Amino Acids Reading Module-1 PDF

Chemistry  41     Course  Pack  1:  Amino  Acids     PROTEINS LECTURE 2: AMINO ACIDS: STRUCTURE AND PROPERTIES Topic Learning Outcomes: At the end of this topic, you should be able to:...

Chemistry  41     Course  Pack  1:  Amino  Acids     PROTEINS LECTURE 2: AMINO ACIDS: STRUCTURE AND PROPERTIES Topic Learning Outcomes: At the end of this topic, you should be able to: 1. Demonstrate knowledge of the structure, chemistry and reactivity of amino acids. 2. Relate the structure of amino acids to their biological roles. 3. Recognize protein from non-protein amino acids. Introduction Amino acids make up the most abundant organic constituent of cells, which are the proteins. Amino acids form the repeating units of these biopolymers that are biosynthesized through the formation of peptide bonds. Thus the structure, and biological functions of proteins are dependent on the amino acids comprising these proteins. Knowing the structure and properties of amino acids, therefore is key to understanding protein structure and biological functions. This section will also tell you about other amino acids and their roles in biological systems. Amino acids Amino acids are organic compounds containing both a carboxyl and an amino group. It is the repeating unit of proteins, which is made up of 20 common protein amino acids arranged in various orders and proportions. These common protein amino acids are coded for by the genetic code. However there are numerous other amino acids, produced by metabolism in cells. What are common protein amino acids? The structural features of these common amino acids are the following: 1. A central carbon atom bonded to 4 substituents 2. A carboxyl functional group as a substituent to the central carbon atom 3. An amino group as another substituent to the central carbon 4. An acidic hydrogen as the 3rd substituent to the central carbon 5. A variable side chain denoted simply as R also attached to the central carbon. The carboxyl, amino and acidic hydrogen are the common substituents to the central carbon that is sp3 hybridized. It means simply that these substituents are found in all protein amino acids. The substituent R is variable for all protein amino acids found in nature. The sp3 hybridization of the central carbon atom   1   Chemistry  41     Course  Pack  1:  Amino  Acids     results in bond angles that are 109o from each other, forming a tetrahedral configuration. Of the 20 common protein amino acids, 19 have the α-structure shown below (Fig. 1.1a) while one, proline (Fig. 1.1b) is an imino acid that has a structure where the central carbon is part of the pyrrolidone ring. a) b) source: https://study.com/academy/lesson/proline-structure-lesson-quiz.html Fig. 2.1a. Structure of an α-amino acid. Fig. 1.1b. The Imino acid, proline Structural features of amino acids and associated properties Amino acids have properties associated with its structural features. Properties due to the central carbon atom 1. Central carbon 1.1. Asymmetry Except for glycine, the central carbon of the 19 common protein amino acid is an asymmetric carbon or a chiral center. This carbon is sp3 hybridized with 4 bonds attached to 4 different substituents. Thus, because of this asymmetry, it can have two different arrangements in space called mirror images or enantiomers. Fig. 2.2 shows the enantiomers of amino acids denoted D and L and the reference compound glyceraldehyde.   2   Chemistry  41     Course  Pack  1:  Amino  Acids     Figs. 2.2 The reference compound, glyceraldehyde and the structure showing the spatial arrangements of the enantiomers of amino acids denoted, D and L. The reference substituent is the position of the hydroxyl group in glyceraldehyde corresponding to the position of the amino group in amino acids. Mirror image isomers, also called enantiomers, have identical physical properties except for their arrangements in space. Naturally occurring amino acids are in the L configuration. It is this spatial arrangement that is recognized by enzymes and naturally occurring biological systems. When the spatial arrangement is changed to the D configuration, the ability of specific groups to fit and/or bind properly with sites in biologically active molecules, e.g. enzymes, is disrupted (Fig. 1.3). Proteins that have protective functions such as immunoglobulins contain J-proteins that have D-amino acids. Similarly, natural antibiotics, e.g. bacitracin also contain D-amino acids so that these peptides and proteins are unrecognizable to hydrolytic enzymes of microorganisms. adapted from: https://wou.edu/chemistry/files/2019/07/table-of-pka-values.jpg Fig. 2.3. Specificity of binding of D and L amino acids 1.2. Optical activity Asymmetry or the difference in the spatial arrangements of the substituents of amino acids attached to the chiral carbon determines the ability of the molecule to rotate plane-polarized light. The difference in rotation is due to the difference in the mass of the substituents and the polarizability of the nucleophilic electron pairs in the substituents of the molecules. The size and degree of polarizability determines the rotation of plane-polarized light, i.e. if rotation is clockwise, the molecule is designated (d) or (+), meaning it is dextrorotatory; if rotation is counter- clockwise, the molecule is levoratory and designated (l) or (-). Fig. 2.4.   3   Chemistry  41     Course  Pack  1:  Amino  Acids     Source: https://chem.libretexts.org/Bookshelves/Biological_Chemistry/Supplemental_Mod ules_(Biological_Chemistry)/Proteins/Amino_Acids/Properties_of_Amino_Acids/ Stereochemistry_of_Amino_Acids Fig. 2.4. Rotation of plane-polarized light due to the different in the spatial configuration of D and L amino acids. Note that optical activity, while dependent on spatial configuration, is not the same as the latter. A D-amino acid can rotate plane-polarized light in the counterclockwise direction and designated (l), i.e. the amino acid is D (l)-amino acid. An example of this is D(l)-alanine. Properties due to the substituents of the central carbon atom 2. α-carboxyl group 2.1. Acidity The carboxyl group is the acidic functional group of organic compounds. This group is acidic because it contains two electronegative oxygen atoms that make the carbonyl atom to which these oxygen atoms are attached partially positive. Of the two oxygen atoms, one is sp2 hybridized, while the other which is bonded to hydrogen is sp3 hybridized. By orbital electronegativity, the sp2-hybridized oxygen is more electronegative hence the electron density is drawn toward it increasing the δ-positivity of the carbonyl carbon. To relieve this, the OH bond increases polarization of electrons towards the carbonyl carbon weakening the bond. In a polar environment such as water, that provides the “pull”, the proton dissociates. Dissociation of the acidic proton is also favored by the stability of the resulting carboxylate anion. This stability is the result of electron delocalization of the negative charges over several atoms (Fig. 2.5).   4   Chemistry  41     Course  Pack  1:  Amino  Acids     Fig. 2.5. Resonance stabilization of the carboxylate anion The pKa value of the carboxyl functional group is below 7.0, thus at physiological pH, the carboxyl functional group is ionized. pKa values refer to the negative logarithm of the dissociation constant, Ka or the logarithm of 1/Ka, For the carboxyl functional group, the range of pKa values for the different amino acids is 1.7-2.6. It is a weaker acid than HCl and the like but a stronger acid than -NH3+. 3. α-amino group 3.1. Basicity The amino group is the basic functional group of amino acids. Amino groups are basic due to the presence of a lone pair in nitrogen that increases electron density in this atom. Amino acids contain the amino functional group hence are also basic; some amino acids also have an amino group in R. At physiological pH, the amino group is positively charged upon protonation resulting in the formation of a conjugate weak acid, -NH3+. The range of pKa values of –NH3+ for the amino acids are above 7.0, hence this is a weaker acid than the carboxyl group. 4. α-hydrogen 4.1. Acidity Amino acids also possess an acidic α-hydrogen that is responsible for some of the chemical reactivities of amino acids. The acidity of the α- hydrogen is due to inductive effect on the α-carbon by a δ-positive carbonyl carbon. The carbonyl carbon is δ-positive hence by inductive effect the α-carbon is also δ-positive. This results in a polar C-H bond with the hydrogen loosely held and therefore weakly acidic. The increased polarity of the C-H bond due to this structural effect weakens this bond facilitating the release of a proton to a receiver or a strong nucleophile in a highly polar environment. Dissociation of the proton leaves a paired electron that can delocalize during Schiff’s base formation or form   5   Chemistry  41     Course  Pack  1:  Amino  Acids     condensation products. 5. Variable side chain, R 5.1. Polarity The 4th substituent is the variable side chain of amino acids denoted simply as “R”. Some side chains are made up of hydrocarbons only or contains heteroatoms that are centrally located in the molecule and are therefore non-polar; others contain atoms that can H-bond with water and are polar, while other R groups are either acidic, i.e. it contains the carboxyl functional group or are basic, i.e. it contains the amino functional group. The R group is essentially the determinant of the classification of amino acids based on the polarity of this group. The polarity of the side chain, R, determines the solubility of the amino acids. Some contains hydrocarbons and are non polar with variable van der waals radii. Still others contain electronegative heteroatorns that are often attached to a hydrogen atom or a less electronegative atom, making them polar. Some R groups also exhibit acidic or basic properties because of the presence of the carboxyl and amino functional group, respectively. In protein molecules, these R groups jut out of the planar structure of the peptide bond and are therefore responsible for many of the intermolecular forces of attraction or non- covalent interactions that shape protein structures. These intermolecular forces include H-bonding, hydrophobic interactions, and ionic bonds or salt bridges. On the basis of the structure and properties of the R groups, amino acids are classified into a) nonpolar, b) polar, uncharged and c) polar, charged amino acids. The last two classifications were made based on its form at the physiological pH, which is 6.7-7.4. Polar uncharged amino acids contain R groups that do not ionize at physiological pH hence are uncharged. Charged amino acids are those containing R group that are either negatively or positively charged at physiological pH. Amino acids containing the carboxyl group in R are negatively charged while those with amino groups are positively charged at physiological pH. Table 2.1 gives the classification of the amino acids based on the polarity of R, the names, abbreviations, and structure of R. 5.2. Acidity and basicity R groups containing carboxyl functional groups adds to the acidity of the amino acid while R groups containing the amino functional group are classified as basic amino acids. Table 2.1 also lists the pKa values of the ionizable protons of the acidic functional groups of the 20 commonly occurring protein amino acids.   6    Chemistry  41   The 20 biological   amino Course  Pacids ack  1:  Amino  Acids     The 20 biological amino acids Aliphatic nonpolar side chains: Aliphatic polar side ch The 20 biological amino acids The 20 biological Table 2.1. The 20 common Aliphatic amino protein amino nonpolar acids sideacids. chains: Aliphatic polar side ch Amino Acid Structure of R 3- 1- pK1 pK2 pK3 Aliphatic nonpolar side chains: Aliphatic letter polar side chains: ogical amino acids Aliphatic nonpolar side chains: abbrev abbrev letter Aliphatic polar side chains: The 20 Non-polar biological Glycine Gly amino acids Alanine Ala Proline Pro Serine Ser T T de chains: Alanine G Aliphatic polar side chains: Ala A A 2.34 P 9.69 S T Glycine Alanine Proline Serine T Gly Ala Pro Ser T Aliphatic nonpolar side G chains: A Aliphatic P polar side chains: S T Glycine Gly The 20 biological ValineAlanine Ala Glycine Alanineamino acids Proline Pro Serine Val Ser Proline V Threonine 2.32 Thr Serine 9.62 Threonine G A Gly PAla ProS T Ser Thr G A P S T Alanine Aliphatic nonpolar side chains: Proline Serine Threonine Aliphatic polar side chains: Ala Leucine Pro Ser Leu Thr L 2.36 9.68 A P Glycine SAlanine T Proline Serine Threonine Asparagine G Valine Isoleucine Leucine Gly Ala Pro Ser Thr Asn G Val Ile Leu G A P S TN Q V I L Valine Isoleucine Leucine Asparagine G Val Ile Leu Asn G V N Q Isoleucine Aromatic side chains: IIle I 2.36 L 9.68 Sulfur containing side Valine Isoleucine Glycine Leucine Alanine Asparagine Proline Glutamine Serine Threonine Val GlyIle Valine Ala Leu Isoleucine Pro Asn Leucine Gln Asparagine Ser ThrGlutamine V G I Val Aromatic AIleL side chains: P N Leu Q SAsn Sulfur containing T Gln side V I L N Q Aromatic Isoleucineside chains: Sulfur Phe containing F side chains: Leucine Asparagine Glutamine Phenylalanine 1.83 9.13 Ile Aromatic Leu side chains: Asn Gln Sulfur containing side chains: I L Valine NIsoleucine Q Leucine Asparagine Glutamine Val Ile Leu Asn Gln V I L N Q s: Sulfur containing side chains: Aromatic side chains: Phenylalanine Tryptophan Sulfur containing sideMethionine Tyrosine chains: Phe Trp Methionine Valine Isoleucine Met Leucine M 2.28Tyr 9.21 Asparagine Met Glutamine Val IleF Phenylalanine W LeuTryptophan Asn Y Tyrosine Gln M Methionine V I Phe L Trp N Q Tyr Met Basic F side chains: W Y Acidic M side chains: Phenylalanine Tryptophan Tyrosine Methionine Cysteine Phe Aromatic Trp side chains: Tyr Met SulfurCys containing side chains: F Phenylalanine Basic side chains: Tryptophan Tyrosine Methionine Acidic side chains: Cysteine Phe W Y Trp MTyr C Met Cys F W Y M C The 20 biological amino Tryptophan acids Basic side chains:Tryptophan Tyrosine Methionine Trp side chains: Acidic Cysteine W 2.38 9.39 Trp Basic side chains: Tyr Met Cys Acidic side chains: W Phenylalanine Y M Tryptophan C Tyrosine Methionine Cysteine Phe Trp Tyr Met Cys Aliphatic nonpolar side F chains: W Aliphatic Y polar side chains: M C Acidic side chains: Basic side chains: Proline Pro P Acidic side 1.99 chains: 10.60 Phenylalanine Tryptophan Lysine Tyrosine Histidine Methionine Arginine Cysteine Glutamic acid As Phe Trp Lys Tyr His Met Arg CysGlu As F W K Y H M R C E D Lysine Polar, unchargedHistidine Arginine Glutamic acid As Glycine Alanine Proline Lys Serine His Threonine Arg Glu As Gly Basic side Ala chains: Pro K HSer AcidicThrside R chains: E D G A Acidic and basic groups are shown PArginine S in their ionizedT form. ©2007 Peter A. Doucette Lysine Histidine Glutamic acid Aspartic acid Lys His Lysine Arg Histidine GluArginine Asp Glutamic acid Aspartic acid K   H Lys R and basic groups E Acidic His areArg D form. ©2007 Peter A. shown in their ionized Glu Asp 7   Doucette K H R E D Acidic and basic groups areArginine Histidine shown in their ionized form. ©2007 Glutamic acid Peter A.Aspartic Doucetteacid  The 20 biological amino acids mino ide acids Proline chains: Pro Serine Chemistry   Ser 41   Threonine Aliphatic Thr polar side chains:   Course  Pack  1:  Amino  Acids   P S T   Aliphatic nonpolar side chains: Aliphatic polar side cha Serine Aliphatic polar side chains: Ser S 2.21 9.15 Valine Isoleucine Leucine Asparagine Glutamine Val Ile Leu Asn Gln V I L N Q Alanine Threonine Proline Serine Thr Threonine T 2.63 10.43 ogical amino acids Ala Pro Ser Thr Aromatic A side chains: P S Sulfur T containing side chains: Glycine Alanine Proline Serine Th Gly Ala Pro Ser Th Leucine Proline Asparagine TyrosineSerine Glutamine GThreonine Tyr A Y 2.20 P 9.11 10.07S T Leu AsnSer Gln de chains: Pro N Aliphatic Q Thr polar side chains: LP S T no acids Sulfur containing side chains: Isoleucine Leucine Asparagine Glutamine Ile Aliphatic Leu Asn polar side chains: Gln Alanine Phenylalanine I Ala Cysteine Proline Tryptophan L Serine N Tyrosine Cys Threonine QMethionine C 1.71 Cysteine10.78 8.33 Pro Ser Thr Asparagine Gl Phe Trp Tyr Valine Met Isoleucine Cys Leucine A P S T Asn Gl F W Y Val M Ile CLeu VGlutamine N Q s: Leucine Asparagine Sulfur containing sideI chains: L Leu Asn Gln Basic side chains:Asparagine Acidic AsN side chains: N 2.02 8.80 L N Q Aromatic side chains: Sulfur containing side c Tyrosine Proline Methionine Serine Cysteine Threonine Tyr Pro Sulfur Met Ser containing side chains: Cys Thr PY MS TC Glutamine Acidic side chains: GlN Q 2.17 9.13 Isoleucine Ile Leucine Leu The Asn 20 biological Asparagine Glutamine Gln amino acids I L N Q Tryptophan Tyrosine Methionine Cysteine Trp Tyr Met Cys : W Y Aliphatic Sulfur M nonpolar containing side side chains: chains: C Aliphatic polar side cha Phenylalanine Tryptophan Tyrosine Methionine C Lysine Histidine Phe Arginine Glutamic Trp acid Aspartic Tyr acid Met C Lys His Acidic F Argside chains: Glu W AspY M C Leucine K Tyrosine Glycine Asparagine HMethionine Glutamine RCysteine E Gly G 2.34 D 9.60 Leu Asn Gln Tyr Met Cys L Y N Q M Basic Cside chains: Acidic side chains: Acidic and basic groups are shown in their ionized form. ©2007 Peter A. Doucette Acidiccontaining side chains: Charged Sulfur side chains: Glycine Alanine Proline Serine Th Arginine Lysine Glutamic acid Aspartic Gly acid Lys Ala K 2.18 Pro 8.95 10.53 Ser Th Arg Glu AspG A P S T R E D Tryptophan Tyrosine Methionine Cysteine Trp nized Tyr form. ©2007 Peter A. Doucette Met Cys W Y M C Histidine Arginine Acidic side Glutamic chains: acid Aspartic acid His Arg Glu Asp H Arginine R E D Arg R 2.17 9.04 12.48 Lysine Histidine Arginine Glutamic acid Asp Tyrosine Methionine Cysteine Lys His Arg GluAsparagine Asp G Valine Isoleucine Leucine Tyr s are shown Met ©2007 Peter A. Doucette in their ionized form. Cys KVal H RLeu E Asn DG Ile Y Arginine MGlutamic acid C Aspartic acid N Q V I L Arg Glu Asp R E D Acidic side chains: Acidic and basic groups are shown in their ionized form. ©2007 Peter A. Doucette Aromatic side chains: Sulfur containing side onized form. ©2007 Peter A. Doucette   8   Histidine Arginine Glutamic acid Aspartic acid His Arg Glu Asp  L N Q he Trp Tyr Met Cys W Y M C Sulfur containing side chains: asic side chains: Chemistry  41   Acidic   side chains:Course  Pack  1:  Amino  Acids   Asparagine Glutamine Isoleucine   Leucine Asn Gln Ile Leu I L N Q Phenylalanine Tryptophan Tyrosine Methionine Cysteine Phe Trp Tyr Met Cys : F Sulfur W containing side chains: Y M C Basic side chains: Acidic side chains: Tyrosine Methionine Cysteine Tyr Met Cys Y M C Lysine Histidine Histidine Arginine His acid Glutamic H 1.82acid Aspartic 9.17 6.00 Lys His Arg Glu Asp K Acidic H side chains: R E D Tryptophan Acidic Tyrosine and basic groups are shown Methionine in their ionized form. Cysteine ©2007 Peter A. Doucette Trp Tyr Met Cys W Aspartic Y acid M C Asp D 2.09 9.82 3.86 Lysine Acidic side chains: Histidine Arginine Glutamic acid Aspartic acid Lys His Arg Glu Asp K H R E D Glutamic acid Glu E 2.19 9.67 4.25 Acidic and basic groups are shown in their ionized form. ©2007 Peter A. Doucette Arginine Glutamic acid Aspartic acid Arg Glu Asp R E D ed form. ©2007 Peter A. Doucette *pKa values from: https://wou.edu/chemistry/files/2019/07/table-of-pka- Histidine values.jpg Arginine Glutamic acid Aspartic acid His Arg Glu Asp H Note: R some biochemists E and authors also D classify amino acids in accordance with the organic classification of the R groups. Thus, the amino acids are grouped are shown in their ionized as 1)Peter form. ©2007 aliphatic, 2) aromatic, 3) hydroxyl-containing, 4) thiol-containing A. Doucette groups, 4) cyclic, 5) carboxyl-containing, 6) amine-containing and 7) amide- containing. Acidity and Basicity of R The pKa values of the ionizing groups of amino acids can be obtained by pH titration, i.e, by adding small amounts of standard base, which will neutralize the H+ released. The pH of the resulting solution will increase. A titration curve is a plot of the pH values of the solution and the amount of OH- added. The concentration of the OH- used is usually in the range 0.1-0.5 M. By convention, pK1 refers to the dissociation of the acidic proton of the – COOH functional group while pK2 is the ionization of the –NH3+ proton. At the pH lower than the pK1 value, the –COOH functional group is electrically neutral, i.e. the acidic proton do not dissociate. Similarly, at pH lower than the pK2 value, the amino group is protonated and assumes the form of the conjugate acid, -NH3+ of the weak base, -NH2. These pKa values determine the ease of ionization of the   9   The pKa values for any polyprotic acid always get progressively higher, because it becomes increasingly difficult to stabilize the additional electron density that results from each successive proton donation. H3PO4 is a strong acid because the (single) Chemistry   negative 41   on its conjugate base H2PO4-  can be delocalized charge Course   overPtwo ack  oxygen 1:  Amino  Acids   atoms.   protons of acidic forms of the functional groups and therefore its form in solution H2PO4- is substantially less acidic, because proton donation now results in the at a particular pH. It is also useful for separating amino acids in an electric field. formation of an additional negative charge: a –2 charge is inherently higher in energy than a –1 Atcharge because that pH, of negative-negative the acidic electrostatic form of the amino group isrepulsion. positivelyThe third Thus, charged. deprotonation, resulting in formation of a third negative charge, has an for amino acids in solution at pH lower than pK1, most amino acids contain even higher two pK a. We will have more to say about the acidity of phosphate ionizing protons, one each from the carboxyl and –NH3+ groups. groups in chapter 9, acid The amino when we study the reactions of phosphate groups on biomolecules. therefore is also a weak polyprotic acid of the form H A. Hence, the titration 2 curve will also show at least two inflection points or plateaus. These are points in Exercise the graph 7.29: In a pH where buffer at physiological = pKa pH, what and [A-] = [HA]. form(s) In these of phosphoric points, note thatacid pH values predominate? What is the average do not vary significantly netaddition even with charge? of small amount of acids (moving to the left) or base (moving to the right from the point where pH = pKa. This is also the point when amino acids, acting as buffers in biological systems will have its Free amino buffering maximum acids are polyprotic, capacity. with The pKa values plateau of approximately gives the range of 2pH forvalues the carboxylic at which acid group and 9-10 for the ammonium group. Alanine, for example, has the the buffer will still have a maximum buffering capacity, i.e. at pH = pKa ± 1.0. acid constants pKa1 = 2.3 and pKa2 = 9.9. CH3 pKa = 2.3 CH3 CH3 pKa = 9.9 OH O O H3N H3N H2N O O O cation at low pH zwitterion at pH 7 anion at high pH fig 56 The Henderson-Hasselbalch equation tells us that alanine is almost fully protonated and positively charged when dissolved in a solution that is buffered to pH 0.5. At pH 7, alanine has lost one proton from the carboxylic acid group, and thus is a zwitterion (it has both a negative and a positive formal charge). At pH levels above 12, the ammonium group is fully deprotonated, and alanine has a negative overall charge. Organic Chemistry With a Biological Emphasis 370 Tim Soderberg Source: Soderberg T. “Organic Chemistry with a Biological Emphasis” Fig. 2.6 shows the forms of alanine, a nonpolar amino acid at different pH of the solution, and the titration curve resulting from the neutralization of the ionizable protons. The pKR values of amino acids provide the hint on which R groups contribute to the acidity of the peptide and protein comprising the amino acids. For amino acids with ionizable protons in R such as the acidic, basic and some polar uncharged amino acids, the amino acid also assumes the form of a weak acid of the form H3A. For example, aspartic acid has the following pKa values: pK1 = 2.1; pK2 = 9.8 and pKR = 3.9.   10   Chemistry  41     Course  Pack  1:  Amino  Acids     Source: https://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/proteins.htm Fig. 2.7. The titration curve of aspartic acid, an acidic amino acid It could be observed that the amino acids with more than two ionizable protons have three inflection points at which points, pKa = pH. All amino acids with ionizable protons In R, including polar, uncharged amino acids with acidic protons in R, such as tyrosine and cysteine have similar titration curves. Like other weak acids with ionizable protons, amino acids will exist in different forms at different pH values. At pH lower than pK1, all amino acids will be positively charged. An amino acid that is nonpolar or have no ionizable protons in R will have the general formula H2A. As pH is increased, the carboxyl proton, being the more acidic proton, ionizes and the amino acid will become zwitterionic, i.e. it has both positive and negative charges but is electrically neutral or its net charge is zero (Fig. 2.8). The net charge of the amino acid is the algebraic sum of all positive and negative charges at the pH indicated. As pH is increased further, the proton from the amino, which has the higher pKa value since the amino group is a weaker acid, will then dissociate and the amino acid becomes negatively charged. At pH = pKa, there is an equilibrium concentration between the weak acid and its conjugate base. The ratio of these forms can be determined using the Henderson-Hasselbalch equation. The forms of the amino acid with the general formula H3A (acidic, basic, some polar uncharged amino acids such as cysteine and tyrosine) will likewise change depending on the pH. The pH at which the amino acid exists primarily in the zwitterionic form is the isoelectric pH or pI of the amino acid. It is computed as the average of the algebraic sum of the pKa values before and after the zwitterionic form. When the pH = pI value of the amino acid, the amino acid, being electrically neutral, will not move in an electric field. The pI of the different amino acids is also variable. The figures below show the zwitterion form an amino acid that has no   11   Chemistry  41     Course  Pack  1:  Amino  Acids     ionizable proton in R. For these groups of amino acids, the structure at physiological pH is shown on the left. Source: https://2012books.lardbucket.org/books/introduction-to-chemistry- general-organic-and-biological/index.html Fig. 2.8. The ionic state of a zwitterion. The right drawing is incorrect since the carboxyl group will only be protonated at a very low pH at which pH the amino group is protonated. Even when the carboxyl group is deprotonated, ionization of this proton occurs at pH ranges of 1.8-2.4 for amino acids. At this pH, the amino group is still protonated since it is a strong base. Its conjugate acid therefore is weak and dissociates only at pH ranges of 9-10.4. Thus amino acids in solution assume its form as protons are ionized in a solution at a particular pH. These forms change charges from positive to zero to negative as pH of the solution increases. The form with a net charge of zero occurs predominantly at the pH equal to the pI of the amino acid. At this pH, the amino acid will not move in an electric field. Fig. 2.9 shows the predominant forms of 2 nonpolar amino acids, an acidic and basic amino acid at pH of 6.0. The pI value is also provided and is variable for the amino acids. The predominant form of an amino acid at pH lower than the pI value of that amino acid is positively charged while the predominant form at pH higher than the pI value is negatively charge. Fig. 2.9. The ionic forms of the amino acids, arginine, alanine, aspartic acid and isoleucine in solution at various pH values; a) alanine, b) cysteine, c) aspartic acid and d) lysine     12   Chemistry  41     Course  Pack  1:  Amino  Acids     Exercise 2.1. At what pH will you be able to separate the amino acids val, his and arg? When amino acids are linked as a result of the formation of the peptide bond, it is the ionizable proton in the R group that will contribute to the acidity and basicity, as well as the form of the peptide in solution. Other properties Molecular size Amino acids have molecular weights raging from 57 to 186 daltons (1 dalton is equivalent to 1 amu). If we take the average weight to be about 110 daltons, a proteIn with a molecular weight of 22.000 daltons (or 22 kDa) contain approximately 220 amino acids residues. Hydrophobicity Hydrophobicity is the most important characteristics of the R groups of amino acids that drives protein folding. When a protein folds, exposed hydrophobic side chains get buried and expels water which cannot interact with these side chains. Protein structure, folding and stability increases the entropy of water. This is called the hydrophobic effect. Table 2.2. gives the hydrophobicity values of the amino acids. Table 2.2. The hydrophobicity values of the amino acids (https://www.web- books.com/MoBio/Free/Ch2A2.htm) Non-polar AA 1-letter H* Polar, uncharged 1-letter H* Amino Acid abbrev value Amino Acid abbrev value Alanine A 1.8 Serine S -0.8 Valine V 4.2 Threonine T -0.7 Leucine L 3.8 Tyrosine Y -1.3 Isoleucine I 4.5 Cysteine C 2.5 Phenylalanine F 2.8 Asparagine N -3.5 Methionine M 1.9 Glutamine N -3.5 Tryptophan W -0.9 Glycine G -0.4 Proline P -1.6 Charged, acidic 1-letter H* Charged, basic 1-letter H* Amino Acid abbrev value Amino Acid abbrev value Aspartic acid D -3.5 Histidine H -3.2 Glutamic acid E -3.5 Lysine K -3.9 Arginine R -4.5 *H value = relative hydrophobicity scale; the higher the value is, the more hydrophobic the R group is and the amino acid is buried in the protein in aqueous solutions; a negative value means that the amino acid is hydrophilic and are found outside the protein structure in aqueous solution.   13   Chemistry  41     Course  Pack  1:  Amino  Acids     Properties unique to structure of some amino acids:  Bridge formation by the side chain of cysteines  rigidity of the pyrrolidone rings of prolines which causes helices to break  flexibility of glycines which causes polypeptides to turn. Reactivities The reactions that amino acids undergo are primarily due to the carboxyl group, the amino group, the a-hydrogen and functional groups in R. These are the same reactions as organic compounds with the same functional groups, e.g. carboxyl groups are acidic and can undergo substitution reactions. Amino groups are basic and are nucleophiles in substitution or addition reactions. The R groups of amino acids contain substituted and unsubstituted arenes, thiols, hydroxyls, amides, amino, and carboxylic groups. These functional groups participate in various reactions including oxidation-reduction reactions transforming them into intermediates and products essential for structural stability and cellular metabolism. Some of these reactions are shown in Figs. 2.10, 2.11, 2.12. Oxidation of thiol groups Fig. 2.10. Oxidation of thiol groups leading to the formation of disulfide bonds between proximal cysteines. Source: https://2012books.lardbucket.org/books/introduction-to-chemistry- general-organic-and-biological/s21-amino-acids-proteins-and-enzym.html#gob- ch18_t01   14   Chemistry  41     Course  Pack  1:  Amino  Acids     The ninhydrin reaction: Paper Chromatography Source: https://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/proteins.htm Fig. 2.11. The ninhydrin reaction is a substitution reaction involving the a-amino group of amino acids and the reagent, 2,2-Dihydroxy-1H-indene-1,3(2H)-dione. The product is a purple colored compound often referred to as Ruhemann’s purple. (https://byjus.com/chemistry/ninhydrin-test/) Acylation of the amino groups Source: https://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/proteins.htm Fig. 2.12. The acylation reaction of amino acids Non-protein amino acids Other than the 20 common protein amino acids there are a number of amino acids occurring in nature. Many of these have functions such as the role of hydroxyproline in maintaining the stability of quaternary structures of collagen, β-alanine is found in microbial cell wall while ornithine, homoserine and homocysteine are intermediates of metabolism. Interestingly, 2 amino acids, selenocysteine (Sec/U) and pyrrolysine (Pyl/O) are non protein amino acids that are regulators of the genetic mechanism; these are incorporated at the stop   15   Chemistry  41     Course  Pack  1:  Amino  Acids     codon, UGA and UAG, respectively. Fig. 2.13. Some non-protein amino acids. See also: https://www.slideshare.net/mprasadnaidu/non-protein-aminoacids In summary, you have been introduced to the following 1. The general structural features of amino acids and their properties 2. The structure, unique properties and reactivities of the 20 common protein amino acids, which are crucial to the structure and functions of proteins, which they comprise. 3. The acidity and basicity properties of amino acids and their forms at varying pH of the solution. 4. The structure and function of some non-protein amino acids that are essential for the genetic regulation, protection of important structures, metabolic processes, and structural stability as well. Additional references: Schmitz,  A.  chapter  18  from  the  book  Introduction  to  Chemistry:  General,  Organic,   and  Biological  (v.  1.0).     https://biochem.oregonstate.edu/content/biochemistry-­‐free-­‐and-­‐easy   https://microbenotes.com/amino-acids-properties-structure-classification-and- functions/       16