures, so for biological molecules that can exist in different stereoisomeric forms, an enzyme that synthesizes it would make only one of the possible isomers. By contrast, the same molecules made chemically (not using enzymes) end up with equal amounts of both isomers, called a racemic mix.
Gibbs free energy
The Gibbs free energy calculation allows us to determine whether a reaction will be spontaneous, by taking into consideration two factors, change in enthalpy (ΔH) and change in entropy (ΔS). The free energy content of a system is given by the Gibbs free energy ($G$) and is equal to the enthalpy ($H$) for a process minus the absolute temperature (T) times the entropy (S)
$G = H = TS$
For a process, the change in the Gibbs free energy ΔG is given by
$ΔG = ΔH - TΔS$
A negative $ΔG$ corresponds to release of free energy. Reactions that release energy are exergonic, whereas those that absorb energy are called endergonic.
The biological standard Gibbs free energy change (ΔG°’) corresponds to the ΔG for a process under standard conditions of temperature, pressure, and at pH = 7. For a reaction
$aA + bB \rightleftharpoons cC + dD,$
the equilibrium constant, $K_{eq}$ is equal to
$K_{eq} = \dfrac{ [C]^c_{eq} [D]^d_{eq}}{[A]^a_{eq} [B]^b_{eq}}$
where $a$, $b$, $c$, and $d$ are integers in the balanced equation. Large values of $K_{eq}$ correspond to favorable reactions (more C and D produced than A and B) and small values of $K_{eq}$ mean the opposite. At equilibrium,
$ΔG^{o\prime} = -RT \ln K_{eq}$
If a process has a $ΔG = Z$ and a second process has a $ΔG = Y$, then if the two processes are linked, $ΔG$ and $ΔG^{o \prime}$ values for the overall reaction will be the sum of the individual ΔG and ΔG°’ values.
$ΔG_{total} = ΔG_1+ ΔG_2 = Z + Y$
$ΔG^{o \prime }_{total} = ΔG_1^{o \prime}+ ΔG_2^{o\prime}$
Catalysis
Catalysis is an increase in the rate of a reaction induced by a substance that is, itself, unchanged by the reaction. Because catalysts remain unchanged at the end of a reaction, a single catalyst molecule can be reused for many reaction cycles. Proteins that catalyze reactions in cells are called enzymes, while ribozymes are RNA molecules that act as catalysts.
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Source: BiochemFFA_1_3.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
When it comes to water, we’re literally drowning in it, as water is by far the most abundant component of every cell. To understand life, we begin the discussion with the basics of water, because everything that happens in cells, even reactions buried deep inside enzymes, away from water, is influenced by water’s chemistry.
The water molecule has wide ‘V’ shape (the HO-H angle is 104°) with uneven sharing of electrons between the oxygen and the hydrogen atoms (Figure 1.23). Oxygen, with its higher electronegativity, holds electrons closer to itself than the hydrogens do. The hydrogens, as a result, are described as having a partial positive charge (typically designated as δ+) and the oxygen has a partial negative charge (written as δ- ). Thus, water is a polar molecule because charges are distributed around it unevenly, not symmetrically.
Water as a solvent
Water (Figure 1.23) is described as a solvent because of its ability to solvate (dissolve) many, but not all, molecules. Molecules that are ionic or polar dissolve readily in water, but non-polar substances dissolve poorly in water, if at all. Oil, for example, which is non-polar, separates from water when mixed with it. On the other hand, sodium chloride, which ionizes, and ethanol, which is polar, are able to form hydrogen bonds, so both dissolve in water. Ethanol’s solubility in water is crucial for brewers, winemakers, and distillers – but for this property, there would be no wine, beer or spirits. As explained in an earlier section, we use the term hydrophilic to describe substances that interact well with water and dissolve in it and the term hydrophobic to refer to materials that are non-polar and do not dissolve in water. Table 1.3 illustrates some polar and non-polar substances. A third term, amphiphilic, refers to compounds that have both properties. Soaps, for example are amphiphilic, containing a long, non-polar aliphatic tail and a head that ionizes.
Table 1.3 Image by Aleia Kim
Solubility
The solubility of materials in water is based in free energy changes, as measured by ΔG. Remember, from chemistry, that H is the enthalpy (heat at constant pressure) and S is entropy. Given this,
\[ΔG = ΔH - TΔS\]
where T is the temperature in Kelvin. For a process to be favorable, the ΔG for it must be less than zero.
From the equation, lowered ΔG values will be favored with decreases in enthalpy and/or increases in entropy. Let us first consider why non-polar materials do not dissolve in water. We could imagine a situation where the process of dissolving involves the “surrounding” of each molecule of the nonpolar solute in water, just like each sodium and each chloride ion gets surrounded by water molecules as salt dissolves.
Water organization
There is a significant difference, though between surrounding a non-polar molecule with water molecules and surrounding ions (or polar compounds) with water molecules.
The difference is that since non-polar molecules don’t really interact with water, the water behaves very differently than it does with ions or molecules that form hydrogen bonds. In fact, around each non-polar molecule, water gets very organized, aligning itself regularly. As any freshman chemistry student probably remembers, entropy is a measure of disorder, so when something becomes ordered, entropy decreases, meaning the ΔS is negative, so the TΔS term in the equation is positive (negative of a negative).
Since mixing a non-polar substance with water doesn’t generally have any significant heat component, the ΔG is positive. This means, then, that dissolving a non-polar compound in water is not favorable and does not occur to any significant extent. Further, when the non-polar material associates with itself and not water, then the water molecules are free to mix, without being ordered, resulting in an increase of entropy. Entropy therefore drives the separation of non-polar substances from aqueous solutions.
Amphiphilic substances
Next, we consider mixing of an amphiphilic substance, such as a soap, with water (Figure 1.24). The sodium ions attached to the fatty acids in soap readily come off in aqueous solution, leaving behind a negatively charged molecule at one end and a non-polar region at the other end. The ionization of the soap causes in an increase in entropy - two particles instead of one. The non-polar portion of the negatively charged soap ion is problematic - if exposed to water, it will cause water to organize and result in a decrease of entropy and a positive ΔG.
Since we know fatty acids dissolve in water, there must be something else at play. There is. Just like the non-polar molecules in the first example associated with each other and not water, so too do the non-polar portions of the soap ions associate with each other and exclude water. The result is that the soap ions arrange themselves as micelles (Figure 1.25) with the non-polar portions on the interior of the structure away from water and the polar portions on the outside interacting with water.
The interaction of the polar heads with water returns the water to its more disordered state. This increase in disorder, or entropy, drives the formation of micelles. As will be seen in the discussion of the lipid bilayer, the same forces drive glycerophospholipids and sphingolipids to spontaneously form bilayers where the non-polar portions of the molecules interact with each other to exclude water and the polar portions arrange themselves on the outsides of the bilayer (Figure 1.28).
Yet another example is seen in the folding of globular proteins in the cytoplasm. Nonpolar amino acids are found in the interior portion of the protein (water excluded). Interaction of the non-polar amino acids turns out to be a driving force for the folding of proteins as they are being made in an aqueous solution.
Hydrogen bonds
The importance of hydrogen bonds in biochemistry (Figure 1.30) is hard to overstate. Linus Pauling himself said,
“ . . . . I believe that as the methods of structural chemistry are further applied to physiological problems it will be found that the significance of the hydrogen bond for physiology is greater than that of any other single structural feature.”
In 2011, an IUPAC task group gave an evidence-based definition of hydrogen bonding that states,
“The hydrogen bond is an attractive interaction between a hydrogen atom from a molecule or a molecular fragment X–H in which X is more electronegative than H, and an atom or a group of atoms in the same or a different molecule, in which there is evidence of bond formation.”
Partial Charges
The difference in electronegativity between hydrogen and the molecule to which it is covalently bound give rise to partial charges as described above. These tiny charges (δ+ and δ- ) result in formation of hydrogen bonds, which occur when the partial positive charge of a hydrogen atom is attracted to the partial negative of another molecule. In water, that means the hydrogen of one water molecule is attracted to the oxygen of another (Figure 1.31). Since water is an asymmetrical molecule, it means also that the charges are asymmetrical. Such an uneven distribution is what makes a dipole. Dipolar molecules are important for interactions with other dipolar molecules and for dissolving ionic substances (Figure 1.32).
Hydrogen bonds are not exclusive to water. In fact, they are important forces holding together macromolecules that include proteins and nucleic acids. Hydrogen bonds occur within and between macromolecules.
The complementary pairing that occurs between bases in opposite strands of DNA, for example, is based on hydrogen bonds. Each hydrogen bond is relatively weak (compared to a covalent bond, for example - Table 1.4), but collectively they can be quite strong.
Table 1.4 Image by Aleia Kim
Benefits of weak interactions
Their weakness, however, is actually quite beneficial for cells, particularly as regards nucleic acids (Figure 1.33). The strands of DNA, for example, must be separated over short stretches in the processes of replication and the synthesis of RNA. Since only a few base pairs at a time need to be separated, the energy required to do this is small and the enzymes involved in the processes can readily take them apart, as needed. Hydrogen bonds also play roles in binding of substrates to enzymes, catalysis, and protein-protein interaction, as well as other kinds of binding, such as protein-DNA, or antibody-antigen.
As noted, hydrogen bonds are weaker than covalent bonds (Table 1.4) and their strength varies form very weak (1-2 kJ/mol) to fairly strong (29 kJ/mol). Hydrogen bonds only occur over relatively short distances (2.2 to 4.0 Å). The farther apart the hydrogen bond distance is, the weaker the bond is.
The strength of the bond in kJ/mol represents the amount of heat that must be put into the system to break the bond - the larger the number, the greater the strength of the bond. Hydrogen bonds are readily broken using heat. The boiling of water, for example, requires breaking of H-bonds. When a biological structure, such as a protein or a DNA molecule, is stabilized by hydrogen bonds, breaking those bonds destabilizes the structure and can result in denaturation of the substance - loss of structure. It is partly for this reason that most proteins and all DNAs lose their native, or folded, structures when heated to boiling.
Image by Aleia Kim Table 1.5
For DNA molecules, denaturation results in complete separation of the strands from each other. For most proteins, this means loss of their characteristic three-dimensional structure and with it, loss of the function they performed. Though a few proteins can readily reassume their original structure when the solution they are in is cooled, most can’t. This is one of the reasons that we cook our food. Proteins are essential for life, so denaturation of bacterial proteins results in death of any microorganisms contaminating the food.
The importance of buffers
Water can ionize to a slight extent (10-7 M) to form H+ (proton) and OH- (hydroxide). We measure the proton concentration of a solution with pH, which is the negative log of the proton concentration.
pH = -Log[H+]
If the proton concentration, [H+]= 10-7 M, then the pH is 7. We could just as easily measure the hydroxide concentration with the pOH by the parallel equation,
pOH = -Log[OH- ]
In pure water, dissociation of a proton simultaneously creates a hydroxide, so the pOH of pure water is 7, as well. This also means that
pH + pOH = 14
Now, because protons and hydroxides can combine to form water, a large amount of one will cause there to be a small amount of the other. Why is this the case? In simple terms, if I dump 0.1 moles of H+ into a pure water solution, the high proton concentration will react with the relatively small amount of hydroxides to create water, thus reducing hydroxide concentration. Similarly, if I dump excess hydroxide (as NaOH, for example) into pure water, the proton concentration falls for the same reason.
Acids vs bases
Chemists use the term “acid” to refer to a substance which has protons that can dissociate (come off) when dissolved in water. They use the term “base” to refer to a substance that can absorb protons when dissolved in water. Both acids and bases come in strong and weak forms. (Examples of weak acids are shown in Table 1.5.) Strong acids, such as HCl, dissociate completely in water. If we add 0.1 moles (6.02x1022 molecules) of HCl to a solution to make a liter, it will have 0.1 moles of H+ and 0.1 moles of Cl- or 6.02x1022 molecules of each . There will be no remaining HCl when this happens. A strong base like NaOH also dissociates completely into Na+ and OH- .
Weak Acids
Weak acids and bases differ from their strong counterparts. When you put one mole of acetic acid (HAc) into pure water, only a tiny percentage of the HAc molecules dissociate into H+ and Ac- . Clearly, weak acids are very different from strong acids. Weak bases behave similarly, except that they accept protons, rather than donate them. Since we can view everything as a form of a weak acid, we will not use the term weak base here.
Students are often puzzled and expect that [H+] = [A- ] because the dissociation equation shows one of each from HA. This is, in fact, true ONLY when HA is allowed to dissociate in pure water. Usually the HA is placed into solution that has protons and hydroxides to affect things. Those protons and /or hydroxides change the H+ and Aconcentration unequally, since A- can absorb some of the protons and/or HA can release H+ when influenced by the OH- in the solution. Therefore, one must calculate the proton concentration from the pH using the Henderson Hasselbalch equation.
\[pH = pKa + log ([Ac- ]/[HAc])\]
Image by Aleia Kim Table 1.6
You may wonder why we care about weak acids. You may never have thought much of weak acids when you were in General Chemistry. Your instructor described them as buffers and you probably dutifully memorized the fact that “buffers are substances that resist change in pH” without really learning what Clearing Confusion - this meant. Buffers are much too important to be thought of in this way.
UPS
Weak acids are critical for life because their affinity for protons causes them to behave like a UPS. We’re not referring to the UPS that is the United Parcel Service, but instead, to the encased battery backup systems for computers called Uninterruptible Power Supplies that kick on to keep a computer running during a power failure. The battery in a laptop computer is a UPS, for example.
We can think of weak acids as Uninterruptible Proton Suppliers within certain pH ranges, providing (or absorbing) protons as needed. Weak acids thus help to keep the H+ concentration (and thus the pH) of the solution they are in relatively constant.
Consider the bicarbonate/carbonic acid system. Figure 1.35 shows what happens when H2CO3dissociates. Adding hydroxide ions (by adding a strong base like NaOH) to the solution causes the H+ ions to react with OH- ions to make water. Consequently, the concentration of H+ ions would go down and the pH would go up.
However, in contrast to the situation with a solution of pure water, there is a backup source of H+ available in the form of H2CO3. Here is where the UPS function kicks in. As protons are taken away by the added hydroxyl ions (making water), they are partly replaced by protons from the H2CO3. This is why a weak acid is a buffer. It resists changes in pH by releasing protons to compensate for those “used up” in reacting with the hydroxyl ions.
Henderson-Hasselbalch
It is useful to be able to predict the response of the H2CO3 system to changes in H+ concentration. The Henderson-Hasselbalch equation defines the relationship between pH and the ratio of HCO3 - and H2CO3. It is
pH = pKa + log ([HCO3- ]/ [H2CO3])
This simple equation defines the relationship between the pH of a solution and the ratio of HCO3- and H2CO3 in it. The new term, called the pKa, is defined as
pKa = -Log Ka,
just as
pH = -Log [H+].
The Ka is the acid dissociation constant and is a measure of the strength of an acid. For a general acid, HA, which dissociates as
HA ⇄ H+ + A -, Ka = [H+][A- ]/[HA]
Thus, the stronger the acid, the more protons that will dissociate from it when added to water and the larger the value its Ka will have. Large values of Ka translate to lower values of pKa. As a result, the lower the pKa value is for a given acid, the stronger the weak acid is.
Constant pKa
Please note that pKa is a constant for a given acid. The pKa for carbonic acid is 6.37. By comparison, the pKa for formic acid is 3.75. Formic acid is therefore a stronger acid than acetic acid. A stronger acid will have more protons dissociated at a given pH than a weaker acid.
Now, how does this translate into stabilizing pH? Figure 1.35 shows a titration curve. In this curve, the titration begins with the conditions at the lower left (very low pH). At this pH, the H2CO3 form predominates, but as more and more OH- is added (moving to the 45 Why do we care about pH? Because biological molecules can, in some cases, be exquisitely sensitive to changes in it. As the pH of a solution changes, the charges of molecules in the solution can change, as you will see. Changing charges on biological molecules, especially proteins, can drastically affect how they work and even whether they work at all right), the pH goes up, the amount of HCO3- goes up and (correspondingly), the amount of H2CO3 goes down. Notice that the curve “flattens” near the pKa (6.37).
Buffering region
Flattening of the curve tells us is that the pH is not changing much (not going up as fast) as it did earlier when the same amount of hydroxide was added. The system is resisting a change in pH (not stopping the change, but slowing it) in the region of about one pH unit above and one pH unit below the pKa. Thus, the buffering region of the carbonic acid/ bicarbonate buffer is from about 5.37 to 7.37. It is maximally strong at a pH of 6.37.
Now it starts to become apparent how the buffer works. HA can donate protons when extras are needed (such as when OH- is added to the solution by the addition of NaOH). Similarly, A- can accept protons when extra H+ are added to the solution (adding HCl, for example). The maximum ability to donate or accept protons comes when
[A- ] = [HA]
This is consistent with the Henderson Hasselbalch equation and the titration curve. When [A- ] = [HA], pH = 6.37 + Log(1). Since Log(1) = 0, pH = 6.37 = pKa for carbonic acid. Thus for any buffer, the buffer will have maximum strength and display flattening of its titration curve when [A- ] = [HA] and when pH = pKa. If a buffer has more than one pKa (Figure 1.36), then each pKa region will display the behavior.
Buffered vs non-buffered
To understand how well a buffer protects against changes in pH, consider the effect of adding .01 moles of HCl to 1.0 liter of pure water (no volume change) at pH 7, compared to adding it to 1.0 liter of a 1M acetate buffer at pH 4.76. Since HCl completely dissociates, in 0.01M (10-2 M) HCl you will have 0.01M H+. For the pure water, the pH drops from 7.0 down to 2.0 (pH = -log(0.01M)).
By contrast, the acetate buffer’s pH after adding the same amount of HCl is 4.74. Thus, the pure water solution sees its pH fall from 7 to 2 (5 pH units), whereas the buffered solution saw its pH drop from 4.76 to 4.74 (0.02 pH units). Clearly, the buffer minimizes the impact of the added protons compared to the pure water.
Buffer capacity
It is important to note that buffers have capacities limited by their concentration. Let’s imagine that in the previous paragraph, we had added the 0.01 moles HCl to an acetate buffer that had a concentration of 0.01M and equal amounts of Ac- and HAc. When we try to do the math in parallel to the previous calculation, we see that there are 0.01M protons, but only 0.005M A- to absorb them. We could imagine that 0.005M of the protons would be absorbed, but that would still leave 0.005M of protons unbuffered. Thus, the pH of this solution would be approximately
pH = -log(0.005M) = 2.30
Exceeding buffer capacity dropped the pH significantly compared to adding the same amount of protons to a 1M acetate buffer. Consequently, when considering buffers, it is important to recognize that their concentration sets their limits. Another limit is the pH range in which one hopes to control proton concentration.
Multiple ionizable groups
Now, what happens if a molecule has two (or more) ionizable groups? It turns out, not surprisingly, that each group will have its own pKa and, as a consequence, will have multiple regions of buffering.
Figure 1.36 shows the titration curve for the amino acid aspartic acid. Note that in- stead of a single flattening of the curve, as was seen for acetic acid, aspartic acid’s titration curve displays three such regions. These are individual buffering regions, each centered on the respective pKa values for the carboxyl group and the amine group.
Aspartic acid has four possible charges: +1 (α-carboxyl group, α-amino group, and Rgroup carboxyl each has a proton), 0 (α- carboxyl group missing proton, α- amino group has a proton, R-group carboxyl has a proton), -1 (α-carboxyl group and R-group carboxyl each lack a proton, α-amino group retains a proton), -2 (α-carboxyl, R-group carboxyl, and α-amino groups all lack extra proton).
Prediction
How does one predict the charge for an amino acid at a given pH? A good rule of thumb for estimating charge is that if the pH is more than one unit below the pKa for a group (carboxyl or amino), the proton is on. If the pH is more than one unit above the pKa for the group, the proton is off. If the pH is NOT more than one or less than one pH unit from the pKa, this simple assumption will not work.
Further, it is important to recognize that these rules of thumb are estimates only. The pI (pH at which the charge of a molecule is zero) is an exact value calculated as the average of the two pKa values on either side of the zero region. It is calculated at the average of the two pKa values around the point where the charge of the molecule is zero. For aspartic acid, this corresponds to pKa1and pKa2.
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Thumbanil: Structure of human hemoglobin. The proteins α and βsubunits are in red and blue, and the iron-containing hemegroups in green. Image used with permission (CC BY-SA 3.0; Richard Wheeler).
02: Structure and Function
Source: BiochemFFA_2_1.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
"The man who does not read good books has no advantage over the man who cannot read them." Mark Twain
In this chapter, we will examine the structures of the major classes of biomolecules, with an eye to understanding how these structures relate to function.
As noted earlier, water is the most abundant molecule in cells, and provides the aqueous environment in which cellular chemistry happens. Dissolved in this water are inorganic ions like sodium, potassium and calcium. But the distinctiveness of biochemistry derives from the vast numbers of complex, large, carbon compounds, that are made by living cells. You have probably learned that the major classes of biological molecules are proteins, nucleic acids, carbohydrates and lipids. The first three of these major groups are macromolecules that are built as long polymers made up of smaller subunits or monomers, like strings of beads. The lipids, while not chains of monomers, also have smaller subunits that are assembled in various ways to make the lipid components of cells, including membranes. The chemical properties and three dimensional conformations of these molecules determine all the molecular interactions upon which life depends. Whether building structures within cells, transferring information, or catalyzing reactions, the activities of biomolecules are governed by their structures. The properties and shapes of macromolecules, in turn, depend on the subunits of which they are built.
Interactive 2.1: The enzyme Hexokinase: as for all enzymes, the activity of hexokinase depends on its structure. Protein Database (PDB)
We will next examine the major groups of biological macromolecules: proteins, polysaccharides, nucleic acids, and lipids. The building blocks of the first three, respectively, are amino acids, monosaccharides (sugars), and nucleotides. Acetyl-CoA is the most common building block of lipids.
2.04: Structure and Function- Proteins II
Source: BiochemFFA_2_3.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
In this section, we hope to bring to life the connection between structure and function of proteins. So far, we have described notable features of the four elements (primary, secondary, tertiary, and quaternary) of protein structure and discussed example proteins/motifs exhibiting them. In this section, we will examine from a functional perspective a few proteins/domains whose function relies on secondary, tertiary, or quaternary structure. It is, of course a bit of a narrow focus to ascribe protein function to any one component of structure, but our hope is by presenting these examples, we can bring to life the way in which a protein’s secondary, tertiary, and quarternary structure lead to the functions it has.
Hemoglobin Wikipedia
Fibrous proteins - secondary structure
Proteins whose cellular or extracellular roles have a strong structural component are composed primarily of primary and second structure, with little folding of the chains. Thus, they have very little tertiary structure and are fibrous in nature. Proteins exhibiting these traits are commonly insoluble in water and are referred to as fibrous proteins (also called scleroproteins). The examples described in this category are found exclusively in animals where they serve roles in flesh, connective tissues and hardened external structures, such as hair. They also contain the three common fibrous protein structures α -helices (keratins), β-strands/sheets (fibroin & elastin) and triple helices (collagen). The fibrous proteins have some commonality of amino acid sequence. Each possesses an abundance of repeating sequences of amino acids with small, non-reactive side groups. Many contain short repeats of sequences, often with glycine.
Keratins
The keratins are a family of related animal proteins that take numerous forms. α-keratins are structural components of the outer layer of human skin and are integral to hair, nails, claws, feathers, beaks, scales, and hooves. Keratins provide strength to tissues, such as the tongue, and over 50 different keratins are encoded in the human genome. At a cellular level, keratins comprise the intermediate filaments of the cytoskeleton. α- keratins primarily contain α-helices, but can also have β-strand/sheet structures. Individual α-helices are often intertwined to form coils of coiled structures and these strands can also be further joined together by disulfide bonds, increasing structural strength considerably. This is particularly relevant for α-keratin in hair, which contains about 14% cysteine. The odor of burned hair and that of the chemicals used to curl/uncurl hair (breaking/re-making disulfide bonds) arise from their sulfurous components. β-keratins are comprised of β-sheets, as their name implies.
Fibroin
An insoluble fibrous protein that is a component of the silk of spiders and the larvae of moths and other insects, fibroin is comprised of antiparallel β-strands tightly packed together to form β- sheets. The primary structure of fibroin is a short repeating sequence with glycine at every other residue (Figure 2.57). The small R-groups of the glycine and alanine in the repeating sequence allows for the tight packing characteristic of the fibers of silk. Wikipedia link HERE Elastin As suggested by its name, elastin is a protein with elastic characteristics that functions in many tissues of the body to allow them to resume their shapes after expanding or contracting. The protein is rich in glycine and proline and can comprise over 50% of the weight of dry, defatted arteries.
Elastin
is made by linking tropoelastin proteins together through lysine residues to make a durable complex crosslinked by desmosine. In arteries, elastin helps with pressure wave propagation for facilitating blood flow.
Collagen
Collagen is the most abundant protein in mammals, occupying up to a third of the total mass. There are at least 16 types of collagen. Its fibers are a major component of tendons and they are also found abundantly in skin. Collagen is also prominent in cornea, cartilage, bone, blood vessels and the gut.
Collagen’s structure is an example of a helix of helices, being composed of three lefthanded helical chains that each are coiled together in a right-handed fashion to make the collagen fiber (Figure 2.60). Each helix is stretched out more than an α-helix, giving it an extended appearance. On the inside of the triple helical structure, only residues of glycine are found, since the side chains of other amino acids are too bulky. Collagen chains have the repeating structure glycinem-n where m is often proline and n is often hydroxyproline (Figure 2.61).
Collagen is synthesized in a pre-procollagen form. Processing of the pre-procollagen in the endoplasmic reticulum results in glycosylation, removal of the ‘pre’ sequence, and hydroxylation of lysine and proline residues (see below). The hydroxides can form covalent cross-links with each other, strengthening the collagen fibers. As pro-collagen is exported out of the cell, proteases trim it, resulting in a final form of collagen called tropocollagen.
Hydroxylation
Hydroxylation of proline and lysine side chains occurs post-translationally in a reaction catalyzed by prolyl-4-hydroxylase and lysyl-hydroxylase (lysyl oxidase), respectively. The reaction requires vitamin C. Since hydroxylation of these residues is essential for formation of stable triple helices at body temperature, vitamin C deficiency results in weak, unstable collagen and, consequently, weakened connective tissues. It is the cause of the disease known as scurvy. Hydrolyzed collagen is used to make gelatin, which is important in the food industry. collagens. Wikipedia link HERE
Lamins
Lamins are fibrous proteins that provide structure in the cell nucleus and play a role in transcription regulation. They are similar to proteins making up the intermediate filaments, but have extra amino acids in one coil of the protein. Lamins help to form the nuclear lamin in the interior of the nuclear envelope and play important roles in assembling and disassembling the latter in the process of mitosis. They also help to position nuclear pores. In the process of mitosis, disassembly of the nuclear envelope is promoted by phosphorylation of lamins by a protein called mitosis promoting factor and assembly is favored by reversing the reaction (dephosphorylation).
Structural domains - tertiary structure
Every globular protein relies on its tertiary structure to perform its function, so rather than trying to find representative proteins for tertiary structure (an almost impossible task!), we focus here on a few elements of tertiary structure that are common to many proteins. These are the structural domains and they differ from the structural motifs of supersecondary structure by being larger (25-500 amino acids), having a conserved amino acid sequence, and a history of evolving and functioning independently of the protein chains they are found in. Structural domains are fundamental units of tertiary structure and are found in more than one protein. A structural domain is selfstabilizing and often folds independently of the rest of the protein chain.
Leucine zipper
A common feature of many eukaryotic DNA binding proteins, leucine zippers are characterized by a repeating set of leucine residues in a protein that interact like a zipper to favor dimerization. Another part of the domain has amino acids (commonly arginine and lysine) that allow it to interact with the DNA double helix (Figure 2.63). Transcription factors that contain leucine zippers include Jun-B, CREB, and AP-1 fos/ jun.
Zinc fingers
The shortest structural domains are the zinc fingers, which get their name from the fact that one or more coordinated zinc ions stabilize their finger-like structure. Despite their name, some zinc fingers do not bind zinc. There are many structural domains classified as zinc fingers and these are grouped into different families. Zinc fingers were first identified as components of DNA binding transcription factors, but others are now known to bind RNA, protein, and even lipid structures. Cysteine and histidine side chains commonly play roles in coordinating the zinc.
Src SH2 domain
The Src oncoprotein contains a conserved SH2structural domain that recognizes and binds phosphorylated tyrosine side chains in other proteins (Figure 2.65). Phosphorylation is a fundamental activity in signaling and phosphorylation of tyrosine and interaction between proteins carrying signals is critically needed for cellular communication. The SH2domain is found in over 100 human proteins.
Helix-turn-helix domain
Helix-turn-helix is a common domain found in DNA binding proteins, consisting of two α-helices separated by a small number of amino acids. As seen in Figure 2.66, the helix parts of the structural domain interact with the bases in the major groove of DNA. Individual α-helices in a protein are part of a helix-turn-helix structure, where the turn separates the individual helices.
Pleckstrin homology domain
Pleckstrin Homology (PH) domains are protein domains with important functions in the process of signaling. This arises partly from the affinity for binding phosphorylated inositides, such as PIP2 and PIP3, found in Figure 2.66 - Helix-Turn-Helix Domain of a Protein Bound to DNA Wikipedia Figure 2.65 - SH2 Domain Wikipedia biological membranes. PH domains can also bind to G-proteins and protein kinase C. The domain spans about amino acids and is found in numerous signaling proteins. These include Akt/Rac Serine/ Threonine Protein Kinases, Btk/ltk/Tec tyrosine protein kinases, insulin receptor substrate (IRS-1), Phosphatidylinositolspecific phospholipase C, and several yeast proteins involved in cell cycle regulation.
Structural globular proteins
Enzymes catalyze reactions and proteins such as hemoglobin perform important specialized functions. Evolutionary selection has reduced and eliminated waste so that we can be sure every protein in a cell has a function, even though in some cases we may not know what it is. Sometimes the structure of the proFigure 2.68 - Relationship of basement membrane to epithelium, endothelium, and connective tissue tein is its primary function because the structure provides stability, organization, connections other important properties. It is with this in mind that we present the following proteins.
Basement membrane
The basement membrane is a layered extracellular matrix of tissue comprised of protein fibers (type IV collagen) and glycosaminoglycans that separates the epithelium from other tissues (Figure 2.68). More importantly, the basement membrane acts like a glue to hold tissues together. The skin, for example, is anchored to the rest of the body by the basement membrane. Basement membranes provide an interface of interaction between cells and the environment around them, thus facilitating signaling processes. They play roles in differentiation during embryogenesis and also in maintenance of function in adult organisms.
Actin
Actin is the most abundant globular protein found in most types of eukaryotic cells, comprising as much as 20% of the weight of muscle cells. Similar proteins have been identified in bacteria (MreB) and archaeons (Ta0583). Actin is a monomeric subunit able to polymerize readily into two different types of filaments. Microfilaments are major component of the cytoskeleton and are acted on by myosin in the contraction of muscle cells (See HERE). Actin will be discussed in more detail in the next section HERE.
Intermediate Filaments
Intermediate filaments are a part of the cytoskeleton in many animal cells and are comprised of over 70 different proteins. They are called intermediate because their size (average diameter = 10 nm) is between that of the microfilaments (7 nm) and the microtubules (25 nm).
The intermediate filament components include fibrous proteins, such as the keratins and the lamins, which are nuclear, as well as cytoplasmic forms. Intermediate filaments give flexibility to cells because of their own physical properties. They can, for example, be stretched to several times of their original length.
Six types
There are six different types of intermediate filaments. Type I and II are acidic or basic and attract each other to make larger filaments. They include epithelial keratins and trichocytic keratins (hair components). Type III proteins include four structural proteins - desmin, GFAP (glial fibrillary acidic protein), peripherin, and vimentin. Type IV also is a grouping of three proteins and one multiprotein structure (neurofilaments). The three proteins are α-internexin, synemin, and syncoilin. Type V intermediate filaments encompass the lamins, which give structure to the nucleus. Phosphorylation of lamins leads to their disassembly and this is important in the process of mitosis. The Type VI category includes only a single protein known as nestin.
Tubulin
A third type of filament found in cells is that of the microbutules. Comprised of a polymer of two units of a globular protein called tubulin, microtubules provide “rails” for motor proteins to move organelles and other “cargo” from one part of a cell to another. Microtubules and tubulin are discussed in more detail HERE.
Vimentin
Vimentin (Figure 2.70) is the most widely distributed protein of the intermediate filaments. It is expressed in fibroblasts, leukocytes, and blood vessel endothelial cells. The protein has a significant role maintaining the position of organelles in the cytoplasm, with attachments to the nucleus, mitochondria, and endoplasmic reticulum (Figure 2.70). Vimentin provides elasticity to cells and resilience that does not arise from the microtubules or microfilaments. Wounded mice that lack the vimentin gene survive, but take longer to heal wounds than wild type mice. Vimentin also controls the movement of cholesterol from lysosomes to the site of esterification. The result is a reduction in the amount of cholesterol stored inside of cells and has implications for adrenal cells, which must have esters of cholesterol.
Mucin
Mucins are a group of proteins found in animal epithelial tissue that have many glycosyl residues on them and typically are of high molecular weight (1 to 10 million Da). They are gel-like in their character and are often used for lubrication. Mucus is comprised of mucins. In addition to lubrication, mucins also help to control mineralization, such as bone formation in vertebrate organisms and calcification in echinoderms. They also play roles in the immune system by helping to bind pathogens. Mucins are commonly secreted onto mucosal surfaces (nostrils, eyes, mouth, ears, stomach, genitals, anus) or into fluids, such as saliva. Because of their extensive mucosylation, mucins hold a considerable amount of water (giving them the “slimy” feel) and are resistant to proteolysis.
Vinculin
Vinculin (Figure 2.72) is a membrane cytoskeletal protein found in the focal adhesion structures of mammalian cells. It is found at cell-cell and cell-matrix junctions and interacts with integrins, talin, paxillins and F-actin. Vinculin is thought to assist (along with other proteins) in anchoring actin microfilaments to the membrane (Figure 2.71). Binding of vinculin to actin and to talin is regulating by polyphosphoinositides and can be inhibited by acidic phospholipids.
Syndecans
Syndecans are transmembrane proteins that make a single pass with a long amino acid chain (24-25 residues) through plasma membranes and facilitate G proteincoupled receptors’ interaction with Figure 2.71 - Actin filaments (green) attached to vinculin in focal adhesion (red) Wikipedia ligands, such as growth factors, fibronectin, collagens (I, III, and IV) and antithrombin-1. Syndecans typically have 3-5 heparan sulfate and chondroitin sulfate chains attached to them.
Heparan sulfate can be cleaved at the site of a wound and stimulate action of fibroblast growth factor in the healing process. The role of syndecans in cell-cell adhesion is shown in mutant cells lacking syndecan I that do not adhere well to each other. Syndecan 4 is also known to adhere to integrin. Syndecans can also inhibit the spread of tumors by the ability of the syndecan 1 ectodomain to suppress growth of tumor cells without affecting normal epithelial cells.
Defensin
Defensins (Figure 2.73) are a group of small cationic proteins (rich in cysteine residues) that serve as host defense peptides in vertebrate and invertebrate organisms. They protect against infection by various bacteria, fungi, and viruses. Defensins contain between 18 and 45 amino acids with (typically) about 6- 8 cysteine residues. In the immune system, defensins help to kill bacteria engulfed by phagocytosis by epithelial cells and neutrophils. They kill 120 Figure 2.72 - Vinculin Wikipedia bacteria by acting like ionophores - binding the membrane and opening pore-like structures to release ions and nutrients from the cells.
Focal adhesions
In the cell, focal adhesions are structures containing multiple proteins that mechanically link cytoskeletal structures (actin bundles) with the extracellular matrix. They are dynamic, with proteins bringing and leaving with signals regarding the cell cycle, cell motility, and more almost constantly. Focal adhesions serve as anchors and as a signaling hub at cellular locations where integrins bind molecules and where membrane clustering events occur. Over 100 different proteins are found in focal adhesions.
Focal adhesions communicate important messages to cells, acting as sensors to update information about the status of the extracellular matrix, which, in turn, adjusts/ affects their actions. In sedentary cells, they are stabler than in cells in motion because when cells move, focal adhesion contacts are established at the “front” and removed at the rear as motion progresses. This can be very important in white blood cells’ ability to find tissue damage.
Ankyrin
Ankyrins (Figure 2.74) are a family of membrane adaptor proteins serving as “anchors” to interconnect integral membrane proteins to the spectrin-actin membrane cytoskeleton. Ankyrins are anchored to the plasma membrane by covalently linked palmitoyl-CoA. They bind to the β subunit of spectrin and at least a dozen groups of integral membrane proteins. The ankyrin proteins contain four functional domains: an N-terminal region with 24 tandem ankyrin repeats, a central spectrin-binding domain, a “death domain” interacting with apoptotic proteins, and a C-terminal regulatory domain that is highly varied significantly among different ankyrins.
Spectrin
Spectrin (Figures 2.75 & 2.76) is a protein of the cellular cytoskeleton that plays an important role in maintaining its structure and the integrity of the plasma membrane. In animals, spectrin gives red blood cells their shape. Spectrin is located inside the inner layer of the eukaryotic plasma membrane where it forms a network of pentagonal or hexagonal arrangements.
Spectrin fibers collect together at junctional complexes of actin and is also attached to ankyrin for stability, as well as numerous integral membrane proteins, such as glycophorin.
Integrins
In multicellular organisms, cells need connections, both to each other and to the extracellular matrix. Facilitating these attachments at the cellular end are transmembrane proteins known as integrins (Figure 2.77). Integrins are found in all metazoan cells. Ligands for the integrins include collagen, fibronectin, laminin, and vitronectin. Integrins function not only in attachment, but also in communication, cell migration, virus linkages (adenovirus, for example), and blood clotting. Integrins are able to sense chemical and mechanical signals about the extracellular matrix and move that information to intracellular domains as part of the process of signal transduction. Inside the cells, responses to the signals affect cell shape, regulation of the cell cycle, movement, or changes in other cell receptors in the membrane. The process is dynamic and allows for rapid responses as may be necessary, for example in the process of blood clotting, where the integrin known as GPIbIIIa (on the surface of blood platelets) attaches to fibrin in a clot as it develops.
Integrins work along with other receptors, including immunoglobulins, other cell adhesion molecules, cadherins, selectins, and syndecans. In mammals the proteins have a large number of subunits - 18 α- and 8 β-chains. They are a bridge between its links outside the cell to the extracellular matrix (ECM) and its links inside the cell to the cytoskeleton. Integrins play central role in formation and stability of focal adhesions. These are large molecular complexes arising from clustering of integrin-ECM connections. In the process of cellular movement, integrins at the “front” of the cell (in the direction of the movement), make new attachments to substrate and release connections to substrate in the back of the cell. These latter integrins are then endocytosed and reused.
Integrins also help to modulate signal transduction through tyrosine kinase receptors in the cell membrane by regulating movement of adapters to the plasma membrane. β1c integrin, for example, recruits the Shp2 phosphatase to the insulin growth factor receptor to cause it to become dephosphorylated, thus turning off the signal it communicates. Integrins can also help to recruit signaling molecules inside of the cell to activated tyrosine kinases to help them to communicate their signals.
Cadherins
Cadherins (Figure 2.78) constitute a type-1 class of transmembrane proteins playing important roles in cell adhesion. They require calcium ions to function, forming adherens junctions that hold tissues together (See Figure 2.69). Cells of a specific cadherin type will preferentially cluster with each other in preference to associating with cells containing a different cadherin type. Caderins are both receptors and places for ligands to attach. They assist in the proper positioning of cells in development, separation of different tissue layers, and cell migration.
Selectins
Selectins (Figure 2.79) are cell adhesion glycoproteins that bind to sugar molecules. As such, they are a type of lectin - proteins that bind sugar polymers (see HERE also). All selectins have an N-terminal calcium-dependent lectin domain, a single transmembrane domain, and an intracellular cytoplasmic tail.
There are three different types of selectins, 1) E-selectin (endothelial); 2) L (lymphocytic; and 3) P (platelets and endothelial cells. Selectins function in lymphocyte homing (adhesion of blood lymphocytes to cells in lymphoid organs), in inflammation processes, and in cancer metastasis. Near the site of inflammation, P-selectin on the surface of blood capillary cells interacts with glycoproteins on leukocyte cell surfaces. This has the effect of slowing the movement of the leukocyte. At the target site of inflammation, E- selectin on the endothelial cells of the blood vessel and L-selectin on the surface of the leukocyte bind to their respective carbohydrates, stopping the leukocyte movement. The leukocyte then crosses the wall of the capillary and begins the immune response. Selectins are involved in the inflammatory processes of asthma, psoriasis, multiple scleroris, and rheumatoid arthritis.
Laminins
Laminins are extracellular matrix glycoproteins that a major components of the basal lamina and affect cell differentiation, migration, and adhesion. They are secreted into the extracellular matrix where they are incorporated and are essential for tissue maintenance and survival. When laminins are defective, muscles may not form properly and give rise to muscular dystrophy.
Laminins are associated with fibronectin, entactin, and perlecan proteins in type IV collagen networks and bind to integrin receptors in the plasma membrane. As a consequence, laminins contribute to cellular attachment, differentiation, shape, and movement. The proteins are trimeric in structure, having one α-chain, a β-chain, and a γ-chain. Fifteen combinations of different chains are known.
Vitronectin
Vitronectin is a glycoprotein (75kDa) found in blood serum (platelets), the extracellular matrix, and in bone. It promotes the process of cell adhesion and spreading and binds to several protease inhibitors (serpins). It is secreted from cells and is believed to play roles in blood clotting and the malignancy of tumors. One domain of vitronectin binds to plasminogen activator inhibitor and acts to stabilize it. Another domain of the protein binds to cellular integrin proteins, such as the vitronectin receptor that anchors cells to the extracellular matrix.
Catenins
Catenins are a family of proteins interacting with cadherin proteins in cell adhesion (Figure 2.69). Four main types of catenins are known, α-, β-, γ-, and δ-catenin. Catenins play roles in cellular organization before development occurs and help to regulate cellular growth. α-catenin and β-catenin are found at adherens junctions with cadherin and help cells to maintain epithelial layers. Cadherins are connected to actin filaments of the cytoskeleton and catenins play the critical role. Catenins are important for the process whereby cellular division is inhibited when cells come in contact with each other (contact inhibition).
When catenin genes are mutated, cadherin cell adhesions can disappear and tumorigenesis may result. Catenins have been found to be associated with colorectal and numerous other forms of cancer.
Glycophorins
All of the membrane proteins described so far are notable for the connections they make to other proteins and cellular structures. Some membrane proteins, though, are designed to reduce cellular connections to proteins of other cells. This is particularly important for blood cells where “stickiness” is undesirable except where clotting is concerned.
Glycophorins (Figure 2.80) are membrane-spanning sialoglycoproteins of red blood cells. They are heavily glycosylated (60%).and rich in sialic acid, giving the cells a very hydrophilic (and negatively charged) coat, which enables them to circulate in the bloodstream without adhering to other cells or the vessel walls.
Five glycophorins have been identified - four (A,B,C,and D) from isolated membranes and a fifth form (E) from coding in the human genome. The proteins are abundant, forming about 2% of the total membrane proteins in these cells. Glycophorins have important roles in regulating RBC membrane mechanical properties and shape. Because some glycophorins can be expressed in various nonerythroid tissues (particularly Glycophorin C), the importance of their interactions with the membrane skeleton may have a considerable biological significance.
Cooperativity and allosterism - quaternary structure
Quaternary structure, of course describes the interactions of individual subunits of a multi-subunit protein (Figure 2.81). The result of these interactions can give rise to important biological phenomena, such as cooperative binding of substrates to a protein and allosteric effects on the action of an enzyme.
Allosteric effects can occur by a series of mechanisms, but a common feature is that binding of an effector to an enzyme subunit causes (or locks) the enzyme in either a Tstate (less activity) or an R-state (more activity). Effectors can be enzyme substrates (homotropic effectors) or non-substrates (heterotropic effectors). Allosterism will be covered in more depth in the Catalysis chapter HERE.
We begin our consideration of quaternary structure with a discussion of cooperativity, how it arises in the multi-subunit protein hemoglobin and how its properties contrast with those of the related, single subunit protein myoglobin.
Cooperativity
Cooperativity is defined as the phenomenon where binding of one ligand molecule by a protein favors the binding of additional molecules of the same type. Hemoglobin, for example, exhibits cooperativity when the binding of an oxygen molecule by the iron of the heme group in one of the four subunits causes a slight conformation change in the subunit. This happens because the heme iron is attached to a histidine side chain and binding of oxygen ‘lifts’ the iron along with the histidine ring (also known as the imidazole ring).
Movie 2.3 - Hemoglobin’s structural changes on binding oxygen Wikipedia
Since each hemoglobin subunit interacts with and influences the other subunits, they too are induced to change shape slightly when the first subunit binds to oxygen (a transition described as going from the T-state to the R-state). These shape changes favor each of the remaining subunits binding oxygen, as well. This is very important in the lungs where oxygen is picked up by hemoglobin, because the binding of the first oxygen molecule facilitates the rapid uptake of more oxygen molecules. In the tissues, where the oxygen concentration is lower, the oxygen leaves hemoglobin and the proteins flips from the R-state back to the Tstate.
CO2 transport
Cooperativity is only one of many fascinating structural aspects of hemoglobin that help the body to receive oxygen where it is needed and pick it up where it is abundant. Hemoglobin also assists in the transport of the product of cellular respiration (carbon dioxide) from the tissues producing it to the lungs where it is exhaled. Like the binding of oxygen to hemoglobin, binding of other molecules to hemoglobin affects its affinity for oxygen. The effect is particularly pronounced when comparing the oxygen binding characteristics of hemoglobin’s four subunits with the oxygen binding of the related protein myoglobin’s single subunit (Figure 2.83).
Different oxygen binding
Like hemoglobin, myoglobin contains an iron in a heme group that binds to oxygen. The structure of the globin protein in myoglobin is very similar to the structure of the globins in hemoglobin and hemoglobin is thought to have evolved from myoglobin in evolutionary history. As seen in Figure 2.83, the binding curve of hemoglobin for oxygen is S-shaped (sigmoidal), whereas the binding curve for myoglobin is hyperbolic. What this tells us is that hemoglobin’s affinity for oxygen is low at a low concentration oxygen, but increases as the oxygen concentration increases. Since myoglobin very quickly saturates with oxygen, even under low oxygen concentrations, it says that its affinity for oxygen is high and doesn’t change.
Because myoglobin has only a single subunit, binding of oxygen by that subunit can’t affect any other subunits, since there are no other subunits to affect. Consequently, cooperativity requires more than one subunit. Therefore, hemoglobin can exhibit cooperativity, but myoglobin can’t. It is worth noting that simply having multiple subunits does not mean cooperativity will exist. Hemoglobin is one protein that exhibits the characteristic, but many multisubunit proteins do not.
Interactive 2.2 - Hemoglobin in the presence (top) and absence (bottom) of oxygen
Storage vs. delivery
The lack of ability of myoglobin to adjust its affinity for oxygen according to the oxygen concentration (low affinity at low oxygen concentration, such as in tissues and high affinity at high oxygen concentration, such as in the lungs) means it is better suited for storing oxygen than for delivering it according to the varying oxygen needs of and animal body. As we shall see, besides cooperativity, hemoglobin has other structural features that allow it to deliver oxygen precisely where it is needed most in the body.
Bohr effect
The Bohr Effect was first described over 100 years ago by Christian Bohr, father of the famous physicist, Niels Bohr. Shown graphically (Figures 2.86, 2.87, and 2.88), the observed effect is that hemoglobin’s affinity for oxygen decreases as the pH decreases and as the concentration of carbon dioxide increases. Binding of the protons and carbon dioxide by amino Figure 2.85 - Sequential model of binding. The sequential model is one way to explain hemoglobin’s cooperativity. Squares represent no oxygen bound. Circles represent subunits bound with oxygen and rounded subunits correspond to units whose affinity for oxygen increases by interacting with a subunit that has bound oxygen. Image by Aleia Kim acid side chains in the globin proteins helps to facilitate structural changes in them. Most commonly, the amino acid affected by protons is histidine #146 of the β strands. When this happens, the ionized histidine can form an ionic bond with the side chain of aspartic acid #94, which has the effect of stabilizing the T-state (reduced oxygen binding state) and releasing oxygen. Other histidines and the amine of the amino terminal amino acids in the α-chains are also binding sites for protons.
2,3-BPG
Another molecule favoring the release of oxygen by hemoglobin is 2,3- bisphosphoglycerate (also called 2,3-BPG or just BPG - Figure 2.89). Like protons and carbon dioxide, 2,3-BPG is produced by actively respiring tissues, as a byproduct of glucose metabolism. The 2,3-BPG mole cule fits into the ‘hole of the donut’ of adult hemoglobin (Figure 2.89). Such binding of 2,3-BPG favors the T-state (tight - low oxygen binding) of hemoglobin, which has a reduced affinity for oxygen. In the absence of 2,3-BPG, hemoglobin can more easily exist in the R-state (relaxed - higher oxygen binding), which has a high affinity for oxygen.
Smokers
Notably, the blood of smokers is higher in the concentration of 2,3-BPG than non-smokers, so more of their hemoglobin remains in the T-state and thus the oxygen carrying capacity of smokers is lower than non-smokers.Another reason why smokers’ oxygen carrying capacity is lower than that of non-smokers is that cigarette smoke contains carbon monoxide and this molecule, which has almost identical dimensions to molecular oxygen, effectively outcompetes with oxygen for binding to the iron atom of heme (Figure 2.90). Part of carbon monoxide’s toxicity is due to its ability to bind hemoglobin and prevent oxygen from binding.
Carbon dioxide
Carbon dioxide binds to form a carbamate when binding the α-amine of each globin chain. The process of forming this structure releases a proton, which helps to further enhance the Bohr effect. Physiologically, the binding of CO2 and H+ has significance because actively respiring tissues (such as contracting muscles) require oxygen and release protons and carbon dioxide. The higher the concentration of protons and carbon dioxide, the more oxygen is released to feed the tissues that need it most.
About 40% of the released protons and about 20% of the carbon dioxide are carried back to the lungs by hemoglobin. The remainder travel as part of the bicarbonate buffering system or as dissolved CO2. In the lungs, the process reverses itself. The lungs have a higher pH than respiring tissues, so protons are released from hemoglobin and CO2 too is freed to be exhaled.
Fetal hemoglobin
Adult hemoglobin releases oxygen when it binds 2,3- BPG. This is in contrast to fetal hemoglobin, which has a slightly different configuration (α2γ2) than adult hemoglobin (α2β2). Fetal hemoglobin has a greater affinity for oxygen than maternal hemoglobin, allowing the fetus to obtain oxygen effectively from the mother’s blood. Part of the reason for fetal hemoglobin’s greater affinity for oxygen is that it doesn’t bind 2,3-BPG. Consequently, fetal hemoglobin remains in the R-state much more than adult hemoglobin and because of this, fetal hemoglobin has greater affinity for oxygen than adult hemoglobin and can take oxygen away from adult hemoglobin. Thus, the fetus can get oxygen from the mother.
Sickle cell disease
Mutations to the globin genes coding for hemoglobin can sometimes have deleterious consequences. Sickle cell disease (also called sickle cell anemia) is a genetically transmitted disease that arises from such mutations. There are different forms of the disease. It is a recessive trait, meaning that to be afflicted with it, an individual must inherit two copies of the mutated gene.
The predominant form of hemoglobin in adults is hemoglobin A, designated HbA (two α chains and two β chains). The mutant form is known as HbS. The most common mutation is an A to T mutation in the middle of the codon for the seventh amino acid (some counting schemes call it the sixth amino acid) of the β-chain. This results in conversion of a GAG codon to GTG and thus changes the amino acid specified at that position from a glutamic acid to a valine. This minor change places a small hydrophobic patch of amino acids on the surface of the β-globin chains.
Polymerization
Under conditions of low oxygen, these hydrophobic patches will associate with each other to make long polymers of hemoglobin molecules. The result is that the red blood cells containing them will change shape from being rounded to forming the shape of a sickle (Figure 2.94). Rounded red blood cells readily make it through tiny capillaries, but sickleshaped cells do not.
Worse, they block the flow of other blood cells. Tissues where these blockages occur are already low in oxygen, so stopping the flow of blood through them causes them to go quickly anaerobic, causing pain and, in some cases, death of tissue. In severe circumstances, sickled red blood cells death may result. The disease is referred to as an anemia because the sickling of the red blood cells targets them for removal by the blood monitoring system of the body, so a person with the disease has chronically reduced numbers of red blood cells.
Heterozygote advantage
Interestingly, there appears to be a selective advantage to people who are heterozygous for the disease in areas where malaria is prominent. Heterozygotes do not suffer obvious ill effects of the disease, but their red blood cells appear to be more susceptible to rupture when infected. As a consequence, the parasite gets less of a chance to reproduce and the infected person has a greater chance of survival.
The protective effect of the mutant gene, though, does not extend to people who suffer the full blown disease (homozygotes for the mutant gene). Treatments for the disease include transfusion, pain management, and avoidance of heavy exertion. The drug hydroxyurea has been linked to reduction in number and severity of attacks, as well as an increase in survival time1,2. It appears to work by reactivating expression of the fetal hemoglobin gene, which typically is not synthesized to any significant extent normally after about 6 weeks of age.
Oxygen binding
Animals have needs for oxygen that differ from all other organisms. Oxygen, of course, is the terminal electron acceptor in animals and is necessary for electron transport to work. When electron transport is functioning, ATP generation by cells is many times more efficient than when it is absent. Since abundant ATP is essential for muscular contraction and animals move around a lot - to catch prey, to exercise, to escape danger, etc., having an abundant supply of oxygen is important.
This is particularly a concern deep inside tissues where diffusion of oxygen alone (as occurs in insects) does not deliver sufficient quantities necessary for long term survival. The issue is not a problem for plants since, for the most part, their motions are largely related to growth and thus don’t have rapidly changing needs/demands for oxygen that animals have. Unicellular organisms have a variety of mechanisms for obtaining oxygen and surviving without it. Two other important oxygen binding proteins besides hemoglobin are myoglobin and hemocyanin.
Myoglobin
Myoglobin is the primary oxygen-storage protein found in animal muscle tissues. In contrast to hemoglobin, which circulates throughout the body, myoglobin protein is only found in muscle tissue and appears in the blood only after injury. Like hemoglobin, myoglobin binds oxygen at a prosthetic heme group it contains.
The red color of meat arises from the heme of myoglobin and the browning of meat by cooking it comes from oxidation of the ferrous (Fe++) ion of myoglobin’s heme to the ferric (Fe+++) ion via oxidation in the cooking process. As meat sits in our atmosphere (an oxygen-rich environment), oxidation of Fe++ to Fe+++ occurs, leaving the brown color noted above. If meat is stored in a carbon monoxide (CO) environment, CO binds to the heme group and reduces the amount of oxidation, keeping meat looking red for a longer period of time.
High affinity
Myoglobin (Figure 2.97) displays higher affinity for oxygen at low oxygen concentrations than hemoglobin and is therefore able to absorb oxygen delivered by hemoglobin under these conditions. Myoglobin’s high affinity for oxygen makes it better suited for oxygen storage than delivery. The protein exists as a single subunit of globin (in contrast to hemoglobin, which contains four subunits) and is related to the subunits found in hemoglobin. Mammals that dive deeply in the ocean, such as whales and seals, have muscles with particularly high abundance of myoglobin. When oxygen concentration in muscles falls to low levels, myoglobin releases its oxygen, thus functioning as an oxygen “battery” that delivers oxygen fuel when needed and holding onto it under all other conditions. Myoglobin holds the distinction of being the first protein for which the 3D structure was determined by X-ray crystallography by John Kendrew in 1958, an achievement for which he later won the Nobel Prize.
Hemocyanin
Hemocyanin is the protein transporting oxygen in the bodies of molluscs and arthropods. It is a coppercontaining protein found not within blood cells of these organisms, but rather is suspended in the circulating hemolymph they possess. The oxygen binding site of hemocyanin contains a pair of copper(I) cations directly coordinated to the protein by the imidazole rings of six histidine side chains.
Most, but not all hemocyanins bind oxygen non-cooperatively and are less efficient than hemoglobin at transporting oxygen. Notably, the hemocyanins of horseshoe crabs and some other arthropods do, in fact, bind oxygen cooperatively. Hemocyanin contains many subunit proteins, each with two copper atoms that can bind one oxygen molecule (O2). Subunit proteins have atomic masses of about 75 kilodaltons (kDa). These may be arranged in dimers or hexamers depending on species. Superstructures comprised of dimer or hexamer complexes are arranged in chains or clusters and have molecular weights of over 1500 kDa.
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textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/02%3A_Structure_and_Function/2.05%3A_Structure_and_Function-_Protein_Function_II.txt
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princeton-nlp/TextbookChapters
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Source: BiochemFFA_2_4.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
To this point, the proteins we have discussed have not been catalysts (enzymes). The majority of proteins in cells, however, catalyze reactions. In this section we begin our discussion of a subclass of proteins that catalyze reactions releasing energy and convert it into mechanical force. These operate at the cellular and organismal level and are known as motor proteins. Motor proteins rely on globular structural proteins, so it is important that we describe how these cellular “railways” are assembled before discussing the motor proteins themselves. There are two relevant fibrous structures serving as rails for motor proteins. They are:
1. microfilaments (composed of an actin polymer) and
2. microtubules (composed of a polymer of tubulin.
Actin
The monomeric unit of actin is called G-actin (globular actin) and the polymer is known as F-actin (filamentous actin). Filaments of Factin comprise the smallest filaments of cells known as microfilaments (Figure 2.101). Actin is essential for muscular contraction and also has diverse roles in cellular signaling and maintenance of cell junctions. In conjunction with other proteins, actin has numerous interactions with the cell membrane. The β- and γ-forms of actin are components of the cytoskeleton and facilitate motility inside of cells. α-actin is important in muscle tissues, where it is used by myosin in the mechanical process of contraction (See HERE).
Monomeric and polymeric forms of actin play roles in cellular activities relating to motion. Two parallel F-actin strands can pair with each other and create a double helical structure with 2.17 subunits per turn of the helix. Helical F-actin in muscles contains tropomyosin, which covers the actin binding sites for myosin in resting muscles to prevent contraction. Other proteins bound to actin muscle filaments include the troponins (I, T, and C).
Actin Cellular Action
Examples of actin action at the cellular level include cell motility, cytokinesis, intracellular transport of vesicles and organelles, and cell shape. Each actin monomer is bound to a molecule of ATP or ADP and the presence of one of these is essential for proper G-actin functioning.
The role of ATP
In the monomer, actin is more commonly bound to ATP, whereas in the filaments, it is typically bound to ADP. Actin is an inefficient ATPase, breaking the molecule down slowly, but the catalysis speeds up as much as 40,000 fold when the monomer begins to polymerize. Actin also has a binding site for divalent cations - either calcium or magnesium. F-
Actin binds to structural proteins at the adherens junction (Figure 2.102). These include α-actinin, vinculin (provides a membrane connection and connections to the catenins and cadherin).
Polymerization
Polymerization of actin begins with a nucleating event (Figure 2.103). One factor known to affect the process is known as the Arp 2/3 complex. It does this by mimicking an actin dimer, starting an autocatalytic process of actin assembly. The Arp 2/3 complex plays roles both in the initiation of polymerization of new actin filaments as well as the formation of branches in the filaments.
Two proteins play roles in modulating polymer growth. Thymosin functions on the end of actin filaments to control growth. Profilin works on G-actin monomers exchanging ADP for ATP, promoting addition of monomers to a growing chain.
F-actin filaments are held together by relatively weak bonds compared to the covalent bonds of the monomers of nucleic acids, thus allowing for easier disassembly when desired. Actin’s amino acid sequence is optimized, having diverged only a relatively small amount (20%) between algae and humans. Mutations in the actin gene result in muscular diseases and/or deafness.
Tubulin
Tubulin proteins are the monomeric building blocks of eukaryotic microtubules (Figure 2.104 & 2.105). Bacterial (TubZ) and archaeon (FtsZ) equivalents are known. The α-tubulin and β-tubulin proteins polymerize to make microtubule structures in the cytoplasm of cells. Microtubules are major components of the cytoskeleton of eukaryotic cells, providing structural support, transport within the cell, and functions necessary for segregation of DNAs during cell division.
Dimerization of the α-tubulin and β-tubulin proteins is necessary for polymerization and requires that the subunits bind to GTP. Microtubules only grow in one direction. β- tubulin is found on the plus end of the tubule (growth end = plus end) and α-tubulin is exposed on the other end (non-growth end = minus end). Dimers of α-tubulin/β-tubulin are incorporated into growing microtubules in this orientation. If a dimer is bound to GDP instead of GTP, it tends to be unstable and fall apart, whereas those bound to GTP stably assemble into microtubules.
Microtubules
Microtubules, along with microfilaments and intermediate filaments (see HERE) constitute the cytoskeleton of cells. Found in the cytoplasm, they are found in eukaryotic cells, as well as some bacteria. Microtubules help to give cells structure. They comprise the inner structure of flagella and cilia and provide rail-like surfaces for the transport of materials within cells.
Polymerization of α- tubulin and β-tubulin to form microtubules occurs after a nucleating event. Individual units get arranged in microtubule organizing centers (MTOCs), an example of which is the centrosome. Centrosomes are focal points of connection of microtubules. Basal bodies of cilia and flagella also help to organize microtubules.
Motor proteins
From the transport of materials within a cell to the process of cytokinesis where one cell splits into two in mitosis, a cell has needs for motion at the molecular level. Secretory vesicles and organelles must be transported. Chromosomes must be separated in mitosis and meiosis.
The proteins dynein and kinesin (Figure 2.106) are necessary for intracellular movement. These motor proteins facilitate the movement of materials inside of cells along microtubule “rails”. These motor proteins are able to move along a portion of the cytoskeleton by converting chemical energy into motion with the hydrolysis of ATP. An exception is flagellar rotation, which uses energy provided from a gradient created by a proton pump.
Kinesins and dyneins
As noted, kinesins and dyneins navigate in cells on microtubule tracks (Figure 2.108 & Movie 2.4). Most kinesins move in the direction of the synthesis of the microtubule (+ end movement), which is generally away from the cell center and the opposite direction of movement of dyneins, which are said to do retrograde transport toward the cell center. Both proteins provide movement functions necessary for the processes of mitosis and meiosis. These include spindle formation, chromosome separation, and shuttling of organelles, such as the mitochondria, Golgi apparatuses, and vesicles.
Kinesins are comprised of two heavy chains and two light chains. The head motor domains of heavy chains (in the feet) use energy of ATP hydrolysis to do mechanical work for the movement along the microtubules. There are at least fourteen distinct kinesin families and probably many related ones in addition.
Dyneins are placed into two groups - cytoplasmic and axonemal (also called ciliary or flagellar dyneins - Figure 2.109). Dyneins are more complex in structure than kinesins with many small polypeptide units. Notably, plants do not have dynein motor proteins, but do contain kinesins.
Movie 2.4 The motor protein kinesin walking down a microtubule. Image used with permission (Public Domain; zp706).
Myosin
An important group of motor proteins in the cell is the myosins. Like kinesins and dyneins, myosins use energy from hydrolysis of ATP for movement. In this case, the movement is mostly not along microtubules, but rather along microfilaments comprised of a polymer of actin (F-actin). Movement of myosin on actin is best known as the driving force for muscular contraction. Myosins are a huge family of proteins, all of which bind to actin and all of which involve motion. Eighteen different classes of myosin proteins are known.
Myosin II is the form responsible for generating muscle contraction. It is an elongated protein formed from two heavy chains with motor heads and two light chains. Each myosin motor head binds actin and has an ATP binding site. The myosin heads bind and hydrolyze ATP. This hydrolysis produces the energy necessary for myosin to walk toward the plus end of an actin filament.
Non-muscle myosin IIs provide contraction needed to power the action of cytokinesis. Other myosin proteins are involved in movement of non-muscle cells. Myosin I is involved in intracellular organization. Myosin V performs vesicle and organelle transport. Myosin XI provides movement along cellular microfilament networks to facilitate organelle and cytoplasmic streaming in a particular direction.
Structure
Myosins have six subunits, two heavy chains and four light chains. Myosin proteins have domains frequently described as a head and a tail (Figure 2.111). Some also describe an intermediate hinge region as a neck. The head portion of myosin is the part that binds to actin. It uses energy from ATP hydrolysis to move along the actin filaments. In muscles, myosin proteins form aggregated structures referred to as thick filaments. Movements are directional.
Structural considerations of muscular contraction
Before we discuss the steps in the process of muscular contraction, it is important to describe anatomical aspects of muscles and nomenclature.
There are three types of muscle tissue - skeletal (striated), smooth, and (in vertebrates) cardiac. We shall concern ourselves mostly here with skeletal muscle tissue. Muscles may be activated by the central nervous system or, in the case of smooth and cardiac muscles, may contract involuntarily. Skeletal muscles may be slow twitch or fast twitch.
Sarcomeres
Sarcomeres are described as the basic units comprising striated muscles and are comprised of thick (myosin) and thin (actin) filaments and a protein called titin. The filaments slide past each other in muscular contraction and then backwards in muscular relaxation. They are not found in smooth muscles.
Under the microscope, a sarcomere is the region between two Z-lines of striated muscle tissue (Figure 2.112). The Z-line is the distinct, narrow, dark region in the middle of an I-band. Within the sarcomere is an entire Aband with its central H-zone. Within the Hzone are located tails of myosin fibers, with the head pointed outwards from there projecting all the way to the I-band. The outside of the Aband is the darkest and it gets lighter moving towards the center.
Within the Iband are located thin filaments that are not occupied with thick myosin filaments. The Aband contains intact thick filaments overlaying thin filaments except in the central H zone, which contains only thick filaments. In the center of the H-zone is a line, known as the M-line. It contains connecting elements of the cellular cytoskeleton. In muscular contraction, myosin heads walk along pulling their tails over the actin thin filaments, using energy from hydrolysis of ATP and pulling them towards the center of the sarcomere.
Sarcolemma
The sarcolemma (also known as the myolemma) is to muscle cells what the plasma membrane is to other eukaryotic cells - a barrier between inside and outside. It contains a lipid bilayer and a glycocalyx on the outside of it. The glyocalyx contains polysaccharides and connects with the basement membrane. The basement membrane serves a s scaffolding to connect muscle fibers to. This connection is made by transmembrane proteins bridging the actin cytoskeleton on the inside of the cell with the basement membrane on the outside. On the ends of the muscle fibers, each sarcolemma fuses with a tendon fiber and these, in turn, adhere to bones.
Sarcoplasmic reticulum
The sarcoplasmic reticulum (Figure 2.114) is a name for the structure found within muscle cells that is similar to the smooth endoplasmic reticulum found in other cells. It contains a specialized set of proteins to meet needs unique to muscle cells. The organelle largely serves as a calcium “battery,” releasing stored calcium to initiate muscular contraction when stimulated and taking up calcium when signaled at the end of the contraction cycle. It accomplishes these tasks using calcium ion channels for release of the ion and specific calcium ion pumps to take it up.
Movement direction
All myosins but myosin VI move towards the + end (the growing end) of the microfilament. The neck portion serves to link the head and the tail. It also a binding site for myosin light chain proteins that form part of a macromolecular complex with regulatory functions. The tail is the point of attachment of molecules or other “cargo” being moved. It can also connect with other myosin subunits and may have a role to play in controlling movement.
Muscular contraction
The sliding filament model has been proposed to describe the process of muscular tension/contraction. In this process a repeating set of actions slide a thin actin filament over a thick myosin filament as a means of creating tension/ shortening of the muscle fiber.
Steps in the process occur as follows:
A. A signal from the central nervous system (action potential) arrives at a motor neuron, which it transmits towards the neuromuscular junction (see more on the neurotransmission part of the process HERE)
B. At the end of the axon, the nerve signal stimulates the opening of calcium channels at the axon terminus causing calcium to flow into the terminal.
C. Movement of calcium into the axon of the nerve causes acetylcholine (a neurotransmitter) in synaptic vesicles to fuse with the plasma membrane. This causes the acetylcholine to be expelled into the synaptic cleft between the axon and the adjacent skeletal muscle fiber.
D. Acetylcholine diffuses across the synapse and then binds to nicotinic acetylcholine receptors on the neuromuscular junction, activating them.
E. Activation of the receptor stimulates opening gates of sodium and potassium channels, allowing sodium to move into the cell and potassium to exit. The polarity of the membrane of the muscle cell (called a sarcolemma - Figure 2.111) changes rapidly (called the end plate potential).
F. Change in the end plate potential results in opening of voltage sensitive ion channels specific for sodium or potassium only to Figure 2.117 - 3. ATP cleavage by myosin allows actin attachment (J) Wikipediaopen, creating an action potential (voltage change) that spreads throughout the cell in all directions.
G. The spreading action potential depolarizes the inner muscle fiber and opens calcium channels on the sarcoplasmic reticulum (Figure 2.115).
H. Calcium released from the sarcoplasmic reticulum binds to troponin on the actin filaments (Figure 2.115).
I. Troponin alters the structure of the tropomyosin to which is it bound. This causes tropomyosin to move slightly, allowing access to myosin binding sites on the microfilament (also called thin filament) that it was covering (Figure 2.116).
J. Myosin (bound to ATP) cleaves the ATP to ADP and Pi, which it holds onto in its head region and then attaches itself to the exposed binding sites on the thin filaments causing inorganic phosphate to be released from the myosin followed by ADP (Figure 2.117).
K. Release of ADP and Pi is tightly coupled to a bending of the myosin hinge, resulting in what is called the power stroke. This causes the thin filament to move relative to the thick fibers of myosin (Figures 2.118 & 2.119).
L. Such movement of the thin filaments causes the Z lines to be pulled closer to each other. This results in shortening of the sarcomere as a whole (Figure 2.122) and narrowing of the I band and the H zones (Figure 2.123). M. If ATP is available, it binds to myosin, allowing it to let go of the actin (Figures 2.120 & 2.121). If ATP is not available, the muscle will remain locked in this state. This is the cause of rigor mortis in death - contraction without release of muscles
.
Figure 2.120 - When ATP is present, it binds to myosin (M). Wikipedia
N. After myosin has bound the ATP, it hydrolyzes it, producing ADP and Pi, which are held by the head. Hydrolysis of ATP resets the hinge region to its original state, unbending it. This unbent state is also referred to as the cocked position.
O.If tropomyosin is still permitting access to binding sites on actin, the process repeats so long as ATP is available and calcium remains at a high enough concentration to permit it to bond to troponin.
Relaxation of the muscle tension occurs as the action potential in the muscle cell dissipates. This happens because all of the following things happen 1) the nerve signal stops; 2) the neurotransmitter is degraded by the enzyme acetylcholinesterase; and 3) the calcium concentration declines because it is taken up by the sarcoplasmic reticulum.
It should be noted that the sarcoplasmic reticulum is always taking up calcium. Only when its calcium gates are opened by the action potential is it unable to reduce cellular calcium concentration. As the action potential decreases, then the calcium gates close and the sarcoplasmic reticulum “catches up” and cellular calcium concentrations fall. At that point troponin releases calcium, tropomyosin goes back to covering myosin binding sites on actin, myosin loses its attachment to actin and the thin filaments slide back to their original positions relative to the myosin thick filaments.
Tropomyosin
Tropomyosins are proteins that interact with actin thin filaments to help regulate their roles in movement, both in muscle cells and non-muscle cells (Figure 2.124). Tropomyosins interact to form head-to-toe dimers and perch along the α-helical groove of an actin filament. The isoforms of tropomyosin that are in muscle cells control interactions between myosin and the actin filament within the sarcomere and help to regulate contraction of the muscle. In other cells, nonmuscle tropomyosins help to regulate the cytoskeleton’s functions.
The interactions of tropomyosin with the cytoskeleton are considerably more complicated than what occurs in muscle cells. Muscle cells have five tropomyosin isoforms, but in the cytoskeleton of non-muscle cells, there are over 40 tropomyosins.
Troponin
The troponins involved in muscular contraction are actually a complex of three proteins known as troponin I, troponin C, and troponin T (Figure 2.125). They associate with each other and with tropomyosin on actin filaments to help regulate the process of muscular contraction. Troponin I prevents binding of myosin’s head to actin and thus prevents the most important step in contraction.
Troponin C is a unit that binds to calcium ions. Troponin T is responsible for binding all three proteins to tropomyosin. Troponins in the bloodstream are indicative of heart disorders. Elevation of troponins in the blood occurs after a myocardial infarction and can remain high for up to two weeks.
Actinin
Actinin is a skeletal muscle protein that attaches filaments of actin to Z-lines of skeletal muscle cells. In smooth muscle cells, it also connects actin to dense bodies.
Titin
Titin (also known as connectin) is the molecular equivalent of a spring that provides striated muscle cells with elasticity. It is the third most abundant protein in muscle cells. The protein is enormous, with 244 folded individual protein domains spread across 363 exons (largest known number), with the largest known exon (17,106 base pairs long), and it is the largest protein known (27,000 to 33,000 amino acids, depending on splicing).
Unstructured sequences
The folded protein domains are linked together by unstructured sequences. The unstructured regions of the protein allow for unfolding when stretching occurs and refolding upon relaxation. Titin connects the M and Z lines in the sarcomere (Figure 2.123). Tension created in titin serves to limit the range of motion of the sarcomere, giving rise to what is called passive stiffness.
Skeletal and cardiac muscles have slight amino acid sequence variations in their ti tin proteins and these appear to relate to differences in the mechanical characteristics of each muscle.
Energy backup for muscle energy
Myoglobin was described as a molecular batter for oxygen. Muscle cells have a better of their own for ATP. The is important for animals, but not for plants because a plant’s need for energy is different than an animal’s. Plants do not need to access energy sources as rapidly as animals do, nor do they have to maintain a constant internal temperature. Plants can neither flee predators, nor chase prey. These needs of animals are much more immediate and require that energy stores be accessible on demand. Muscles, of course, enable the motion of animals and the energy required for muscle contraction is ATP. To have stores of energy readily available, muscles have, in addition to ATP, creatine phosphate for energy and glycogen for quick release of glucose to make more energy. The synthesis of creatine phosphate is a prime example of the effects of concentration on the synthesis of high energy molecules. For example, creatine phosphate has an energy of hydrolysis of -43.1 kJ/mol whereas ATP has an energy of hydrolysis of -30.5 kJ/mol. Creatine phosphate, however, is made from creatine and ATP in the reaction shown in Figure 2.126. How is this possible?
The ∆G°’ of this reaction is +12.6 kJ/mol, reflecting the energies noted above. In a resting muscle cell, ATP is abundant and ADP is low, driving the reaction downward, creating creatine phosphate. When muscular contraction commences, ATP levels fall and ADP levels climb. The above reaction then reverses and proceeds to synthesize ATP immediately. Thus, creatine phosphate acts like a battery, storing energy when ATP levels are high and releasing it almost instantaneously to create ATP when its levels fall.
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textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/02%3A_Structure_and_Function/2.06%3A_Structure_and_Function_-_Nucleic_Acids.txt
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princeton-nlp/TextbookChapters
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Source: BiochemFFA_2_5.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
The nucleic acids, DNA and RNA, may be thought of as the information molecules of the cell. In this section, we will examine the structures of DNA and RNA, and how these structures are related to the functions these molecules perform.
We will begin with DNA, which is the hereditary information in every cell, that is copied and passed on from generation to generation. The race to elucidate the structure of DNA was one of the greatest stories of 20th century science. Discovered in 1869 by Friedrich Miescher, DNA was identified as the genetic material in experiments in the 1940s led by Oswald Avery, Colin MacLeod, and Maclyn McCarty. X-ray diffraction work of Rosalind Franklin and the observations of Erwin Chargaff were combined by James Watson and Francis Crick to form a model of DNA that we are familiar with today. Their famous paper, in the April 25, 1953 issue of Nature, opened the modern era of molecular biology. Arguably, that one-page paper has had more scientific impact per word than any other research article ever published. Today, every high school biology student is familiar with the double helical structure of DNA and knows that G pairs with C and A with T.
The double helix, made up of a pair of DNA strands, has at its core, bases joined by hydrogen bonds to form base pairs - adenine always paired with thymine, and guanine invariably paired with cytosine. Two hydrogen bonds are formed between adenine and thymine, but three hydrogen bonds hold together guanine and cytosine (Figure 2.127).
The complementary structure immediately suggested to Watson and Crick how DNA might be (and in fact, is) replicated and it further explains how information is DNA is transmitted to RNA for the synthesis of proteins. In addition to the hydrogen bonds between bases of each strand, the double helix is held together by hydrophobic interactions of the stacked, non-polar bases. Crucially, the sequence of the bases in DNA carry the information for making proteins. Read in groups of three, the sequence of the bases directly specifies the sequence of the amino acids in the encoded protein.
Structure
A DNA strand is a polymer of nucleoside monophosphates held together by phosphodiester bonds. Two such paired strands make up the DNA molecule, which is then twisted into a helix. In the most common Bform, the DNA helix has a repeat of 10.5 base pairs per turn, with sugars and phosphate forming the covalent phosphodiester “backbone” of the molecule and the adenine, guanine, cytosine, and thymine bases oriented in the middle where they form the now familiar base pairs that look like the rungs of a ladder.
Building blocks
The term nucleotide refers to the building blocks of both DNA (deoxyribonucleoside triphosphates, dNTPs) and RNA (ribonucleoside triphosphates, NTPs). In order to discuss this important group of molecules, it is necessary to define some terms.
Nucleotides contain three primary structural components. These are a nitrogenous base, a pentose sugar, and at least one phosphate. Molecules that contain only a sugar and a nitrogenous base (no phosphate) are called nucleosides. The nitrogenous bases found in nucleic acids include adenine and guanine (called purines) and cytosine, uracil, or thymine (called pyrimidines). There are two sugars found in nucleotides - deoxyribose and ribose (Figure 2.128). By convention, the carbons on these sugars are labeled 1’ to 5’. (This is to distinguish the carbons on the sugars from those on the bases, which have their carbons simply labeled as 1, 2, 3, etc.) Deoxyribose differs from ribose at the 2’ position, with ribose having an OH group, where deoxyribose has H.
Nucleotides containing deoxyribose are called deoxyribonucleotides and are the forms found in DNA. Nucleotides containing ribose are called ribonucleotides and are found in RNA. Both DNA and RNA contain nucleotides with adenine, guanine, and cytosine, but with very minor exceptions, RNA contains uracil nucleotides, whereas DNA contains thymine nucleotides. When a base is attached to a sugar, the product, a nucleoside, gains a new name.
• uracil-containing = uridine (attached to ribose) / deoxyuridine (attached to deoxyribose)
• thymine-containing = ribothymidine (attached to ribose) / thymidine (attached to deoxyribose)
• cytosine-containing = cytidine (attached to ribose - Figure 2.129) / deoxycytidine (attached to deoxyribose)
• guanine-containing = guanosine (attached to ribose) / deoxyguanosine (attached to deoxyribose)
• adenine-containing = adenosine (attached to ribose) / deoxyadenosine (attached to deoxyribose)
Of these, deoxyuridine and ribothymidine are the least common. The addition of one or more phosphates to a nucleoside makes it a nucleotide. Nucleotides are often referred to as nucleoside phosphates, for this reason. The number of phosphates in the nucleotide is indicated by the appropriate prefixes (mono, di or tri).
Thus, cytidine, for example, refers to a nucleoside (no phosphate), but cytidine monophosphate refers to a nucleotide (with one phosphate). Addition of second and third phosphates to a nucleoside monophosphate requires energy, due to the repulsion of negatively charged phosphates and this chemical energy is the basis of the high energy triphosphate nucleotides (such as ATP) that fuel cells.
Note: Ribonucleotides as Energy Sources
Though ATP is the most common and best known cellular energy source, each of the four ribonucleotides plays important roles in providing energy. GTP, for example, is the energy source for protein synthesis (translation) as well as for a handful of metabolic reactions. A bond between UDP and glucose makes UDP-glucose, the building block for making glycogen. CDP is similarly linked to several different molecular building blocks important for glycerophospholipid synthesis (such as CDP-diacylglycerol).
The bulk of ATP made in cells is not from directly coupled biochemical metabolism, but rather by the combined processes of electron transport and oxidative phosphorylation in mitochondria and/or photophosphorylation that occurs in the chloroplasts of photosynthetic organisms. Triphosphate energy in ATP is transferred to the other nucleosides/nucleotides by action of enzymes called kinases. For example, nucleoside diphosphokinase (NDPK) catalyzes the following reaction
\[\ce{ATP + NDP <-> ADP + NTP}\]
where ‘N’ of “NDP” and “NTP corresponds to any base. Other kinases can put single phosphates onto nucleosides or onto nucleoside monophosphates using energy from ATP.
Deoxyribonucleotides
Individual deoxyribonucleotides are derived from corresponding ribonucleoside diphosphates via catalysis by the enzyme known as ribonucleotide reductase (RNR). The deoxyribonucleoside diphosphates are then converted to the corresponding triphosphates (dNTPs) by the addition of a phosphate group. Synthesis of nucleotides containing thymine is distinct from synthesis of all of the other nucleotides and will be discussed later.
Building DNA strands
Each DNA strand is built from dNTPs by the formation of a phosphodiester bond, catalyzed by DNA polymerase, between the 3’OH of one nucleotide and the 5’ phosphate of the next. The result of this directional growth of the strand is that the one end of the strand has a free 5’ phosphate and the other a free 3’ hydroxyl group (Figure 2.130). These are designated as the 5’ and 3’ ends of the strand.
Figure 2.131 shows two strands of DNA (left and right). The strand on the left, from 5’ to 3’ reads T-C-G-A, whereas the strand on the right, reading from 5’ to 3’ is T-C-G-A. The strands in a double-stranded DNA are arranged in an anti-parallel fashion with the 5’ end of one strand across from the 3’ end of the other.
Hydrogen bonds
Hydrogen bonds between the base pairs hold a nucleic acid duplex together, with two hydrogen bonds per A-T pair (or per A-U pair in RNA) and three hydrogen bonds per G-C pair. The B-form of DNA has a prominent major groove and a minor groove tracing the path of the helix (Figure 2.132). Proteins, such as transcription factors bind in these grooves and access the hydrogen bonds of the base pairs to “read” the sequence therein.
Other forms of DNA besides the B-form (Movie 2.5) are known (Figure 2.133). One of these, the A-form, was identified by Rosalind Franklin in the same issue of Nature as Watson and Crick’s paper. Though the A-form structure is a relatively minor form of DNA and resembles the B-form, it turns out to be important in the duplex form of RNA and in RNA-DNA hybrids. Both the A form and the B-form of DNA have the helix oriented in what is termed the right-handed form.
Movie 2.5 - B-form DNA duplex rotating in space Wikipedia
Z-DNA
The A-form and the B-form stand in contrast to another form of DNA, known as the Z-form. ZDNA, as it is known, has the same base-pairing rules as the B and A forms, but instead has the helices twisted in the opposite direction, making a left-handed helix (Figure 2.133). The Z-form has a sort of zig-zag shape, giving rise to the name Z-DNA.
In addition, the helix is rather stretched out compared to the A- and B-forms. Why are there different topological forms of DNA? The answer relates to both superhelical tension and sequence bias. Sequence bias means that certain sequences tend to favor the “flipping” of Bform DNA into other forms. ZDNA forms are favored by long stretches of alternating Gs and Cs. Superhelical tension is discussed below.
Superhelicity
Short stretches of linear DNA duplexes exist in the B-form and have 10.5 base pairs per turn. Double helices of DNA in the cell can vary in the number of base pairs per turn they contain. There are several reasons for this. For example, during DNA replication, strands of DNA at the site of replication get unwound at the rate of 6000 rpm by an enzyme called helicase. The effect of such local unwinding at one place in a DNA has the effect increasing the winding ahead of it. Unrelieved, such ‘tension’ in a DNA duplex can result in structural obstacles to replication.
Such adjustments can occur in three ways. First, tension can provide the energy for ‘flipping’ DNA structure. Z-DNA can arise as a means of relieving the tension. Second, DNA can ‘supercoil’ to relieve the tension (Figures 2.134 & 2.135). In this method, the duplex crosses over itself repeatedly, much like a rubber band will coil up if one holds one section in place and twists another part of it. Third, enzymes called topoisomerases can act to relieve or, in some cases, increase the tension by adding or removing twists in the DNA.
Topological isomers
As noted, so-called “relaxed” DNA has 10.5 base pairs per turn. Each turn corresponds to one twist of the DNA. Using enzymes, it is possible to change the number of base pairs per turn. In either the case of increasing or decreasing the twists per turn, tension is introduced into the DNA structure. If the tension cannot be relieved, the DNA duplex will act to relieve the strain, as noted. This is most easily visualized for circular DNA, though long linear DNA (such as found in eukaryotic chromosomes) or DNAs constrained in other ways will exhibit the same behavior.
Parameters
To understand topologies, we introduce the concepts of ‘writhe’ and ‘linking number’. First, imagine either opening a closed circle of DNA and either removing one twist or adding one twist and then re-forming the circle. Since the strands have no free ends, they cannot relieve the induced tension by re-adding or removing the twists at their ends, respectively. Instead, the tension is relieved by “superhelices” that form with crossing of the double strands over each other (figure 8 structures in Figure 2.136). Though it is not apparent to visualize, each crossing of the double strands in this way allows twists to be increased or decreased correspondingly. Thus, superhelicity allows the double helix to reassume 10.5 base pairs per turn by adding or subtracting twists as necessary and replacing them with writhes.
We write the equation L= T + W where T is the number of twists in a DNA, W is the number of writhes, and L is the linking number. The linking number is therefore the sum of the twists and writhes. Interestingly, inside of cells, DNAs typically are in a supercoiled form. Supercoiling affects the size of the DNA (compacts it) and also the expression of genes within the DNA, some having enhanced expression and some having reduced expression when supercoiling is present. Enzymes called topoisomerases alter the superhelical density of DNAs and play roles in DNA replication, transcription, and control of gene expression. They work by making cuts in one strand (Type I topoisomerases) or both strands (Type II topoisomerases) and then add or subtract twists as appropriate to the target DNA. After that process is complete, the topoisomerase re-ligates the nick/cut it had made in the DNA in the first step.
Topoisomerases may be the targets of antibiotics. The family of antibiotics known as fluoroquinolones work by interfering with the action of bacterial type II topoisomerases. Ciprofloxacin also preferentially targets bacterial type II topoisomerases. Other topoisomerase inhibitors target eukaryotic topoisomerases and are used in anti-cancer treatments.
RNA
The structure of RNA (Figure 2.137) is very similar to that of a single strand of DNA. Built of ribonucleotides, joined together by the same sort of phosphodiester bonds as in DNA, RNA uses uracil in place of thymine. In cells, RNA is assembled by RNA polymerases, which copy a DNA template in the much same way that DNA polymerases replicate a parental strand. During the synthesis of RNA, uracil is used across from an adenine in the DNA template. The building of messenger RNAs by copying a DNA template is a crucial step in the transfer of the information in DNA to a form that directs the synthesis of protein. Additionally, ribosomal and transfer RNAs serve important roles in “reading” the information in the mRNA codons and in polypeptide synthesis. RNAs are also known to play important roles in the regulation of gene expression.
RNA world
The discovery, in 1990, that RNAs could play a role in catalysis, a function once thought to be solely the domain of proteins, was followed by the discovery of many more so-called ribozymes- RNAs that functioned as enzymes. This suggested the answer to a long-standing chicken or egg puzzle - if DNA encodes proteins, but the replication of DNA requires proteins, how did a replicating system come into being? This problem could be solved if the first replicator was RNA, a molecule that can both encode information and carry out catalysis. This idea, called the “RNA world” hypothesis, suggests that DNA as genetic material and proteins as catalysts arose later, and eventually prevailed because of the advantages they offer. The lack of a 2’OH on deoxyribose makes DNA more stable than RNA. The double-stranded structure of DNA also provides an elegant way to easily replicate it. RNA catalysts, however, remain, as remnants of that early world. In fact, the formation of peptide bonds, essential for the synthesis of proteins, is catalyzed by RNA.
Secondary structure
With respect to structure, RNAs are more varied than their DNA cousin. Created by copying regions of DNA, cellular RNAs are synthesized as single strands, but they often have self-complementary regions leading to “foldbacks” containing duplex regions. These are most easily visualized in the ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) (Figure 2.138), though other RNAs, including messenger RNAs (mRNAs), small nuclear RNAs (snRNAs), microRNAs (Figure 2.139), and small interfering RNAs (siRNAs) may each have double helical regions as well.
Base pairing
Base pairing in RNA is slightly different than DNA. This is due to the presence of the base uracil in RNA in place of thymine in DNA. Like thymine, uracil forms base pairs with adenine, but unlike thymine, uracil can, to a limited extent, also base pair with guanine, giving rise to many more possibilities for pairing within a single strand of RNA.
These additional base pairing possibilities mean that RNA has many ways it can fold upon itself that single-stranded DNA cannot. Folding, of course, is critical for protein function, and we now know that, like proteins, some RNAs in their folded form can catalyze reactions just like enzymes. Such RNAs are referred to as ribozymes. It is for this reason scientists think that RNA was the first genetic material, because it could not only carry information, but also catalyze reactions. Such a scheme might allow certain RNAs to make copies of themselves, which would, in turn, make more copies of themselves, providing a positive selection.
Stability
RNA is less chemically stable than DNA. The presence of the 2’ hydroxyl on ribose makes RNA much more prone to hydrolysis than DNA, which has a hydrogen instead of a hydroxyl. Further, RNA has uracil instead of thymine. It turns out that cytosine is the least chemically stable base in nucleic acids. It can spontaneously deaminate and in turn is converted to a uracil. This reaction occurs in both DNA and RNA, but since DNA normally has thymine instead of uracil, the presence of uracil in DNA indicates that deamination of cytosine has occurred and that the uracil needs to be replaced with a cytosine. Such an event occurring in RNA would be essentially undetectable, since uracil is a normal component of RNA. Mutations in RNA have much fewer consequences than mutations in DNA because they are not passed between cells in division.
Catalysis
RNA structure, like protein structure, has importance, in some cases, for catalytic function. Like random coils in proteins that give rise to tertiary structure, single-stranded regions of RNA that link duplex regions give these molecules a tertiary structure, as well. Catalytic RNAs, called ribozymes, catalyze important cellular reactions, including the formation of peptide bonds in ribosomes (Figure 2.114). DNA, which is usually present in cells in strictly duplex forms (no tertiary structure, per se), is not known to be involved in catalysis.
RNA structures are important for reasons other than catalysis. The 3D arrangement of tRNAs is necessary for enzymes that attach amino acids to them to do so properly. Further, small RNAs called siRNAs found in the nucleus of cells appear to play roles in both gene regulation and in cellular defenses against viruses. The key to the mechanisms of these actions is the formation of short foldback RNA structures that are recognized by cellular proteins and then chopped into smaller units. One strand is copied and used to base pair with specific mRNAs to prevent the synthesis of proteins from them.
Denaturing nucleic acids
Like proteins, nucleic acids can be denatured. Forces holding duplexes together include hydrogen bonds between the bases of each strand that, like the hydrogen bonds in proteins, can be broken with heat or urea. (Another important stabilizing force for DNA arises from the stacking interactions between the bases in a strand.) Single strands absorb light at 260 nm more strongly than double strands. This is known as the hyperchromic effect (Figure 2.141)and is a consequence of the disruption of interactions among the stacked bases. The changes in absorbance allow one to easily follow the course of DNA denaturation. Denatured duplexes can readily renature when the temperature is lowered below the “melting temperature” or Tm, the temperature at which half of the DNA strands are in duplex form. Under such conditions, the two strands can re-form hydrogen bonds between the complementary sequences, returning the duplex to its original state. For DNA, strand separation and rehybridization are important for the technique known as the polymerase chain reaction (PCR). Strand separation of DNA duplexes is accomplished in the method by heating them to boiling. Hybridization is an important aspect of the method that requires single stranded primers to “find” matching sequences on the template DNA and form a duplex. Considerations for efficient hybridization (also called annealing) include temperature, salt concentration, strand concentration, and magnesium ion levels (for more on PCR, see HERE).
DNA packaging
DNA is easily the largest macromolecule in a cell. The single chromosome in small bacterial cells, for example, can have a molecular weight of over 1 billion Daltons. If one were to take all of the DNA of human chromosomes from a single cell and lay them end to end, they would be over 7 feet long. Such an enormous molecule demands careful packaging to fit within the confines of a nucleus (eukaryotes) or a tiny cell (bacteria). The chromatin system of eukaryotes is the best known, but bacteria, too, have a system for compacting DNA.
DNA in Bacteria
In bacteria, there is no nucleus for the DNA. Instead, DNA is contained in a structure called a nucleoid (Figure 2.142). It contains about 60% DNA with much of the remainder comprised of RNAs and transcription factors. Bacteria do not have histone proteins that DNA wrap around, but they do have proteins that help organize the DNA in the cell - mostly by making looping structures.
These proteins are known as Nucleoid Associated Proteins and include ones named HU, H-NS, Fis, CbpA, and Dps. Of these, HU most resembles eukaryotic histone H2B and binds to DNA non-specifically. The proteins associate with the DNA and can also cluster, which may be the origin of the loops. It is likely these proteins play a role in helping to regulate transcription and respond to DNA damage. They may also be involved in recombination.
Eukaryotes
The method eukaryotes use for compacting DNA in the nucleus is considerably different, and with good reason - eukaryotic DNAs are typically much larger than prokaryotic DNAs, but must fit into a nucleus that is not much bigger than a prokaryotic cell. Human DNA, for example, is about 1000 times longer than c DNA. The strategy employed in eukaryotic cells is that of spooling - DNA is coiled around positively charged proteins called histones. These proteins, whose sequence is very similar in cells as diverse as yeast and humans, come in four types, dubbed H1, H2a, H2b, H3, and H4. A sixth type, referred to as H5 is actually an isoform of H1 and is rare. Two each of H2a, H2b, H3, and H4 are found in the core structure of what is called the fundamental unit of chromatin - the nucleosome (Figure 2.143).
Octamer
The core of 8 proteins is called an octamer. The stretch of DNA wrapped around the octamer totals about 147 base pairs and makes 1 2/3 turns around it. This complex is referred to as a core particle (Figure 2.144). A linker region of about 50-80 base pairs separate core particles. The term nucleosome then refers to a a core particle plus a linker region (Figure 2.143). Histone H1 sits near the junction of the incoming DNA and the histone core. It is often referred to as the linker histone. In the absence of H1, non-condensed nucleosomes resemble “beads on a string” when viewed in an electron microscope.
Histones
Histone proteins are similar in structure and are rich in basic amino acids, such as lysine and arginine (Figure 2.145). These amino acids are positively charged at physiological pH, with enables them to form tight ionic bonds with the negatively charged phosphate backbone of DNA.
For DNA, compression comes at different levels (Figure 2.146). The first level is at the nucleosomal level. Nucleosomes are stacked and coiled into higher order structures. 10 nm fibers are the simplest higher order structure (beads on a string) and they grow in complexity. 30 nm fibers consist of stacked nucleosomes and they are packed tightly. Higher level packing produces the metaphase chromosome found in meiosis and mitosis.
The chromatin complex is a logistical concern for the processes of DNA replication and (particularly) gene expression where specific regions of DNA must be transcribed. Altering chromatin structure is therefore an essential function for transcriptional activation in eukaryotes. One strategy involves adding acetyl groups to the positively charged lysine side chains to “loosen their grip” on the negatively charged DNA, thus allowing greater access of proteins involved in activating transcription to gain access to the DNA. The mechanisms involved in eukaryotic gene expression are
Ames test
The Ames test (Figure 2.147) is an analytical method that allows one to determine whether a compound causes mutations in DNA (is mutagenic) or not. The test is named for Dr. Bruce Ames, a UC Berkeley emeritus professor who was instrumental in creating it. In the procedure, a single base pair of a selectable marker of an organism is mutated in a plasmid to render it nonfunctional. In the example, a strain of Salmonella is created that lacks the ability to grow in the absence of histidine. Without histidine, the organism will not grow, but if that one base in the plasmid’s histidine gene gets changed back to its original base, a functional gene will be made and the organism will be able to grow without histidine.
A culture of the bacterium lacking the functional gene is grown with the supply of histidine it requires. It is split into two vials. To one of the vials, a compound that one wants to test the mutagenicity of is added. To the other vial, nothing is added. The bacteria in each vial are spread onto plates lacking histidine. In the absence of mutation, no bacteria will grow. The more colonies of bacteria that grow, the more mutation happened. Note that even the vial without the possible mutagenic compound will have a few colonies grow, as a result of mutations unlinked to the potential mutagen.
Mutation happens in all cells at a low level. If the plate with the cells from the vial with the compound has more colonies than the cells from the control vial (no compound), then that would be evidence that the compound causes more mutations than would normally occur and it is therefore a mutagen. On the other hand, if there was no significant difference in the number of colonies on each plate, then that would suggest it is not mutagenic. The test is not perfect - it identifies about 90% of known mutagens - but its simplicity and inexpensive design make it an excellent choice for an initial screen of a compound.
2.07: Structure and Function- Carbohydrates
Endogenous glycation, on the other hand, arises with a frequency that is proportional to the concentration of free sugar in the body. These occur most frequently with fructose, galactose, and glucose in that decreasing order and are detected in the bloodstream. Both proteins and lipids can be glycated and the accumulation of endogenous advanced glycation endproducts (AGEs) is associated with Type 2 diabetes, as well as in increases in cardiovascular disease (damage to endothelium, cartilage, and fibrinogen), peripheral neuropathy (attack of myelin sheath), and deafness (loss of myelin sheath).
The formation of AGEs increases oxidative stress, but is also thought to be exacerbated by it. Increased oxidative stress, in turn causes additional harm. Damage to collagen in blood cells causes them to stiffen and weaken and is a factor in hardening of the arteries and formation of aneurysms, respectively. One indicator of diabetes is increased glycation of hemoglobin in red blood cells, since circulating sugar concentration are high in the blood of diabetics. Hemoglobin glycation is measured in testing for blood glucose control in diabetic patients.
Homopolymer Monomeric Unit
Glycogen Glucose
Cellulose Glucose
Amylose Glucose
Callose Glucose
Chitin N-acetylglucosamine
Xylan Xylose
Mannan Mannose
Chrysolaminarin Glucose
Function in skin
Hyaluronic acid is a major component of skin and has functions in tissue repair. With exposure to excess UVB radiation, cells in the dermis produce less hyaluronan and increase its degradation.
For some cancers the plasma level of hyaluronic acid correlates with malignancy. Hyaluronic acid levels have been used as a marker for prostate and breast cancer and to follow disease progression. The compound can to used to induce healing after cataract surgery. Hyaluronic acid is also abundant in the granulation tissue matrix that replaces a fibrin clot during the healing of wounds. In wound healing, it is thought that large polymers of hyaluronic acid appear early and they physically make room for white blood cells to mediate an immune response.
Breakdown
Breakdown of hyaluronic acid is catalyzed by enzymes known as hyaluronidases. Humans have seven types of such enzymes, some of which act as tumor suppressors. Smaller hyaluronan fragments can induce inflammatory response in macrophages and dendritic cells after tissue damage. They can also perform proangiogenic functions.
Proteoglycans
Glycosaminoglycans are commonly found attached to proteins and these are referred to as proteoglycans. Linkage between the protein and the glycosaminoglycan is made through a serine side-chain. Proteoglycans are made by glycosylation of target proteins in the Golgi apparatus.
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textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/02%3A_Structure_and_Function/2.08%3A_Structure_and_Function_-_Lipids_and_Membranes.txt
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Source: BiochemFFA_2_7.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
Lipids are a diverse group of molecules that all share the characteristic that at least a portion of them is hydrophobic. Lipids play many roles in cells, including serving as energy storage (fats/oils), constituents of membranes (glycerophospholipids, sphingolipids, cholesterol), hormones (steroids), vitamins (fat soluble), oxygen/ electron carriers (heme), among others. For lipids that are very hydrophobic, such as fats/ oils, movement and storage in the aqueous environment of the body requires special structures. Other, amphipathic lipids, such as glycerophospholipids and sphingolipids spontaneously organize themselves into lipid bilayers when placed in water. Interestingly, major parts of many lipids can be derived from acetyl-CoA.
Fatty acids
The most ubiquitous lipids in cells are the fatty acids. Found in fats, glycerophospholipids, sphingolipids and serving as as membrane anchors for proteins and other biomolecules, fatty acids are important for energy storage, membrane structure, and as precursors of most classes of lipids. Fatty acids, as can be seen from Figure 2.190 are characterized by a polar head group and a long hydrocarbon tail. Fatty acids with hydrocarbon tails that lack any double bonds are described as saturated, while those with one or more double bonds in their tails are known as unsaturated fatty acids. The effect of double bonds on the fatty acid tail is to introduce a kink, or bend, in the tail, as shown for oleic acid.
Stearic acid, a saturated fatty acid, by contrast has a straight hydrocarbon tail. Figures 2.190-2.194 show the most common saturated and unsaturated fatty acids. Fatty acids with unsaturated tails have a lower melting temperature than those with saturated tails of the same length. Shorter tails also decrease melting temperature. These properties carry over to the fats/oils containing them.
Fatty acids with more than one double bond are called polyunsaturated. Plants are excellent sources of unsaturated and polyunsaturated fatty acids. The position of the double bond(s) in fatty acids has important considerations both for their synthesis and for their actions in the body. Biochemically, the double bonds found in fatty acids are predominantly in the cis configuration. So-called trans fats arise as a chemical by-product of partial hydrogenation of vegetable oil.
In humans, consumption of trans fat raises low density lipoprotein (LDL) levels and lowers high density lipoprotein (HDL) levels. Each is thought to contribute to the risk of developing coronary artery disease. The most Figure 2.194 - Fatty acid models. Carboxyl end labeled in red Wikipedia common fatty acids in our body include palmitate, stearate, oleate, linolenate, linoleate, and arachidonate. Two notable shorter fatty acids are nonanoic (9 carbons) and decanoic acid (10 carbons), both of which appear to have anti-seizure effects. Decanoic acid directly inhibits excitatory neurotransmission in the brain and may contribute to the anticonvulsant effect of the ketogenic diet.
Numbering
Figure 2.195 shows two different systems for locating double bonds in a fatty acid. The ω system counts carbons starting with the methyl end (shown in red) while the Δ system counts from the carboxyl end (shown in blue). For example, an ω-3 (omega 3) fatty acid would have a double bond at the third carbon from the methyl end. In the Δ system, a fatty acid that has a cis double bond at carbon 6, counting from the carboxyl end, would be written as cis-Δ6.
Fatty acids are described as essential fatty acids if they must be in the diet (can’t be synthesized by the organism) and nonessential fatty acids if the organism can synthesize them. Humans and other animals lack the desaturase enzymes necessary to make double bonds at positions greater than Δ-9, so fatty acids with double bonds beyond this position must be obtained in the diet. Linoleic acid and linolenic acid, both fall in this category. Related unsaturated fatty acids can be made from these fatty acids, so the presence of linoleic and linolenic acids in the diet eliminates the need to have all unsaturated fatty acids in the diet. Both linoleic and linolenic acid contain 18 carbons, but linoleic acid is an ω-6 fatty acid, whereas linolenic acid is an ω-3 fatty acid. Notably, ω-6 fatty acids tend to be proinflammatory, whereas ω-3 fatty acids are lesser so.
Fats/oils
Fats and oils are the primary energy storage forms of animals and are also known as triacylglycerols and triglycerides, since they consist of a glycerol molecule linked via ester bonds to three fatty acids (Figure 2.196). Fats and oils have the same basic structure. We give the name fat to those compounds that are solid at room temperature and the name oil to those that are liquid at room temperature. Note that biological oils are not the same as petroleum oils.
Increasing the number of unsaturated fatty acids (and the amount of unsaturation in a given fatty acid) in a fat decreases the melting temperature of it. Organisms like fish, which live in cool environments, have fats with more unsaturation and this is why fish oil contains polyunsaturated fatty acids.
Adipocytes
Fats are stored in the body in specialized cells known as adipocytes. Enzymes known as lipases release fatty acids from fats by hydrolysis reactions (Figure 2.197). Triacylglycercol lipase (pancreatic - Figure 2.198) is able to cleave the first two fatty acids from the fat. A second enzyme, monoacylglycerol lipase, cleaves the last fatty acid. Fats can be synthesized by replacing the phosphate on phosphatidic acid with a fatty acid.
Glycerophospholipids
Glycerophospholipids (phosphoglycerides) are important components of the lipid bilayer of cellular membranes. Phosphoglycerides are structurally related to fats, as both are derived from phosphatidic acid (Figure 2.199). Phosphatidic acid is a simple glycerophospholipid that is usually converted into phosphatidyl compounds. These are made by esterifying various groups, such as ethanolamine, serine, choline, inositol, and others (Figure 2.200) to the phosphate of phosphatidic acid. All of these compounds form lipid bilayers in aqueous solution , due to their amphiphilic nature.
Phosphatidylethanolamines
Since all glycerolipids can have a variety of fatty acids at positions 1 and 2 on the glycerol, they all are families of compounds. The phosphatidylethanolamines are found in all living cells and are one of the most common phosphatides, making up about 25% of them. They are common constituents of brain tissue and in the spinal cord, making up as much as 45% of the total phospholipids. Phosphatidylethanolamines are asymmetrically distributed across membranes, being preferentially located on the inner leaflet (closest to the cytoplasm) of the plasma membrane. Metabolically, phosphatidylethanloamines are precursors of phosphatidylcholines. Phosphatidylserines Phosphatidylserines are another group of phosphatidyl compounds that are preferentially distributed across the lipid bilayer of the plasma membrane. Like the phosphatidylethanolamines, phosphatidylserines are preferentially located on the inner leaflet of the plasma membrane. When apoptosis (cell suicide) occurs, the preferential distribution is lost and the phosphatidylserines appear on the outer leaflet where they serve as a signal to macrophages to bind and destroy the cell.
Phosphatidylcholines
Phosphatidylcholines (Figure 2.201) are another group of important membrane components. They tend to be found more commonly on the outer leaflet of the plasma membrane. Nutritionally, the compounds are readily obtained from eggs and soybeans. Phosphatidylcholines are moved across membranes by Phosphatidylcholine transfer protein (PCTP). This protein, which is sensitive to the levels of phosphatidylcholines, acts to stimulate the activity of a thioesterase (breaks thioester bonds, such as acyl-CoAs) and activates PAX3 transcription factors.
Cardiolipins
Cardiolipins are an unusual set of glycerophospholipids in containing two diacylglycerol backbones joined in the middle by a diphosphoglycerol (Figure 2.202). It is an important membrane lipid, constituting about 20% of the inner mitochondrial membrane and is found in organisms from bacteria to humans. In both plants and animals, it is found almost totally in the inner mitochondrial membrane.
The molecules appear to be required for both Complex IV and Complex III of the electron transport chain to maintain its structure. The ATP synthase enzyme (Complex V) of the oxidative phosphorylation system also binds four molecules of cardiolipin. It has been proposed that cardiolipin functions as a proton trap in the process of proton pumping by Complex IV.
Cardiolipin also plays a role in apoptosis. As shown in Figure 2.203, oxidation of cardiolipin by a cardiolipin-specific oxygenase causes cardiolipin to move from the inner mitochondrial membrane to the outer one, helping to form a permeable pore and facilitating the transport of cytochrome c out of the intermembrane space and into the cytoplasm - a step in the process of apoptosis.
Diacylglycerol
Diacylglycerol (also called diglyceride and DAG - Figure 2.204) is an important intermediate in metabolic pathways. It is produced, for example, in the first step of the hydrolysis of fat and is also produced when membrane lipids, such as PIP2 (phosphatidylinositol-4,5-bisphosphate) are hydrolyzed by phospholipase C in a signaling cascade.
DAG is itself a signaling compound, binding to protein kinase C to activate it to phosphorylate substrates. Synthesis of DAG begins with glycerol-3-phosphate, which gains two fatty acids from two acyl-CoAs to form phosphatidic acid. Dephosphorylation of phosphatidic acid produces DAG. DAG can also be rephosphorylated by DAG kinase to re-make phosphatidic acid or another fatty acid can be added to make fat.
Inositol
Though technically not a lipid itself, inositol is found in many lipids. Inositol is a derivative of cyclohexane containing six hydroxyl groups - one on each carbon (Figure 2.205. It has nine different stereoisomers of which one, cis-1,2,3,5-trans-4,6- cyclohexanehexol (called myo-inositol) is the most common. It has a sweet taste (half that of sucrose).
Numerous phosphorylated forms of the compound exist, from a single phosphate to six (one on each carbon). Phytic acid, for example, in plants, has six phosphates (Figure 2.206) that it uses to store phosphate. Inositol is produced from glucose and was once considered vitamin B8, but is made by the body in adequate amounts, so it is not now considered a vitamin. Phosphorylated forms of inositol are found in phosphoinositides, such as PIP2 and PIP3, both of which are important in signaling processes. Some of these include insulin signaling, fat catabolism, calcium regulation, and assembly of the cytoskeleton.
Phosphoinositides
Compounds based on phosphatidylinositol (PI) are often called phosphoinositides. These compounds have important roles in signaling and membrane trafficking. Hydroxyls on carbons 3,4, and 5 of the inositol ring are targets for phosphorylation by a variety of kinases. Seven different combinations are used. Steric hindrance inhibits phosphorylation of carbons 2 or 6. Naming of these phosphorylated compounds follows generally as PI(#P)P, PI(#P, #P)P, or PI(#P, #P, #P)P where #P refers to the number of the carbon where a phosphate is located. For example, PI(3)P refers to a phosphatidyl compound with a phosphate added to carbons 3 of the inositol ring, whereas PI(3,4,5)P is a phosphatidyl compound with a phosphate added to carbons 3,4,and 5.
Phosphatidylinositol-4,5- bisphosphate
Phosphatidylinositol-4,5-bisphosphate (PIP2 - Figure 2.207) is a phospholipid of plasma membranes that functions in the phospholipase C signaling cascade. In this signaling pathway, hydrolysis catalyzed by phospholipase C releases inositol-1,4,5- trisphosphate (IP3) and diacylglycerol. Synthesis of PIP2 begins with phosphatidylinositol, which is phosphorylated at position 4 followed by phosphorylation at position 5 by specific kinases.
PIP2 can be phosphorylated to form the signaling molecule known as phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Along with PIP3, PIP2 serves as a docking phospholipid for the recruitment of proteins that play roles in signaling cascades. Binding of PIP2 is also required by inwardly directed potassium channels.
Phosphatidylinositol (3,4,5)- trisphosphate
Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is an important molecule for the activation of signaling proteins, such as AKT, which activates anabolic signaling pathways related to growth and survival. PIP3 can be dephosphorylated by phosphatase PTEN to yield PIP2 and can be synthesized from PIP2 by kinase action of Class I PI 3- kinases. Kinase activity to synthesize PIP3 results in movement of PIP3-binding proteins to the plasma membrane. They include Akt/ PKB, PDK1, Btk1, and ARNO and each is activated by binding to PIP3.
Plasmalogens
A special class of the glycerophospholipids are the plasmalogens (Figure 2.209). They differ in containing a vinyl ether linkage at position 1 of glycerol, in contrast to other glycerophopsholipids, which have an ester linkage at this position. Position 2 of each is an ester. The precursor for the ether linkage is typically a 16 or 18 carbon saturated alcohol or an 18 carbon unsaturated alcohol.
At the phosphate tail, the most commonly attached groups are ethanolamine or choline. Plasmalogens are found abundantly in humans in heart (30-40% of choline phospholipids). 30% of the glycerophospholipids in brain are plasmalogens and 70% of the ethanolamine lipids of the myelin sheath of nerve cells are plasmalogens.
Though their function is not understood, it is believed that plasmalogens may provide some protection against reactive oxygen species and have roles in signaling.
Lecithin
Lecithin is a generic term for a combination of lipid substances that includes phosphoric acid, glycerol, glycolipids, triglycerides, and phospholipids. Lecithin is a wetting agent helpful with emulsification and encapsulation and is even used as an anti-sludge additive in motor lubricants. Lecithin is used in candy bars to keep cocoa and cocoa butter from separating. Though considered safe as a food ingredient, lecithin can be converted by gut bacteria to trimethylamine-N-oxide which may contribute to cholesterol deposition and atherosclerosis.
Sphingolipids
Fatty acids are also components of a broad class of molecules called sphingolipids. Sphingolipids are structurally similar to glycerophospholipids, though they are synthesized completely independently of them starting with palmitic acid and the amino acid serine. Sphingolipids are named for the amino alcohol known as sphingosine (Figure 2.210), though they are not directly synthesized from it. Figure 2.211 shows the generalized structure of sphingolipids.
If the R-group is a hydrogen, the molecule is called a ceramide. When the R-group is phosphoethanolamine the resulting molecule is sphingomyelin, an important component of the myelin sheath and lipid membranes. If a single, simple sugar is instead added, a cerebroside is created (Figure 2.212). Addition of a complex oligosaccharide creates a ganglioside.
Complex sphingolipids may play roles in cellular recognition and signaling. Sphingolipids are found most abundantly in plasma membrane and are almost completely absent from mitochondrial and endoplasmic reticulum membranes. In animals, dietary sphingolipids have been linked to reduced colon cancer, reductions in LDLs, and increases in HDLs. Like the glycerophospholipids, sphingolipids are amphiphilic. Most sphingolipids except sphingomyelin do not contain phosphate.
Eicosanoids
Fatty acids made from omega-6 and omega-3 fatty acids include three important fatty acids containing 20 carbons. They include arachidonic acid (an ω-6 fatty acid with four double bonds (Δ-5,8,11,14) - Figure 2.213), eicosapentaenoic acid (an ω-3 fatty acid with five double bonds, and dihomo-γ-linolenic acid (an ω-6 fatty acid with three double bonds). The class of compounds known as eicosanoids is made by oxidation of these compounds. Subclasses include include prostaglandins, prostacyclins, thromboxanes, lipoxins, leukotrienes, and endocannabinoids (Figures 2.214-2.219). Eicosanoids play important roles affecting inflammation, immunity, mood, and behavior.
Prostaglandins
A collection of molecules acting like hormones, prostaglandins are derived from arachidonic acid and have many differing (even conflicting) physiological effects. These include constriction or dilation of vascular smooth muscle cells, induction of labor, regulation of inflammation, and action on the thermoregulatory center of the hypothalamus to induce fever, among others.
Prostaglandins are grouped with the thromboxanes (below) and prostacyclins (below), as prostanoids. The prostanoids, which all contain 20 carbons are a subclass of the eicosanoids. Prostaglandins are found in most tissues of higher organisms. They are autocrine or paracrine compounds produced from essential fatty acids. The primary precursor of the prostaglandins is the fatty acid known as arachidonic acid and the prostaglandin made from it is known as PGH2 (Figure 2.214), which, in turn is a precursor of other prostaglandins, as well as the prostacyclins and thromboxanes.
Interesting prostaglandins
PGD2 - inhibits hair follicle growth, vasodilator, causes bronchial constriction, higher in lungs of asthmatics than others.
PGE2 (Figure 2.215) - exerts effects in labor (soften cervix, uterine contraction), stimulates bone resorption by osteoclasts, induces fever, suppresses T-cell receptor signaling, vasodilator, inhibits release of noradrenalin from sympathetic nerve terminals. It is a potent activator of the Wnt signaling pathway.
A prostaglandin can have opposite effects, depending on which receptor it binds to. Binding of PGE2 to the EP1 receptor causes bronchoconstriction and smooth muscle contraction, whereas binding of the same molecule to the EP2 receptor causes bronchodilation and smooth muscle relaxation.
PGF (Figure 2.216)- uterine contractions, induces labor, bronchoconstriction.
PGI2 - vasodilation, bronchodilation, inhibition of platelet aggregation.
Thromboxanes
Thromboxanes play roles in clot formation and named for their role in thrombosis. They are potent vasoconstrictors and facilitate platelet aggregation. They are synthesized in platelets, as well. The anti-clotting effects of aspirin have their roots in the inhibition of synthesis of PGH2, which is the precursor of the thromboxanes. The most common thromboxanes are A2 (Figure 2.217) and B2.
Prostacyclin
Prostacyclin (also known as prostaglandin I2 or PGI2 - Figure 2.218) counters the effects of thromboxanes, inhibiting platelet activation and acting as vasodilators. It is produced from PGH2 by action of the enzyme prostacyclin synthase.
Leukotrienes
Another group of eicosanoid compounds are the leukotrienes (Figure 2.219). Like prostaglandins, leukotrienes are made from arachidonic acid. The enzyme catalyzing their formation is a dioxygenase known as arachidonate 5-lipoxygenase. Leukotrienes are involved in regulating immune responses. They are found in leukocytes and other immunocompetent cells, such as neutrophils, monocytes, mast cells, eosinophils, and basophils. Leukotrienes are associated with production of histamines and prostaglandins, which act as mediators of inflammation. Leukotrienes also trigger contractions in the smooth muscles of the bronchioles. When overproduced, they may pay a role in asthma and allergic reactions. Some treatments for asthma aim at inhibiting production or action of leukotrienes.
Cholesterol
Arguably, no single biomolecule has generated as much discussion and interest as has cholesterol (Figure 2.220). Certainly, from the perspective of the Nobel Prize committee, no small molecule even comes close, with 13 people having been awarded prizes for work on it. Evidence for cholesterol’s importance comes from the study of brain tissue where it comprises 10-15% of the dry mass.
Membrane flexibility
In animal cells, cholesterol provides for membrane flexibility that allows for cellular movement that is in contrast to plant and bacterial cells with fixed structures. Cholesterol is made in many cells of the body, with the liver making the greatest amount. The anabolic pathway leading to synthesis of cholesterol is known as the isoprenoid pathway and branches of it lead to other molecules including other fat-soluble vitamins.
Cholesterol is only rarely found in prokaryotes (Mycoplasma, which requires it for growth, is an exception) and is found in only trace amounts in plants. Instead, plants synthesize similar compounds called phytosterols (Figure 2.221). On average, the body of a 150 pound adult male makes about 1 gram of cholesterol per day, with a total content of about 35 grams.
Packaging
Cholesterol’s (and other lipids’) hydrophobicity requires special packaging into lipoprotein complexes (called chylomicrons, VLDLs, IDLs, LDLs, and HDLs) for movement in the lymph system and bloodstream. Though cholesterol can be made by cells, they also take it up from the blood supply by absorbing cholesterol-containing LDLs directly in a process called receptor-mediated endocytosis.
Oxidative damage to LDLs can lead to formation of atherosclerotic plaques and this is why cholesterol has gotten such a negative image in the public eye. The liver excretes cholesterol through the bile for elimination into the digestive system, but the compound is recycled there.
Reducing cholesterol levels
Strategies for reducing cholesterol in the body focus primarily on three areas - reducing consumption, reducing endogenous synthesis, and reducing the recycling. Dietary considerations, such as saturated fat versus unsaturated fat consumption are currently debated. Dietary trans fats, though, correlate with incidence of coronary heart disease. Consumption of vegetables may provide some assistance with reducing levels of cholesterol recycled in the digestive system, because plant phytosterols compete with cholesterol for reabsorption and when this happens, a greater percentage of cholesterol exits the body in the feces. Drugs related to penicillin are also used to inhibit cholesterol recycling. One of these is ezetimibe, shown in Figure 2.224.
Genetic defects in the cholesterol movement system are a cause of the rare disease known as familial hypercholesterolemia in which the blood of afflicted individuals contains dangerously high levels of LDLs. Left untreated, the disease is often fatal in the first 10-20 years of life. While LDLs have received (and deserve) a bad rap, another group of lipoprotein complexes known as the HDLs (high density lipoprotein complexes) are known as “good cholesterol” because their levels correlate with removal of debris (including cholesterol) from arteries and reduce inflammation.
Membrane function
In membranes, cholesterol is important as an insulator for the transmission of signals in nerve tissue and it helps to manage fluidity of membranes over a wide range of temperatures. Stacked in the lipid bilayer, cholesterol decreases a membrane’s fluidity and its permeability to neutral compounds, as well as protons and sodium ions. Cholesterol may play a role in signaling by helping with construction of lipid rafts within the cell membrane.
Vitamin A
Vitamin A comes in three primary chemical forms, retinol (storage in liver - Figure 2.225), retinal (role in vision - Figure 2.226), and retinoic acid (roles in growth and development). All vitamin A forms are diterpenoids and differ only in the chemical form of the terminal group. Retinol is mostly used as the storage form of the vitamin.
Retinol is commonly esterified to a fatty acid and kept in the liver. In high levels, the compound is toxic. Retinol is obtained in the body by hydrolysis of the ester or by reduction of retinal. Importantly, neither retinal nor retinol can be made from retinoic acid. Retinoic acid is important for healthy skin and teeth, as well as bone growth. It acts in differentiation of stem cells through a specific cellular retinoic acid receptor.
Sources
Good sources of vitamin A are liver and eggs, as well as many plants, including carrots, which have a precursor, β-carotene (Figure 2.227) from which retinol may be made by action of a dioxygenase.
Light sensitivity The conjugated double bond system in the side chain of vitamin A is sensitive to light and can flip between cis and trans forms on exposure to it. It is this response to light that makes it possible for retinal to have a role in vision in the rods and cones of the eyes. Here, the aldehyde form (retinal) is bound to the protein rhodopsin in the membranes of rod and cone cells.
When exposed to light of a particular wavelength, the “tail” of the retinal molecule will flip back and forth from cis to trans at the double bond at position 11 of the molecule. When this happens, a nerve signal is generated that signals the brain of exposure to light. Slightly different forms of rhodopsin have different maximum absorption maxima allowing the brain to perceive red, green and blue specifically and to assemble those into the images we see (Figure 2.228). Cones are the cells responsible for color vision, whereas rods are mostly involved in light detection in low light circumstances.
Deficiency and surplus
Deficiency of vitamin A is common in developing countries and was inspiration for the design and synthesis of the geneticallymodified golden rice, which is used as a source of vitamin A to help prevent blindness in children. Overdose of vitamin A, called hypervitaminosis A is dangerous and can be fatal. Excess vitamin A is also suspected to be linked to osteoporosis. In smokers, excess vitamin A is linked to an increased rate of lung cancer, but non-smokers have a reduced rate.
Vitamin D
The active form of vitamin D plays important roles in the intestinal absorption of calcium and phosphate and thus in healthy bones. Technically, vitamin D isn’t even a vitamin, as it is a compound made by the body. Rather, it behaves more like a hormone.
Derived from ultimately from cholesterol, vitamin D can be made in a reaction catalyzed by ultraviolet light. In the reaction, the intermediate 7-dehydrocholesterol is converted to cholecalciferol (vitamin D3) by the uv light (Figure 2.229). The reaction occurs most readily in the bottom two layers of the skin shown in Figure 2.230.
Forms of vitamin D
Five different compounds are referred to as vitamin D. They are
Vitamin D1 - A mixture of ergocalciferol and lumisterol
Vitamin D2 - Ergocalciferol
Vitamin D3 - Cholecalciferol Vitamin
D4 - 22-Dihydroergocalciferol Vitamin
D5 - Sitocalciferol
Vitamin D3 is the most common form used in vitamin supplements and it and vitamin D2 are commonly obtained in the diet, as well. The active form of vitamin D, calcitriol (Figure 2.231), is made in the body in controlled amounts. This proceeds through two steps from cholecalciferol. First, a hydroxylation in the liver produces calcidiol and a second hydroxylation in the kidney produces calcitriol. Monocyte macrophages can also synthesize vitamin D and they use is as a cytokine to stimulate the innate immune system.
Mechanism of action
Calcitriol moves in the body bound to a vitamin D binding protein, which delivers it to target organs. Calcitriol inside of cells acts by binding a vitamin D receptor (VDR), which results in most of the vitamin’s physiological effects. After binding calcitriol, the VDR migrates to the nucleus where it acts as a transcription factor to control levels of expression of calcium transport proteins (for example) in the intestine. Most tissues respond to VDR bound to calcitriol and the result is moderation of calcium and phosphate levels in cells.
Deficiency/excess
Deficiency of vitamin D is a cause of the disease known as rickets, which is characterized by soft, weak bones and most commonly is found in children. It is not common in the developed world, but elsewhere is of increasing concern.
Excess of vitamin D is rare, but has toxic effects, including hypercalcemia, which results in painful calcium deposits in major organs. Indications of vitamin D toxicity are increased urination and thirst. Vitamin D toxicity can lead to mental retardation and many other serious health problems.
Vitamin E
Vitamin E comprises a group of two compounds (tocopherols and tocotrienols - Figure 2.232) and stereoisomers of each. It is commonly found in plant oils. The compounds act in cells as fat-soluble antioxidants. α-tocopherol (Figure 2.233), the most active form of the vitamin, works 1) through the glutathione peroxidase protective system and 2) in membranes to interrupt lipid peroxidation chain reactions. In both actions, vitamin E reduces levels of reactive oxygen species in cells.
Action
Vitamin E scavenges oxygen radicals (possessing unpaired electrons) by reacting with them to produce a tocopheryl radical. This vitamin E radical can be converted back to its original form by a hydrogen donor. Vitamin C is one such donor. Acting in this way, Vitamin E helps reduce oxidation of easily oxidized compounds in the lipid peroxidation reactions (Figure 2.234).
Vitamin E also can affect enzyme activity. The compound can inhibit action of protein kinase C in smooth muscle and simultaneously activate catalysis of protein phosphatase 2A to remove phosphates, stopping smooth muscle growth.
Deficiency/excess
Deficiency of vitamin E can lead to poor conduction of nerve signals and other issues arising from nerve problems. Low levels of the vitamin may be a factor in low birth weights and premature deliveries. Deficiency, however, is rare, and not usually associated with diet.
Excess Vitamin E reduces vitamin K levels, thus reducing the ability to clot blood. Hypervitaminosis of vitamin E in conjunction with aspirin can be life threatening. At lower levels, vitamin E may serve a preventative role with respect to atherosclerosis by reducing oxidation of LDLs, a step in plaque formation.
Vitamin K
Like the other fat-soluble vitamins, Vitamin K comes in multiple forms (Figure 2.235) and is stored in fat tissue in the body. There are two primary forms of the vitamin - K1 and K2 and the latter has multiple sub-forms . Vitamins K3, K4, and K5 are made synthetically, not biologically.
Action
Vitamin K is used as a co-factor for enzymes that add carboxyl groups to glutamate side chains of proteins to increase their affinity for calcium. Sixteen such proteins are known in humans. They include proteins involved in blood clotting (prothrombin (called Factor II), Factors VII, IX, and X), bone metabolism (osteocalcin, also called bone Gla protein (BGP), matrix Gla protein (MGP), and periostin) and others.
Modification of prothrombin is an important step in the process of blood clotting (see HERE). Reduced levels of vitamin K result in less blood clotting, a phenomenon sometimes referred to as blood thinning. Drugs that block recycling of vitamin K (Figure 2.236) by inhibiting the vitamin K epoxide reductase, produce lower levels of the vitamin and are employed in treatments for people prone to excessive clotting. Warfarin (coumadin) is one such compound that acts in this way and is used therapeutically. Individuals respond to the drug differentially, requiring them to periodically be tested for levels of clotting they possess, lest too much or too little occur.
Sources
Vitamin K1 is a stereoisomer of the plant photosystem I electron receptor known as phylloquinone and is found abundantly in green, leafy vegetables. Phylloquinone is one source of vitamin K, but the compound binds tightly to thylakoid membranes and tends to have low bioavailability. Vitamin K2 is produced by microbes in the gut and is a primary source of the vitamin. Infants in the first few days before they establish their gut flora and people taking broad spectrum antibiotics may have reduced levels, as a result. Dietary deficiency is rare in the absence of damage to the small bowel. Others at risk of deficiency include people with chronic kidney disease and anyone suffering from a vitamin D deficiency. Deficiencies produce symptoms of easy bruising, heavy menstrual bleeding, anemia, and nosebleeds.
Steroids
Steroids, such as cholesterol are found in membranes and act as signaling hormones in traveling through the body.
Steroid hormones are all made from cholesterol and are grouped into five categories - mineralocorticoids (21 carbons), glucocorticoids (21 carbons), progestagens (21 carbons), androgens (19 carbons), and estrogens (18 carbons).
Mineralocorticoids
Mineralocorticoids are steroid hormones that influence water and electrolyte balances. Aldosterone (Figure 2.238) is the primary mineralocorticoid hormone, though other steroid hormones (including progesterone) have some functions like it. Aldosterone stimulates kidneys to reabsorb sodium, secrete potassium, and passively reabsorb water. These actions have the effect of increasing blood pressure and blood volume. Mineralocorticoids are produced by the zona glomerulosa of the cortex of the adrenal gland.
Glucocorticoids
Glucocorticoids (GCs) bind to glucocorticoid receptors found in almost every vertebrate animal cell and act in a feedback mechanism in the immune system to reduce its activity. GCs are used to treat diseases associated with overactive immune systems. These include allergies, asthma, and autoimmune dis- Figure 2.237 - Steroid numbering scheme Image by Pehr Jacobson eases. Cortisol (Figure 2.239) is an important glucocorticoid with cardiovascular, metabolic, and immunologic functions. The synthetic glucocorticoid known as dexamethasone has medical applications for treating rheumatoid arthritis, bronchospasms (in asthma), and inflammation due to its increased potency (25-fold) compared to cortisol. Glucocorticoids are produced primarily in the zona fasciculata of the adrenal cortex.
Progestagens
Progestagens (also called gestagens) are steroid hormones that work to activate the progesterone receptor upon binding to it. Synthetic progestagens are referred to as progestins. The most common progestagen is progesterone (also called P4 - Figure 2.240) and it has functions in maintaining pregnancy. Progesterone is produced primarily in the diestrus phase of the estrous cycle by the corpus luteum of mammalian ovaries. In pregnancy, the placenta takes over most progesterone production.
Androgens
Androgens are steroid hormones that act by binding androgen receptors to stimulate development and maintenance of male characteristics in vertebrates. Androgens are precursors of estrogens (see below). The primary androgen is testosterone (Figure 2.241). Other important androgens include dihydrotestosterone (stimulates differentiation of penis, scrotum, and prostate in embryo) and androstenedione (common precursor of male and female hormones).
Estrogens
The estrogen steroid hormones are a class of compounds with important roles in menstrual and estrous cycles. They are the most important female sex hormones. Estrogens act by activating estrogen receptors inside of cells. These receptors, in turn, affect expression of many genes. The major estrogens in women include estrone (E1), estradiol (E2 - Figure 2.242), and estriol (E3). In the reproductive years, estradiol predominates. During pregnancy, estriol predominates and during menopause, estrone is the major estrogen.
Estrogens are made from the androgen hormones testosterone and androstenedione in a reaction catalyzed by the enzyme known as aromatase. Inhibition of this enzyme with aromatase inhibitors, such as exemestane, is a strategy for stopping estrogen production. This may be part of a chemotherapeutic treatment when estrogenresponsive tumors are present.
Cannabinoids
Cannabinoids are a group of chemicals that bind to and have effects on brain receptors (cannabinoid receptors), repressing neurotransmitter release. Classes of these compounds include endocannabinoids (made in the body), phytocannabinoids (made in plants, such as marijuana), and synthetic cannabinoids (man-made).
Endocannabinoids are natural molecules derived from arachidonic acid. Cannabinoid receptors are very abundant, comprising the largest number of G-protein- 247 Figure 2.243 - Tetrahydrocannabinol - Active ingredient in marijuana coupled receptors found in brain. The best known phytocannabinoid is Δ-9- tetrahydrocannabinol (THC), the primary psychoactive ingredient (of the 85 cannabinoids) of marijuana (Figure 2.243).
Anandamide
Anandamide (N-arachidonoylethanolamine - Figure 2.244) is an endocannabinoid neurotransmitter derived from arachidonic acid. It exerts its actions primarily through the CB1 and CB2 cannabinoid receptors, the same ones bound by the active ingredient in marijuana, Δ9-tetrahydrocannabinol. Anandamide has roles in stimulating eating/appetite and affecting motivation and pleasure. It has been proposed to play a role in “runners high,” an analgesic effect experienced from exertion, especially among runners. Anandamide appears to impair memory function in rats.
Anandamide has been found in chocolate and two compounds that mimic its effects (N-oleoylethanolamine and Nlinoleoylethanolamine) are present as well. The enzyme fatty acid amide hydrolase (FAAH) breaks down anandamide into free arachidonic acid and ethanolamine.
Lipoxins
Lipoxins (Figure 2.245) are eicosanoid compounds involved in modulating immune responses and they have anti-inflammatory effects. When lipoxins appear in inflammation it begins the end of the process. Lipoxins act to attract macrophages to apoptotic cells at the site of inflammation and they are engulfed. Lipoxins further act to start the resolution phase of the inflammation process.
At least one lipoxin (aspirin-triggered LX4) has its synthesis stimulated by aspirin. This occurs as a byproduct of aspirin’s acetylation of COX-2. When this occurs, the enzyme’s catalytic activity is re-directed to synthesis of 15R-hydroxyeicosatetraenoic acid (HETE) instead of prostaglandins. 15R-HETE is a procursor of 15-epimer lipoxins, including aspirin-triggered LX4.
Heme
Heme groups are a collection of protein/ enzyme cofactors containing a large heterocyclic aromatic ring known as a porphyrin ring with a ferrous (Fe++) ion in the middle. An example porphyrin ring with an iron (found in Heme B of hemoglobin), is shown in Figure 2.246. When contained in a protein, these are known collectively as hemoproteins (Figure 2.247).
Heme, of course, is a primary component of hemoglobin, but it is also found in other proteins, such as myoglobin, cytochromes, and the enzymes catalase and succinate dehydrogenase. Hemoproteins function in oxygen transport, catalysis, and electron transport. Heme is synthesized in the liver and bone marrow in a pathway that is conserved across a wide range of biology.
Porphobilinogen
Porphobilinogen (Figure 2.248) is a pyrrole molecule involved in porphyrin metabolism. It is produced from aminolevulinate by action of the enzyme known as ALA dehydratase. Porphobilinogen is acted upon by the enzyme porphobilinogen deaminase. Deficiency of the latter enzyme (and others in porphyrin metabolism) can result in a condition known as porphyria, which results in accumulation of porphobilinogen in the cytoplasm of cells.
The disease can manifest itself with acute abdominal pain and numerous psychiatric issues. Both Vincent van Gogh and King ` George III are suspected to have suffered from porphyria, perhaps causing the “madness of King George III.” Porphyria is also considered by some to be the impetus for the legend of vampires seeking blood from victims, since the color of the skin in non-acute forms of the disease can be miscolored, leading some to perceive that as a deficiency of hemoglobin and hence the “thirst” for blood imagined for vampires.
Dolichols
Dolichol is a name for a group of non-polar molecules made by combining isoprene units together. Phosphorylated forms of dolichols play central roles in the N-glycosylation of proteins. This process, which occurs in the endoplasmic reticulum of eukaryotic cells, begins with a membrane-embedded dolichol pyrophosphate (Figure 2.249) to which an oligosaccharide is attached (also see HERE). This oligosaccharide contains three molecules of glucose, nine molecules of mannose and two molecules of N-acetylglucosamine.
Interestingly, the sugars are attached to the dolichol pyrophosphate with the pyrophosphate pointing outwards (away from) the endoplasmic reticulum, but after attachment, the dolichol complex flips so that the sugar portion is situated on the inside of the endoplasmic reticulum. There, the entire sugar complex is transferred to the amide of an asparagine side chain of a target protein.
The only asparagine side chains to which the attachment can be made are in proteins where the sequences Asn-X-Ser or Asn-X-Thr occur. Sugars can be removed/added after the transfer to the protein. Levels of dolichol in the human brain increase with age, but in neurodegenerative diseases, they decrease.
Terpenes
Terpenes are members of a class of nonpolar molecules made from isoprene units. Terpenes are produced primarily by plants and by some insects. Terpenoids are a related group of molecules that contain functional groups lacking in terpenes.
Terpenes have a variety of functions. In plants, they often play a defensive role protecting from insects. The name of terpene comes from turpentine, which has an odor like some of the terpenes. Terpenes are common components of plant resins (think pine) and they are widely used in medicines and as fragrances. Hops, for example, gain some of their distinctive aroma and flavor from terpenes. Not all terpenes, however have significant odor.
Synthesis
Terpenes, like steroids, are synthesized starting with simple building blocks known as isoprenes. There are two of them - dimethylallyl pyrophosphate and the related isopentenyl pyrophosphate and (Figures 2.252 and 2.253) which combine 1-2 units at a time to make higher order structures. Terpene synthesis overlaps and includes steroid synthesis.
Terpenes and terpenoids are classified according to how many isoprene units they contain. They include hemiterpenes (one unit), monoterpenes (two units), sesquiterpenes (three units), diterpenes (four units), sesterterpenes (five units), triterpenes (six units), sesquarterpenes (seven units), tetraterpenes (eight units), polyterpenes (many units). Another class of terpene-containing molecules, the norisoterpenoids arise from peroxidase-catalyzed reactions on terpene molecules.
Examples
Common terpenes include monoterpenes of terpineol (lilacs), limonene (citrus), myrcene (hops), linalool (lavender), and pinene (pine). Higher order terpenes include taxadiene (diterpene precursor of taxol), lycopene (tetraterpenes), carotenes (tetraterpenes), and natural rubber (polyterpenes).
Steroid precursors geranyl pyrophosphate (monoterpene derivative), farnesyl pyrophosphate (sesquiterpene derivative), and squalene (triterpene) are all terpenes or derivatives of them. Vitamin A and phytol are derived from diterpenes.
Caffeine
Caffeine is the world’s most actively consumed psychoactive drug (Figure 2.255). A methylxanthine alkaloid, caffeine is closely related to adenine and guanine and this is responsible for many effects on the body. Caffeine blocks the binding of adenosine on its receptor and consequently prevents the onset of drowsiness induced by adenosine. Caffeine readily crosses the blood-brain barrier and stimulates release of neurotransmitters. Caffeine stimulates portions of the autonomic nervous system and inhibits the activity of phosphodiesterase. The latter has the result of raising cAMP levels in cells, which activates protein kinase A and activates glycogen breakdown, inhibits TNF-α and leukotriene synthesis, which results in reduction of inflammation and innate immunity.
Caffeine also has effects on the cholinergic system (acetylcholinesterase inhibitor), is an inositol triphosphate receptor 1 antagonist, and is a voltage independent activator of ryanodin receptors (a group of calcium channels found in skeletal muscle, smooth muscle, and heart muscle cells).
The half-life of caffeine in the body varies considerably. In healthy adults, it has a half-life of about 3-7 hours. Nicotine decreases the half-life and contraceptives and pregnancy can double it. The liver metabolizes caffeine, so the health of the liver is a factor in the halflife. CYP1A2 of the cytochrome P450 oxidase enzyme is primarily responsible. Caffeine is a natural pesticide in plants, paralyzing predator bugs.
Lipoprotein complexes and lipid movement in the body
Lipoprotein complexes are combinations of apolipoproteins and lipids bound to them that solubilize fats and other non-polar molecules, such as cholesterol, so they can travel in the bloodstream between various tissues of the body. The apolipoproteins provide the emulsification necessary for this. Lipoprotein complexes are formed in tiny “balls” with the water soluble apolipoproteins on the outside and non-polar lipids, such as fats, cholesteryl esters, and fat soluble vitamins on the inside.
They are categorized by their densities. These include (from highest density to the lowest) high density lipoproteins (HDLs), Low Density Lipoproteins (LDLs), Intermediate Density Lipoproteins (IDLs), Very Low Density Lipoproteins (VLDLs) and the chylomicrons. These particles are synthesized in the liver and small intestines.
Apolipoproteins
Each lipoprotein complex contain a characteristic set of apolipoproteins, as shown in Figure 2.256. ApoC-II and ApoC-III are notable for their presence in all the lipoprotein complexes and the roles they play in activating (ApoC-II) or inactivating (ApoC-III) lipoprotein lipase. Lipoprotein lipase is a cellular enzyme that catalyzes the breakdown of fat from the complexes. ApoE (see below) is useful for helping the predict the likelihood of the occurrence of Alzheimer's disease in a patient.
Gene editing
ApoB-48 and ApoB-100 are interesting in being coded by the same gene, but a unique mRNA sequence editing event occurs that converts one into the other. ApoB-100 is made in the liver, but ApoB-48 is made in the small intestine. The small intestine contains an enzyme that deaminates the cytidine at nucleotide #2153 of the common mRNA. This changes it to a uridine and changes the codon it is in from CAA (codes for glutamine) to UAA (stop codon). The liver does not contain this enzyme and does not make the change in the mRNA. Consequently, a shorter protein is synthesized in the intestine (ApoB-48) than the one that is made in the liver (ApoB-100) using the same gene sequence in the DNA.
Movement
The movement of fats in the body is important because they are not stored in all cells. Only specialized cells called adipocytes store fat. There are three relevant pathways in the body for moving lipids. As described below, they are 1) the exogenous pathway; 2) the endogenous pathway, and 3) the reverse transport pathway.
Exogenous pathway
Dietary fat entering the body from the intestinal system must be transported, as appropriate, to places needing it or storing it. This is the function of the exogenous pathway of lipid movement in the body. All dietary lipids (fats, cholesterol, fat soluble vitamins, and other lipids) are moved by it. In the case of dietary fat, it begins its journey after ingestion first by being solubilized by bile acids in the intestinal tract. After passing through the stomach, pancreatic lipases clip two fatty acids from the fat, leaving a monoacyl glycerol. The fatty acids and monoacyl glycerol are absorbed by intestinal cells (enterocytes) and reassembled back into a fat, and then this is mixed with phospholipids, cholesterol esters, and apolipoprotein B-48 and processed to form chylomicrons (Figures 2.258 & 2.259) in the Golgi apparatus and smooth endoplasmic reticulum.
Exocytosis
These are exocytosed from the cell into lymph capillaries called lacteals. The chylomicrons pass through the lacteals and enter the bloodstream via the left subclavian vein. Within the bloodstream, lipoprotein lipase breaks down the fats causing the chylomicron to shrink and become what is known as a chylomicron remnant. It retains its cholesterol and other lipid molecules.
The chylomicron remnants travel to the liver where they are absorbed (Figure 2.260). This is accomplished by receptors in the liver that recognize and bind to the ApoE of the chylomicrons. The bound complexes are then internalized by endocytosis, degraded in the lysosomes, and the cholesterol is disbursed in liver cells.
Endogenous pathway
The liver plays a central role in managing the body’s needs for lipids. When lipids are needed by the body or when the capacity of the liver to contain more lipids than is supplied by the diet, the liver packages up fats and cholesteryl esters into Very Low Density Lipoprotein (VLDL) complexes and exports them via the endogenous pathway. VLDL complexes contain ApoB-100, ApoC-I, ApoC-II, ApoC-III, and ApoE apolipoproteins. VLDLs enter the blood and travel to muscles and adipose tissue where lipoprotein lipase is activated by ApoC-II. In the muscle cells, the released fatty acids are taken up and oxidized. By contrast, in the adipoctyes, the fatty acids are taken up and reassembled back into triacylglycerides (fats) and stored in fat droplets. Removal of fat from the VLDLs causes them to shrink, first to Intermediate Density Lipoprotein (IDL) complexes (also called VLDL remnants) and then to Low Density Lipoprotein (LDL) complexes.
Shrinking of VLDLs is accompanied by loss of apolipoproteins so that LDLs are comprised primarily of ApoB-100. This lipoprotein complex is important because cells have receptors for it to bind and internalize it by receptor-mediated endocytosis (Figure 2.261). Up until this point, cholesterol and cholesteryl esters have traveled in chylomicrons, VLDLs, and IDLs as fat has been stripped stripped away. For cholesterol compounds to get into the cell from the lipoprotein complexes, they must be internalized by cells and that is the job of receptormediated endocytosis.
Reverse transport pathway
Another important consideration of the movement of lipids in the body is the reverse transport pathway (Figure 2.260). It is also called the reverse cholesterol transport pathway, since cholesterol is the primary molecule involved. This pathway involves the last class of lipoprotein complexes known as the High Density Lipoproteins (HDLs). In contrast to the LDLs, which are commonly referred to as “bad cholesterol” (see below also), the HDLs are known as “good cholesterol.”
HDLs are synthesized in the liver and small intestine. They contain little or no lipid when made (called depleted HDLs), but serve the role of “scavenger” for cholesterol in the blood and from remnants of other (damaged) lipoprotein complexes in the blood. To perform its task, HDLs carry the enzyme known as lecithincholesterol acyl transferase (LCAT), which they use to form cholesteryl esters using fatty acids from lecithin (phosphatidylcholine) and then they internalize them.
The cholesterol used for this purpose comes from the bloodstream, from macrophages, and from foam cells (macrophage-LDL complexes - Figure 2.262). Addition of cholesteryl esters causes the HDL to swell and Figure 2.261 - The process of receptor-mediated endocytosis Image by Aleia Kim when it is mature, it returns its load of cholesterol back to the liver or, alternatively, to LDL molecules for endocytosis. HDLs have the effect of lowering levels of cholesterol and it is for that reason they are described as “good cholesterol.”
Regulation of lipid transfer
It is important that cells get food when they need it so some control of the movement of nutrients is critical. The liver, which plays the central role in modulating blood glucose levels, is also important for performing the same role for lipids. It accomplishes this task the use of specialized LDL receptors on its surface. Liver LDL receptors bind LDLs that were not taken up by other cells in their path through the bloodstream. High levels of LDLs are a signal to the liver to reduce the creation of VLDLs for release.
People with the genetic disease known as familial hypercholesterolemia, which manifests with dangerously high levels of LDLs, lack properly functioning LDL receptors on their liver cells.Figure 2.263 - Progression of atherosclerosis Wikipedia Figure 2.263 - Progression of atherosclerosis Wikipedia Figure 2.263 - Progression of atherosclerosis Wikipedia
In sufferers of this disease, the liver never gets the signal that the LDL levels are high. In fact, to the liver, it appears that all VLDLs and LDLs are being taken up by peripheral tissues, so it creates more VLDLs to attempt to boost levels. Untreated, the disease used to be fatal early, but newer drugs like the statins have significantly increased life spans of patients. Cellular needs for the contents of LDLs are directly linked to the levels of synthesis of LDL receptors on their membranes. As cells are needing more cholesterol, their synthesis of components for receptors goes up and it decreases as need diminishes.
Good cholesterol / bad cholesterol
It is commonly accepted that “high cholesterol” levels are not healthy. This is due, at least indirectly, to the primary carriers of cholesterol, the LDLs. A primary function of the LDLs is to deliver cholesterol and other lipids directly into cells by receptor mediated endocytosis (Figure 2.237). High levels of LDLs, though, are correlated with formation of atherosclerotic plaques (Figure 2.263 & 2.264) and incidence of atherosclerosis, leading to the description of them as “bad cholesterol.” This is because when LDL levels are very high, plaque formation begins. It is thought that reactive oxygen species (higher in the blood of smokers) causes partial oxidation of fatty acid groups in the LDLs. When levels are high, they tend to accumulate in the extracellular matrix of the epithelial cells on the inside of the arteries. Macrophages of the immune system take up the damaged LDLs (including the cholesterol).
Since macrophages can’t control the amount of cholesterol they take up, cholesterol begins to accumulate in them and they take on appearance that leads to their being described as “foam cells.” With too much cholesterol, the foam cells, however, are doomed to die by the process of programmed cell death (apoptosis). Accumulation of these, along with scar tissue from inflammation result in formation of a plaque. Plaques can grow and block the flow of blood or pieces of them can break loose and plug smaller openings in the blood supply, ultimately leading to heart attack or stroke.
Good cholesterol
On the other hand, high levels of HDL are inversely correlated with atherosclerosis and arterial disease. Depleted HDLs are able to remove cholesterol from foam cells. This occurs as a result of contact between the ApoA-I protein of the HDL and a transport protein on the foam cell (ABC-G1). Another transport protein in the foam cell, ABCA-1 transports extra cholesterol from inside the cell to the plasma membrane where it is taken up into the HDL and returned to the liver or to LDLs by the reverse transport cholesterol pathway.
Deficiency of the ABCA-1 gene leads to Tangier disease. In this condition, HDLs are almost totally absent because they remain empty as a result of not being able to take up cholesterol from foam cells, so they are destroyed by the body.
ApoE and Alzheimer’s disease
ApoE is a component of the chylomicrons and is also found in brain, macrophages, kidneys, and the spleen. In humans, it is found in three different alleles, E2, E3, and E4. The E4 allele (present at about 14% of the population) is associated with increased likelihood of contracting Alzheimer's disease. People heterozygous for the allele are 3 times as likely to contract the disease and those homozygous for it are 15 times as likely to do so. It is not known why this gene or allele is linked to the disease. The three alleles differ only slightly in amino acid sequence, but the changes do cause notable structural differences. The E4 allele is associated with increased calcium ion levels and apoptosis after injury. Alzheimer’s disease is associated with accumulation of aggregates of the β- amyloid peptide. ApoE does enhance the proteolytic breakdown of it and the E4 isoform is not as efficient in these reactions as the other isoforms.
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