Note that side chains serve as a) stabilizers of protein structure, b) reactive centers, and c) micro-environments. * + H 3 N-C-COOH H 2 N-C-COO -

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1 BIOCEMISTRY I AMINO ACIDS I. Amino Acid Structure One of the most important macromolecules (chains of distinct molecular units) in the biosphere is protein. Proteins are needed for catalysis, reaction, support, stability, binding and thousands of other functions. The distinct molecular units, or building blocks, that make up proteins are called amino acids. amino >> 3 N -C"-COO << carboxyl terminal terminal (N-term) R (C-term) An amino acid is an organic molecule with a central carbon, called the "-carbon, connected to 1) a hydrogen, 2) an amino group (called the "-amino group), 3) a carboxylic acid group (called the "-carboxyl group), and 4) a variable group (or side chain) designated -R. The "-amino and "-carboxyl (also referred to as "-carbonyl) groups are the terminals of the amino acid. The "-amino, called the N-terminal, is considered the beginning, or front, of the amino acid. The "-carbonyl, called the C-terminal, is the end, or back, of the amino acid. L-amino acid D-amino acid R 3 N -C-COO 3 N -C-COO R There are 20 common amino acids and all have R-groups distinct from the other "-carbon attachments except one (whose R-group is hydrogen). This means that the "-carbon of nineteen of the common amino acids is chiral (or asymmetric and optically active). The one common amino acid that is not chiral, glycine, is achiral. For any asymmetric center that is tetrahedral, there are two possible arrangements of atoms on the chiral atom. The For many possible structures are not the same, but are mirror images of each other. biochemical building blocks, these are designated D- and L-configurations. Note that side chains serve as a) stabilizers of protein structure, b) reactive centers, and c) micro-environments. II. Amino Acids Ionization The terminal functional groups of amino acids are weak acids. The "-carboxyl group is a carboxylic acid. Oxygen is highly electronegative, and its attraction for the electrons that hold the hydrogen is great. ence, the bond between the oxygen and the hydrogen has fair ionic character. The hydrogen is easily stripped from the oxygen (dissociable) as hydrogen ion ( ) at higher p levels. Low p Form igh p Form 3 N-C-COO 2 N-C-COO - R R At low p everything is protonated. high p everything is deprotonated. At In a solution with a low p, there are so many hydrogen ions that the probability that the "-carboxyl group will permanently lose its hydrogen (be deprotonated) is almost zero. But at p levels above 2 or so, is lower and the carbonyl group can be deprotonated, leaving a negative charge on the oxygen. A carboxyl group will have a low pk a. The "-amino group is an amine; an even weaker acid than the carboxylic acid type. Nitrogen is only fairly electronegative, and its attraction for the electrons that hold the hydrogen is just a bit stronger than hydrogen s attraction for them. ence, the bond has poor ionic character. ydrogen ion is not easily stripped from the nitrogen and tt p levels below 9 or so, an amino group will be protonated, and have a positive charge. An amino group will have a high pk a. The p of the solution determines the overall charge of an amino acid. At median p levels (between about 6 to 8), the "-carboxyl group of most amino acid molecules in a solution will be deprotonated, and have a negative charge). Under the same conditions, the "-amino group of most amino acids in solution will be protonated (and have a positive charge). This form of an amino acid with no dissociable hydrogen on the R-group is called a zwitterion (a neutral molecule that has cationic and anionic functional groups). Zwitterion 3 N-C-COO - R

2 The R-group of several amino acids is polar and contains dissociable hydrogens. Like the terminal groups, the charge on these groups is a function of p. Acidic functional groups will deprotonate (lose hydrogen ion) in solutions with a lower p than basic or other nonacidic functional groups. The ionization (protonation or deprotonation) of various amino acid functional groups is important when considering biological reactions. We can examine what happens during protonation/deportonation by analyzing the titration of an amino acids. When an amino acid is titrated, we typically start with an amino acid solution at one extreme of the p scale or the other, then raise or lower the p by the addition of strong base or acid. Lets look at the titration curve of a simple amino acid like glycine. Glycine 3 N-C-COO Glycine has dissociable hydrogens on the N and C-terminals. The "-carbonyl group has a pk of about 2.4 and the "-amino group has a pk of about 9.8. In a solution with a p lower than 2.4, most of the molecules will be protonated, at all functional groups; hence most will have a 1 charge: MOSTLY 3 N -C 2 -COO below this pk As the p is raised (by the addition of base), the in solution is lowered because it reacts with O - to make 2 O; glycine starts to deprotonate. By the time the solution reaches a p of 2.4, half the glycine molecules will have a protonated carbonyl group while the other half will be deprotonated. alf will have a 1 charge, the other half will have no net charge: [ 3 N -C 2 -COO - ]/[ 3 N -C 2 -COO] = 1.0 As the p is raised more, the concentration eventually falls so low that by a p of 6.1, every molecule s carbonyl group will be deprotonated, and all molecules will have no net charge; there will only be zwitterions in solution. This p is the isoelectric point of the amino acid glycine. The isoelectric point is the p at which all molecules have no net charge. The symbol for isoelectric point is pi: ONLY 3 N -C 2 -COO - exists at this p Above the isoelectric point, most of the molecules have no charge, but we start to see deprotonation of the "-amino group. The N-term group starts to deprotonate and some molecules acquire an overall negative charge: MOSTLY 3 N -C 2 -COO - exists above the pi By the time the p of the solution is the same as the pk of the "-amino group, 9.8, there are equal amounts of molecules with no net charge and molecules with a -1 charge: [ 2 N-C 2 -COO - ]/[ 3 N -C 2 -COO - ] = 1.0 At a p above the pk of the "-amino group (9.8 for glycine), most of the molecules will have a negative charge: MOSTLY 2 N-C 2 -COO - exists above this pk In solutions with a p much above (~3 units) the pk of the "-amino group, there will only be molecules with an overall negative charge. For glycine, this is around 12 or more: ONLY 2 N-C 2 -COO - exists above this p Be any of this as it may, when a molecule deprotonates, it is acting as a weak acid, and the deprotonation of a weak acid produces its conjugate base. The relation between the p of a solution and relative amounts of conjugates in solution may be discerned using the enderson- asselbalch equation: p = pk log([conjugate Base]/[Weak Acid]) = pk log([a - ]/[A]) The relative amounts of the conjugates is therefore given by: [A - ]/[A] = 10 p-pk

3 A certain amino acid has a functional group with a pk of If 60% of amino acids in solution must be protonated at that group for some reaction to work properly, determine the p required to ensure the reaction. The protonated form is the acid, A, while the deprotonated form is the conjugate base, A -. If 60% must be protonated, [A - ] = 60, and [A] = 40, p = pk log([a - ]/[A]) = 4.50 log(60/40) ~ 4.68 The solution p should be 4.68 for the reaction to work properly. III. The Relation Between p and Charge The strength of the bond between a dissociable proton and the atom it is attached to is a function of the attraction of the atom to the electrons in the bond with the proton (electronegativity). The greater the atoms attraction for the electrons (its electronegativity), the weaker its bond to the proton, the easier it is to lose the proton! Some environmental factors interact with the functional group, knocking off or pulling away the proton. The lower the electronegativity of the atom holding the proton, the harder the environment must act to remove it. A. At low p, there is an abundance of hydrogen ions in solution. Groups with dissociable hydrogens are acted upon by the environment and hydrogens are stripped off. owever, there are so many hydrogen ions in solution that as soon as a proton is removed or pulled away, its replaced by another from solution. The functional group, for all practical purposes, is never without the hydrogen; everything is always protonated! Therefore, amino acids have a positive charge at low p (in acidic solution). B. At high p there is almost no hydrogen ions in solution. Groups with dissociable hydrogens are acted upon by the environment and protons are stripped off. Since there are no hydrogen ions in solution, the protons are not replaced. The functional group, for all practical purposes, never has the hydrogen; everything is always deprotonated! Therefore, amino acids have a negative charge at high p (in basic solution). A. The Quantitative Relation Between p and Charge For any weak acid, A, the dissociable proton is removed according to the equation: A(aq) 2 O(l) º 3 O (aq) A - (aq) T h e e q u i l i b r i u m c o n s t a n t, K a ( t h e a c i d dissociation constant), for the dissociation of the proton from the acid is equal to [ ][A - ]/[A]. Note that is simply short for 3 O. This equation may be rearranged to produce: p = pk a log([a - ]/[A]), the so-called enderson-asselbalch equation. This expresses the relation between the ratio of the conjugates and the p of the system. Each conjugate represents a form of the substance that has a particular charge. The ratio of those forms is given by the equation: [A - ]/[A] = 10 p-pk where the pk is the -log of the dissociation constant for the acid (A). For simple amino acids that don t have dissociable hydrogens on the side chains, the acid is the form of the molecule with a net charge of 1, 3 N -CR-COO. The conjugate base of the acid is the zwitterion, 3 N -CR-COO -, a molecule with no net charge. So [A - ]/[A] is the ratio of the 0 charge form to the 1 charge form: [ 3 N -CR-COO - ]/[ 3 N -CR-COO]. Note, if p = pk, [ 3 N -CR-COO - ]=[ 3 N -CR-COO]. If the zwitterion form loses a hydrogen ion, it becomes, 2 N-CR-COO - ; a molecule with a -1 charge. The ratio of acid, 0 charge form, to conjugate base, -1 charge form, is still given by 10 p-pk. But now we d use the pk for the zwitterion, the pk for the dissociation of the hydrogen ion from the "-amino group.

4 The ratio of the conjugates is only determined for a valid p range of the particular conjugates in question. For example, for the 1 and 0 forms, 3 N -CR-COO and 3 N -CR-COO -, this is for any p up to the average of the pk s for the 1 and the 0 forms: (pk COO pk N3 )/2. Below that p there are only the 1 charge and 0 charge forms. At that p there is only the 0 charge form; there is no 1 charge form or -1 charge form. Above that p up to maybe 3-5 p units above the pk N3, there is only the 0 charge form and the -1 charge form (there is no 1 charge form). So you d only be concerned with ratios for those forms. At any p above 3-5 p units of that pk, there is only the -1 form. 1) For example, glycine has a pk COO ~ 2.4 and a pk N3 ~ 9.8. What fraction of glycine molecules will have a -1 charge in a solution with a p of 5? 1 form 0 form -1 form 3 N -CR-COO º 3 N -CR-COO - º 2 N-CR-COO - The average of the two pk s is (9.82.4)/2 ~ 6.1. Below a p of 6.1, the population consists only of the 1 form and the 0 form. At a p of 6.1 the population is all molecules with no charge (so 6.1 is the isoelectric point). And above a p of 6.1, the population consists only of molecules with 0 charge and molecules with a -1 charge. So there are no molecules with a -1 charge at a p of 5. 2) What fraction of glycine molecules will have a 1 charge at a p of 8.0? Above a p of 6.1, the population consists only of molecules with 0 charge and molecules with a -1 charge. So there are no molecules with a 1 charge at a p of 8. 3) What fraction of glycine molecules will have a 1 charge at a p of 3.0? The 1 form only occurs below a p of 6.1, and for p values less than 6.1, we only use the pk for the "-carboxyl group. So the form with the protonated "-carboxyl group is acid ( 3 N -CR-COO; the 1 form), and the form with the deprotonated "-carboxyl group is its conjugate base ( 3 N -CR-COO - ; the 0 form). [ 3 N -CR-COO - ]/[ 3 N -CR-COO] = = 3.98/1 The 1 form = 1 and the 0 form = Thus the fraction that is 1 is 1/(3.981) = 0.2 or about 20%. 4) istidine s "-carboxyl and "-amino groups have pks of 1.80 and 9.33 respectively. It has a side chain with a dissociable amino group (-N ) whose pk is If this amino acid is required in a reaction where the hydrogen of the side chain is transferred to some acceptor, what fraction of histidine molecules may participate in the reaction in a solution that has a p of 7.2? N -CN -COO º 3 N -CN -COO - º 3 N -CN-COO - º 2 N-CN-COO No more 2 No more 1 Up to a p of 3.92, ( )/2, only 2 and 1 molecules exists. Up to a p of 7.69, ( )/2, only the 1 and 0 molecules exists. Above this p, there are no molecules with hydrogens to donate on the side chain, so we work with the previous pk, 6.04 (the pk for the 1 and 0 forms). [ 3 N -CN-COO - ]/[ 3 N -CN -COO - ] = = 14.5/1 The concentration of the deprotonated form is 14.5 times greater than that of the protonated form. The fraction protonated is 1/(14.51) ~ or about 6.5% of the histidine molecules in solution may be used in the reaction.

5 B. The p of Amino Acid Solutions (this section is in development) When amino acids are dissolved in water, they act as weak acids and weak bases. The p of pure water is 7, so the predominant form of amino acid in pure water is: 3 N -CR-COO - Some fraction of these molecules will take a proton from water to reprotonate the carboxyl terminal while another fraction will donate hydrogen to water, deprotonating the amino terminal. 3 N -CR-COO - (aq) 2 O(l) º 3 N -CR-COO(aq) O - (aq) 3 N -CR-COO - (aq) 2 O(l) º 2 N-CR-COO - (aq) 3 O (aq) K b K a the sum of these reactions yields: 2 3 N -CR-COO - (aq)2 2 O(l) º 2 N-CR-COO - (aq) 3 N -CR-COO(aq)O - (aq) 3 O (aq) K a K b K a K b = [ 3 O ][O - ][ 2 N-CR-COO - ][ 3 N -CR-COO]/[ 3 N -CR-COO - ] 2 = K w [ 2 N-CR-COO - ][ 3 N -CR-COO]/[ 3 N -CR-COO - ] 2 so that K a K b /K w = [ 2 N-CR-COO - ][ 3 N -CR-COO]/[ 3 N -CR-COO - ] 2 So for a solution of amino acid with two dissociable functional groups, the equilibrium amounts will be: 2 3 N -CR-COO - (aq)2 2 O(l) º 2 N-CR-COO - (aq) 3 N -CR-COO(aq)O - (aq) 3 O (aq) n-2x x x K a K b = [3O ][O - ]x 2 /(n-2x) 2 = x 2 /(n 2-4nx 4x 2 ) where [3O ][O - ] = K w K a K b /K w = x 2 /(n-2x) 2 = x 2 /(n 2-4nx 4x 2 ) (K a K b /K w )n 2 - (K a K b /K w )4nx (K a K b /K w )4x 2 = x 2 [4(K a K b /K w )-1]x 2 - (K a K b /K w )4nx (K a K b /K w )n 2 = 0 x = (K a K b /K w )4n ± SQRT([(K a K b /K w )4n] 2 - [16(K a K b /K w )-4](K a K b /K w )n 2 ])/[8(K a K b /K w )-2] So for a 0.1M solution of glycine (K COO = 0.005, K N3 = 2.5x10-10 ) 3 N -C 2 -COO - (aq) 2 O(l) º 3 N -C 2 -COO(aq) O - (aq) K b = 2.0x N -C 2 -COO - (aq) 2 O(l) º 2 N-C 2 -COO - (aq) 3 O (aq) K a = 2.5x10-10 K a K b /K w = 5.0x10-8 = [ 2 N-C 2 -COO - ][ 3 N -C 2 -COO]/[ 3 N -CR-COO - ] 2 = x 2 /(0.1-2x) 2 = x 2 / x4x 2 x 2 = 5x x10-8 x 2x10-7 x 2 0 ~ 1x 2 2x10-8 x - 5x10-10 x ~ 2.24x10-5 = [ ]??? so p ~ 4.65 IV. Isoelectric Points The isoelectric point, pi, is the p where 100% of the molecules have a net charge of zero. Operationally, this is the average of the pk values for the acid of the zwitterion form and the pk of the zwitterion form. Consider glycine below: 1-form 0-form -1-form pk COO pk N3 3 N -C-COO º 3 N -C-COO - º 2 N-C-COO - The pk of the "-carboxyl group (pk COO ) is 2.34 while that of the "-amino group (pk N3 ) is The acid of the zwitterion is the 1 form, its pk is pk COO. The zwitterion s pk is pk N3. The isoelectric point is the average of these pks: pi = (pk COO pk N3 )/2 = ( )/2 = 5.97 In a solution whose p is 5.97, all glycine molecules will have a net charge of zero. Many amino acids have more than 2 pks because the side chain contains functional groups that have dissociable hydrogens (like amines or carboxylic acids).

6 Lysine 3 N-C-COO (C 2 ) 4 )N 3 This is the low p form of the amino acid lysine; it has a 2 net charge. Lysine has an amine side chain with a pk a of 10.53; it has three dissociable hydrogens. The zwitterion form has deprotonated "-carbonyl and "-amino groups. The acid of that form simply has a deprotonated "-carbonyl group. Its pi is: (pk "-N3 pk R-N3 )/2 = ( )/2 = 9.74 Aspartic Acid 3 N-C-COO C 2 )COO On the left is the low p form of the amino acid aspartic acid; it has a 1 net charge. Aspartic acid has a carboxylic side chain with a pk a of 3.65; it also has three dissociable hydrogens. The zwitterion form has a deprotonated "-carbonyl group. The acid of that form has no deprotonated groups. Its pi is: (pk "-COO pk R-COO )/2 = ( )/2 = 2.77