16/CHAPTER3Table3-1.L-α-Aminoacidspresent inproteins.(continued)NameSymbolStructuralFormulaPKPK,PK-α-COOHQ-NH,WithSideChains Containing Basic GroupsRGroup1.89.0Arg [R]12.5ArginineH-N-CH, —CH, — CH,-CH-COO"1C=NH,*NH,*NH2CH2—CH,CH,—CH,CH-COO2.2Lys [K]9.210.8LysineNH,*NH*-CH-COOCH21.89.36.0HistidineHis [H]HNNNH+Containing Aromatic RingsHis [H]See above.HistidineCH-COO"CH.2.29.2PhenylalaninePhe [F]NH*Tyr[M]2.29.110.1TyrosineHO-CH-COOTH-1NH,*2.49.4TryptophanTrp [W]CH-COO1NH,*HIminoAcid2.010.6ProlinePro [P]COO"NAmino Acids MayHavePositive,Negative,Molecules that contain an equal number of ioniz-able groups of opposite charge and that therefore bearorZeroNetChargeno net charge are termed zwitterions. Amino acids inCharged and uncharged forms of the ionizableblood and most tissues thus should be represented as in-COOHand-NH,weakacid groupsexistin solu-A, below.tion in protonic equilibrium:NHs*NH2R-COOH=R-COO-+HR-NH3* = R-NH2+ H*O0ABWhile both R-COOH and RNH,+are weak acidsR-COOH is a far stronger acid than RNH,t.AtStructure B cannot exist in aqueous solution because atphysiologic pH (pH 7.4), carboxyl groups exist almostentirely as R-COO-and amino groups predomi-any pH low enough to protonate the carboxyl groupnantly as R—NH,+.Figure 3-1 illustrates the effect ofthe amino group would also be protonated. SimilarlypH on the charged state ofaspartic acid.at any pH sufficientlyhigh for an uncharged amino
16 / CHAPTER 3 Table 3–1. L-α-Amino acids present in proteins. (continued) Name Symbol Structural Formula pK1 pK2 pK3 With Side Chains Containing Basic Groups -COOH -NH3 + R Group Arginine Arg [R] 1.8 9.0 12.5 Lysine Lys [K] 2.2 9.2 10.8 Histidine His [H] 1.8 9.3 6.0 Containing Aromatic Rings Histidine His [H] See above. Phenylalanine Phe [F] 2.2 9.2 Tyrosine Tyr [Y] 2.2 9.1 10.1 Tryptophan Trp [W] 2.4 9.4 Imino Acid Proline Pro [P] 2.0 10.6 Amino Acids May Have Positive, Negative, or Zero Net Charge Charged and uncharged forms of the ionizable COOH and NH3 + weak acid groups exist in solution in protonic equilibrium: While both RCOOH and RNH3 + are weak acids, RCOOH is a far stronger acid than RNH3 +. At physiologic pH (pH 7.4), carboxyl groups exist almost entirely as RCOO− and amino groups predominantly as RNH3 +. Figure 3–1 illustrates the effect of pH on the charged state of aspartic acid. R COOH R COO H R NH NH H — — — R— = = − + + + + + 3 2 CH2 C CH2 NH2 NH2 + N CH2 CH NH3 + COO– H NH3 + CH2 CH2 CH2 CH2 CH NH3 + COO– CH NH3 + COO– CH2 HN N CH NH3 + COO– CH NH3 + COO– CH NH3 + COO– CH2 N H CH2 HO CH2 + N H2 COO– Molecules that contain an equal number of ionizable groups of opposite charge and that therefore bear no net charge are termed zwitterions. Amino acids in blood and most tissues thus should be represented as in A, below. Structure B cannot exist in aqueous solution because at any pH low enough to protonate the carboxyl group the amino group would also be protonated. Similarly, at any pH sufficiently high for an uncharged amino O OH NH2 R O A B O– NH3 + R ch03.qxd 2/13/2003 1:35 PM Page 16
17AMINOACIDS&PEPTIDES7OOo0国国H0OOHOHpK, = 2.09pK, = 3.86pKg = 9.82NHNH2NH,*NH(β-COOH)(α-COOH)(-NHa')HOOOooOocDABIn strong acidAround pH 3;Around pH 6-8:In strong alkali(below pH 1);net charge =-1(above pH 11);net charge = 0net charge = +1net charge = -2Figure3-1.Protonic equilibria of aspartic acid.AtIts IsoelectricpH (pl),anAminoAcidgroup to predominate,a carboxyl group will be presentas R-COO-.Theuncharged representation B (above)Bears NoNetChargeis,however,oftenusedforreactionsthatdonot involveThe isoelectric species is the form of a molecule thatprotonic equilibria.has an equal number of positive and negative chargesand thus is electricallyneutral.Theisoelectric pH,alsopK,ValuesExpresstheStrengthscalled the pl, is the pH midway berween pK, values onofWeakAcidseither side of the isoelectric species.For an amino acidsuch as alanine that has only two dissociating groups,The acid strengths of weak acids are expressed as theirthere is no ambiguity.Thefirst pK(R-COOH)ispK, (Table 3-1).The imidazole group of histidine and2.35 and the second pK, (R-NH,+) is 9.69.The iso-the guanidino group of arginine exist as resonance hy-electricpH(pl)ofalaninethus isbrids with positive charge distributed between both nitrogens (histidine) or all three nitrogens (arginine) (Fig)pl= PK1 +pK2 _ 2.35+9.69ure 3-2).The net charge on an amino acidthe=6.0222algebraic sum of all the positively and negativelycharged groups present-depends upon the pK, valuesof its functional groups and on the pH of the surround-Forpolyfunctional acids,pl is alsothepH midwaybeing medium.Altering the charge on amino acids andtween the pK, values on either side of the isoionictheir derivatives by varying the pH facilitates the physi-species.For example,the pl for aspartic acid iscal separation of amino acids,peptides, and proteins(seeChapter 4)pK+pK2_2.09+3.96pl==3.0222For lysine, pI is calculated from::pl= PK2 + pK+2Similar considerations apply to all polyprotic acids (eg,HHproteins), regardless of the number of dissociatinggroups present.In the clinical laboratory,knowledge ofRRR-11theplguides selection of conditionsfor electrophoreticNHNHONHseparations.Forexample,electrophoresis atpH7.0will-?Iseparate two molecules with pl values of 6.0 and 8.0C-NH2C=NH2-NH2C=because at pH 8.0 the molecule with a pl of 6.0 willNH2ONH2NH2have a net positive charge, and that withpl of 8.0a netnegative charge. Similar considerations apply to under-Figure3-2.Resonancehybridsoftheprotonatedstanding chromatographic separations on ionic sup-ports such as DEAE cellulose (see Chapter 4).formsoftheRgroups ofhistidineandarginine
AMINO ACIDS & PEPTIDES / 17 R N H N H R N H N H NH R C NH2 NH2 NH R C NH2 NH2 NH R C NH2 NH2 Figure 3–2. Resonance hybrids of the protonated forms of the R groups of histidine and arginine. group to predominate, a carboxyl group will be present as RCOO− . The uncharged representation B (above) is, however, often used for reactions that do not involve protonic equilibria. pKa Values Express the Strengths of Weak Acids The acid strengths of weak acids are expressed as their pKa (Table 3–1). The imidazole group of histidine and the guanidino group of arginine exist as resonance hybrids with positive charge distributed between both nitrogens (histidine) or all three nitrogens (arginine) (Figure 3–2). The net charge on an amino acid—the algebraic sum of all the positively and negatively charged groups present—depends upon the pKa values of its functional groups and on the pH of the surrounding medium. Altering the charge on amino acids and their derivatives by varying the pH facilitates the physical separation of amino acids, peptides, and proteins (see Chapter 4). At Its Isoelectric pH (pI), an Amino Acid Bears No Net Charge The isoelectric species is the form of a molecule that has an equal number of positive and negative charges and thus is electrically neutral. The isoelectric pH, also called the pI, is the pH midway between pKa values on either side of the isoelectric species. For an amino acid such as alanine that has only two dissociating groups, there is no ambiguity. The first pKa (RCOOH) is 2.35 and the second pKa (RNH3 +) is 9.69. The isoelectric pH (pI) of alanine thus is For polyfunctional acids, pI is also the pH midway between the pKa values on either side of the isoionic species. For example, the pI for aspartic acid is For lysine, pI is calculated from: Similar considerations apply to all polyprotic acids (eg, proteins), regardless of the number of dissociating groups present. In the clinical laboratory, knowledge of the pI guides selection of conditions for electrophoretic separations. For example, electrophoresis at pH 7.0 will separate two molecules with pI values of 6.0 and 8.0 because at pH 8.0 the molecule with a pI of 6.0 will have a net positive charge, and that with pI of 8.0 a net negative charge. Similar considerations apply to understanding chromatographic separations on ionic supports such as DEAE cellulose (see Chapter 4). pl p p = K K 2 3 + 2 pl p p = + = + = K K 1 2 2 2 09 3 96 2 3 02 . . . pl p p = + = + = K K 1 2 2 2 35 9 69 2 6 02 . . . O HO NH3 + OH O H+ pK1 = 2.09 (α-COOH) A In strong acid (below pH 1); net charge = +1 O – O NH3 + O H+ pK2 = 3.86 (β-COOH) B Around pH 3; net charge = 0 O – O O H+ pK3 = 9.82 (— NH3 + ) C Around pH 6–8; net charge = –1 O – O NH2 O– O D In strong alkali (above pH 11); net charge = –2 OH NH3 + O– Figure 3–1. Protonic equilibria of aspartic acid. ch03.qxd 2/13/2003 1:35 PM Page 17
18/CHAPTER3pK,Values VaryWith the EnvironmentTHEQ-RGROUPSDETERMINETHEPROPERTIESOFAMINOACIDSThe environment ofa dissociable group affects its pKThe pK, values of the R groups of free amino acids inSince glycine, the smallest amino acid, can beaccommo-aqueous solution (Table 3-1) thus provide only an ap-datedinplaces inaccessibletootheraminoacids,itoftenproximateguideto thepKvaluesof thesameaminooccurs where peptides bend sharply.Thehydrophobic Racids when present in proteins.A polar environmentgroups ofalanine, valine, leucine, and isoleucine and thefavors the charged form (RCOO- or RNH,)aromatic Rgroups ofphenylalanine, tyrosine,and tryp-and a nonpolar environmentfavors theuncharged formtophan typically occur primarily in the interior of cy-(RCOOHorRNH).Anonpolar environmenttosolic proteins. The charged R groups of basic andthus raises thepK, of a carboxyl group (making itaacidic amino acids stabilize specific protein conforma-weakeracid)but lowersthat of an aminogroup (makingtions viaionic interactions,or salt bonds.These bondsit a stronger acid).The presence of adjacent chargedalso function in “charge relay" systems during enzymaticgroupscan reinforceor counteractsolventeffects.Thecatalysis and electron transport in respiring mitochon-pK, of a functional group thus will depend upon its lo-dria. Histidine plays unique roles in enzymatic catalysis.cation within a given protein.Variations in pK can en-ThepK,ofits imidazoleprotonpermits ittofunctionatcompass whole pH units (Table 3-2).pK, values thatneutral pH as either a base or an acid catalyst.The pri-diverge from those listed by as much as three pH unitsmary alcohol group of serine and the primary thioalco-arecommonattheactivesitesofenzymes.Anextremehol—SH)group of cysteine areexcellent nucleophilesexample,a buried aspartic acid of thioredoxin,hasaand can function as such during enzymatic catalysis.pK, above 9-a shift of over six pH units!However, the secondary alcohol group of threonine,while a good nucleophile, does not fulfill an analogousThe Solubilityand MeltingPointsrole in catalysis. The-—OH groups of serine, tyrosine,ofAminoAcidsReflectand threonine also participate in regulation of the activ-TheirlonicCharacterity of enzymes whose catalytic activity depends on thephosphorylation state of these residues.The charged functional groups of amino acids ensurethat they are readily solvated by-and thus soluble in-FUNCTIONALGROUPSDICTATETHEpolar solvents such as water and ethanol but insolubleCHEMICALREACTIONSOFAMINOACIDSin nonpolar solvents such as benzene, hexane, or ether.Similarly, the high amount of energy required to dis-Each functional group of an amino acid exhibits all ofrupt the ionic forces that stabilize the crystal latticeits characteristic chemical reactions. For carboxylic acidaccount for the high melting points of amino acidsgroups, these reactions include the formation of esters,(>200°C).amides, and acid anhydrides; for amino groups, acyla-Amino acids do not absorb visible light and thus aretion,amidation,and esterification; and forOH andcolorless.However,tyrosine,phenylalanine,and espe-SH groups,oxidation and esterification.The mostcially tryptophan absorb high-wavelength (250-290important reaction of amino acids is theformation ofanm)ultraviolet light.Tryptophan therefore makes thepeptide bond (shaded blue).major contribution to the ability of most proteins toabsorb light in the region of 280 nm.THNTable3-2.Typical rangeofpK,valuesforOUSHionizablegroupsinproteins.AlanylValineCysteinylDissociating GrouppK,Range3.54.0α-CarboxylAminoAcid SequenceDetermines4.04.8Non-α COOH of Asp or GluPrimary Structure6.57.4Imidazoleof HisSH of Cys8.59.0The number and order of all of the amino acid residuesOHofTyr9.510.5in a polypeptide constitute its primary structureα-Amino8.09.0Amino acids present in peptides are called aminoacyle-Amino of Lys9.810.4residues and arenamed byreplacing the-ate or-inesuf-Guanidinium of Arg~12.0fixes of free amino acids with -yl (eg, alanyl, aspartyl, ty
18 / CHAPTER 3 Table 3–2. Typical range of pKa values for ionizable groups in proteins. Dissociating Group pKa Range α-Carboxyl 3.5–4.0 Non-α COOH of Asp or Glu 4.0–4.8 Imidazole of His 6.5–7.4 SH of Cys 8.5–9.0 OH of Tyr 9.5–10.5 α-Amino 8.0–9.0 ε-Amino of Lys 9.8–10.4 Guanidinium of Arg ~12.0 pKa Values Vary With the Environment The environment of a dissociable group affects its pKa. The pKa values of the R groups of free amino acids in aqueous solution (Table 3–1) thus provide only an approximate guide to the pKa values of the same amino acids when present in proteins. A polar environment favors the charged form (RCOO− or RNH3 +), and a nonpolar environment favors the uncharged form (RCOOH or RNH2). A nonpolar environment thus raises the pKa of a carboxyl group (making it a weaker acid) but lowers that of an amino group (making it a stronger acid). The presence of adjacent charged groups can reinforce or counteract solvent effects. The pKa of a functional group thus will depend upon its location within a given protein. Variations in pKa can encompass whole pH units (Table 3–2). pKa values that diverge from those listed by as much as three pH units are common at the active sites of enzymes. An extreme example, a buried aspartic acid of thioredoxin, has a pKa above 9—a shift of over six pH units! The Solubility and Melting Points of Amino Acids Reflect Their Ionic Character The charged functional groups of amino acids ensure that they are readily solvated by—and thus soluble in— polar solvents such as water and ethanol but insoluble in nonpolar solvents such as benzene, hexane, or ether. Similarly, the high amount of energy required to disrupt the ionic forces that stabilize the crystal lattice account for the high melting points of amino acids (> 200 °C). Amino acids do not absorb visible light and thus are colorless. However, tyrosine, phenylalanine, and especially tryptophan absorb high-wavelength (250–290 nm) ultraviolet light. Tryptophan therefore makes the major contribution to the ability of most proteins to absorb light in the region of 280 nm. THE -R GROUPS DETERMINE THE PROPERTIES OF AMINO ACIDS Since glycine, the smallest amino acid, can be accommodated in places inaccessible to other amino acids, it often occurs where peptides bend sharply. The hydrophobic R groups of alanine, valine, leucine, and isoleucine and the aromatic R groups of phenylalanine, tyrosine, and tryptophan typically occur primarily in the interior of cytosolic proteins. The charged R groups of basic and acidic amino acids stabilize specific protein conformations via ionic interactions, or salt bonds. These bonds also function in “charge relay” systems during enzymatic catalysis and electron transport in respiring mitochondria. Histidine plays unique roles in enzymatic catalysis. The pKa of its imidazole proton permits it to function at neutral pH as either a base or an acid catalyst. The primary alcohol group of serine and the primary thioalcohol (SH) group of cysteine are excellent nucleophiles and can function as such during enzymatic catalysis. However, the secondary alcohol group of threonine, while a good nucleophile, does not fulfill an analogous role in catalysis. The OH groups of serine, tyrosine, and threonine also participate in regulation of the activity of enzymes whose catalytic activity depends on the phosphorylation state of these residues. FUNCTIONAL GROUPS DICTATE THE CHEMICAL REACTIONS OF AMINO ACIDS Each functional group of an amino acid exhibits all of its characteristic chemical reactions. For carboxylic acid groups, these reactions include the formation of esters, amides, and acid anhydrides; for amino groups, acylation, amidation, and esterification; and for OH and SH groups, oxidation and esterification. The most important reaction of amino acids is the formation of a peptide bond (shaded blue). Amino Acid Sequence Determines Primary Structure The number and order of all of the amino acid residues in a polypeptide constitute its primary structure. Amino acids present in peptides are called aminoacyl residues and are named by replacing the -ate or -ine suffixes of free amino acids with -yl (eg, alanyl, aspartyl, tyO O O O– H N N H SH Cysteinyl +H3N Alanyl Valine ch03.qxd 2/13/2003 1:35 PM Page 18
19AMINOACIDS&PEPTIDES1SHrosyl). Peptides are then named as derivatives of thecarboxyl terminal aminoacyl residue.For example,LysCH2O=HLeu-Tyr-Gln is called lysyl-leucyl-tyrosyl-glutamine.The -ine ending on glutamine indicates that its α-car-boxyl group is not involved in peptide bond formation.CH2CH,=0Hcoo-CH2PeptideStructuresAreEasytoDraw--NH3-Prefixes like tri-or octa- denote peptides with three oreight residues, respectively, not those with three orCOO"eight peptide bonds. By convention, peptides are writ-ten with the residue that bears the free a-amino groupFigure3-3.Glutathione (y-glutamyl-cysteinyl-at the left.To draw a peptide, use a zigzag to representglycine). Note the non-α peptide bond that linksthe main chain or backbone.Add the main chain atoms,GlutoCys,which occur in the repeating order: α-nitrogen, α-car-bon, carbonyl carbon.Now add a hydrogen atom toeach α-carbon and to each peptide nitrogen, and anreleasing hormone (TRH) is cyclized to pyroglutamicoxygen to the carbonyl carbon.Finally,add the appro-acid, and thecarboxyl group of thecarboxyl terminalpriate Rgroups (shaded)to eacha-carbon atom.prolyl residue is amidated. Peptides elaborated by fungi,bacteria,and lower animals can contain nonproteinamino acids.Theantibiotics tyrocidin and gramicidin Sare cyclic polypeptides that contain D-phenylalanineand ornithine.The heptapeptide opioids dermorphinHand deltophorin in the skin of South American treeHNCOOfrogs contain D-tyrosine and D-alanine.h-CH2CH,PeptidesArePolyelectrolytes10OH-00CThe peptide bond is uncharged at any pH of physiologicinterest.Formation of peptides from amino acids isThree-letter abbreviations linked by straight linestherefore accompanied by a net loss of one positive andrepresent an unambiguous primary structure. Lines areone negativechargeper peptidebond formed.Peptidesomitted for single-letter abbreviations.nevertheless are charged at physiologic pH owing to theirGlu-Ala-Lys -Gly- Tyr-Alacarboxylandaminoterminalgroupsand,wherepresenttheir acidic or basic R groups. Asfor amino acids, the netEAKGYAcharge on a peptide depends on the pH of its environ-Where there is uncertainty about the order of a portionment and on thepK values of its dissociating groups.ofa polypeptide, the questionable residues are enclosedin brackets and separated by commas.ThePeptideBond Has PartialDouble-Bond CharacterGlu- Lys -(Ala, Gly, Tyr)- His - AlaAlthough peptides are written as if a single bond linkedthe α-carboxyl and α-nitrogen atoms, this bond in factSomePeptidesContain Unusualexhibits partial double-bond character:AminoAcids0In mammals,peptidehormonestypically containonlythe aα-amino acids of proteins linked by standard pep-tide bonds.Other peptides may,however,contain nonprotein amino acids, derivatives of the protein aminoHHacids, or amino acids linked by an atypical peptidebond.Forexample,theaminoterminalglutamateofThere thus is no freedom of rotation about the bondglutathione, which participates in protein folding andthat connects the carbonyl carbon and the nitrogen of ainthemetabolism of xenobiotics (Chapter53),ispeptide bond. Consequently, all four of the coloredlinked to cysteine by a non-α peptide bond (Figureatoms of Figure3-4 are coplanar.The imposed semi-3-3).The amino terminal glutamate of thyrotropin-rigidity of the peptide bond has important conse-
AMINO ACIDS & PEPTIDES / 19 CH2 C N O C O CH N CH2 SH CH2 CH2 C COO– COO– H H NH3 + H Figure 3–3. Glutathione (γ-glutamyl-cysteinylglycine). Note the non-α peptide bond that links Glu to Cys. rosyl). Peptides are then named as derivatives of the carboxyl terminal aminoacyl residue. For example, LysLeu-Tyr-Gln is called lysyl-leucyl-tyrosyl-glutamine. The -ine ending on glutamine indicates that its α-carboxyl group is not involved in peptide bond formation. Peptide Structures Are Easy to Draw Prefixes like tri- or octa- denote peptides with three or eight residues, respectively, not those with three or eight peptide bonds. By convention, peptides are written with the residue that bears the free α-amino group at the left. To draw a peptide, use a zigzag to represent the main chain or backbone. Add the main chain atoms, which occur in the repeating order: α-nitrogen, α-carbon, carbonyl carbon. Now add a hydrogen atom to each α-carbon and to each peptide nitrogen, and an oxygen to the carbonyl carbon. Finally, add the appropriate R groups (shaded) to each α-carbon atom. Three-letter abbreviations linked by straight lines represent an unambiguous primary structure. Lines are omitted for single-letter abbreviations. Where there is uncertainty about the order of a portion of a polypeptide, the questionable residues are enclosed in brackets and separated by commas. Some Peptides Contain Unusual Amino Acids In mammals, peptide hormones typically contain only the α-amino acids of proteins linked by standard peptide bonds. Other peptides may, however, contain nonprotein amino acids, derivatives of the protein amino acids, or amino acids linked by an atypical peptide bond. For example, the amino terminal glutamate of glutathione, which participates in protein folding and in the metabolism of xenobiotics (Chapter 53), is linked to cysteine by a non-α peptide bond (Figure 3–3). The amino terminal glutamate of thyrotropinGlu Lys Ala Gly Tyr His Ala - - (, , ) Glu - Ala - Lys - Gly - Tyr - Ala E A K G Y A Cα N N NC C Cα C Cα O O C CH2 H N H C C +H3N H N COO– – OOC H3C H C C CH2 OH H releasing hormone (TRH) is cyclized to pyroglutamic acid, and the carboxyl group of the carboxyl terminal prolyl residue is amidated. Peptides elaborated by fungi, bacteria, and lower animals can contain nonprotein amino acids. The antibiotics tyrocidin and gramicidin S are cyclic polypeptides that contain D-phenylalanine and ornithine. The heptapeptide opioids dermorphin and deltophorin in the skin of South American tree frogs contain D-tyrosine and D-alanine. Peptides Are Polyelectrolytes The peptide bond is uncharged at any pH of physiologic interest. Formation of peptides from amino acids is therefore accompanied by a net loss of one positive and one negative charge per peptide bond formed. Peptides nevertheless are charged at physiologic pH owing to their carboxyl and amino terminal groups and, where present, their acidic or basic R groups. As for amino acids, the net charge on a peptide depends on the pH of its environment and on the pKa values of its dissociating groups. The Peptide Bond Has Partial Double-Bond Character Although peptides are written as if a single bond linked the α-carboxyl and α-nitrogen atoms, this bond in fact exhibits partial double-bond character: There thus is no freedom of rotation about the bond that connects the carbonyl carbon and the nitrogen of a peptide bond. Consequently, all four of the colored atoms of Figure 3–4 are coplanar. The imposed semirigidity of the peptide bond has important conseC N O C H N + O– H ch03.qxd 2/13/2003 1:35 PM Page 19
20/CHAPTER3mixture of free amino acids is then treated with 6-aminoN-hydroxysuccinimidyl carbamate, which reacts withtheir α-amino groups, forming fluorescent derivativesthat are then separated and identified using high-pressureliquid chromatography (see Chapter 5). Ninhydrin, alsowidely used for detecting amino acids, forms a purpleproduct with α-amino acids and a yellow adduct withthe imine groups of proline and hydroxyproline.SUMMARY.Both D-amino acids and non-α-amino acids occurin nature, but only L-α-amino acids are present inFigure3-4.Dimensions ofafullyextendedpolyproteins.peptide chain.Thefouratoms ofthepeptidebond.All amino acids possess at least two weaklyacidic(coloredblue)arecoplanar.Theunshadedatomsarefunctional groups,RNH,+and R-COOH.the a-carbon atom, the α-hydrogen atom, and the α-RMany also possess additional weakly acidicfunctionalgroup of theparticularaminoacid.Free rotation cangroups suchasOH,SH,guanidino,orimid-occuraboutthebondsthatconnecttheot-carbonwithazolegroups.theαt-nitrogenand withtheα-carbonyl carbon (blue.The pK values of all functional groups ofan aminoarrows).Theextendedpolypeptidechainisthusasemiacid dictate its net charge at a given pH.pl is the pHrigid structure with two-thirds of theatoms of the backat which an amino acid bears no net charge and thusboneheldinafixedplanarrelationshiponetoanotherdoes not move in a direct current electrical field.The distance between adjacent α-carbon atoms is 0.36?Of the biochemical reactions of amino acids,thenm (3.6 A).The interatomic distances and bond angles,most important is theformation of peptide bonds.whicharenotequivalent,arealsoshown.(Redrawnand.The R groups of amino acids determine their uniquereproduced,withpermission,fromPaulingL,CoreyLPbiochemical functions.Amino acids are classified asBransonHR:Thestructureofproteins:Twohydrogen-basic,acidic,aromatic,aliphatic,or sulfur-containingbonded helical configurations of the polypeptide chain.based on the properties of their R groups.ProcNatlAcadSciUSA1951:37:205.).Peptides are named for the number of amino acidresidues present, and as derivatives of the carboxylterminal residue.Theprimary structure of a peptidequences for higher orders of protein structure. Encir-is its amino acid sequence, startingfrom the amino-cling arrows (Figure 3-4) indicate free rotation aboutterminal residue.the remaining bonds of the polypeptide backbone..The partial double-bond character of the bond thatlinks the carbonyl carbon and the nitrogen of a pep-Noncovalent Forces Constrain Peptidetide renders four atoms of the peptide bond coplanarConformationsand restricts the numberof possiblepeptideconfor-Folding of a peptide probably occurs coincident withmations.its biosynthesis (see Chapter 38).The physiologicallyactive conformation reflects the amino acid sequence,REFERENCESsteric hindrance, and noncovalent interactions (eg,hy-Doolittle RF: Reconstructing history with amino acid sequences.drogen bonding, hydrophobic interactions)betweenProteinSci1992;1:191.residues.Common conformations include α-helicesKreil G: D-Amino acids in animal peptides. Annu Rev Biochemand β-pleated sheets (see Chapter 5).1997;66:337.Nokihara K, Gerhardt J: Development of an improved automatedANALYSISOFTHEAMINOACIDgas-chromatographic chiral analysis system: application toCONTENTOFBIOLOGICMATERIALSnon-natural amino acids and natural protein hydrolysates.Chirality 2001;13:431.In order to determine the identity and quantity of eachSanger F: Sequences, sequences, and sequences. Annu Rev Biochemamino acid in a sample of biologic material, it is first nec-1988:57:1.essary to hydrolyze the peptide bonds that link the aminoWilson NA et al: Aspartic acid 26 in reduced Eschericbia coli thioreacids together by treatment with hot HCl. The resultingdoxin has a pK, greater than 9. Biochemistry 1995:34:8931
20 / CHAPTER 3 O C C R′ H H N C O C C N H H O H R′′ 122° 120° N 117° 121° 120° 120° 110° 0.36 nm 0.147 nm 0.153 nm 0.1 nm 0.123 nm 0.132 nm Figure 3–4. Dimensions of a fully extended polypeptide chain. The four atoms of the peptide bond (colored blue) are coplanar. The unshaded atoms are the α-carbon atom, the α-hydrogen atom, and the α-R group of the particular amino acid. Free rotation can occur about the bonds that connect the α-carbon with the α-nitrogen and with the α-carbonyl carbon (blue arrows). The extended polypeptide chain is thus a semirigid structure with two-thirds of the atoms of the backbone held in a fixed planar relationship one to another. The distance between adjacent α-carbon atoms is 0.36 nm (3.6 Å). The interatomic distances and bond angles, which are not equivalent, are also shown. (Redrawn and reproduced, with permission, from Pauling L, Corey LP, Branson HR: The structure of proteins: Two hydrogenbonded helical configurations of the polypeptide chain. Proc Natl Acad Sci U S A 1951;37:205.) quences for higher orders of protein structure. Encircling arrows (Figure 3 – 4) indicate free rotation about the remaining bonds of the polypeptide backbone. Noncovalent Forces Constrain Peptide Conformations Folding of a peptide probably occurs coincident with its biosynthesis (see Chapter 38). The physiologically active conformation reflects the amino acid sequence, steric hindrance, and noncovalent interactions (eg, hydrogen bonding, hydrophobic interactions) between residues. Common conformations include α-helices and β-pleated sheets (see Chapter 5). ANALYSIS OF THE AMINO ACID CONTENT OF BIOLOGIC MATERIALS In order to determine the identity and quantity of each amino acid in a sample of biologic material, it is first necessary to hydrolyze the peptide bonds that link the amino acids together by treatment with hot HCl. The resulting mixture of free amino acids is then treated with 6-aminoN-hydroxysuccinimidyl carbamate, which reacts with their α-amino groups, forming fluorescent derivatives that are then separated and identified using high-pressure liquid chromatography (see Chapter 5). Ninhydrin, also widely used for detecting amino acids, forms a purple product with α-amino acids and a yellow adduct with the imine groups of proline and hydroxyproline. SUMMARY • Both D-amino acids and non-α-amino acids occur in nature, but only L-α-amino acids are present in proteins. • All amino acids possess at least two weakly acidic functional groups, RNH3 + and RCOOH. Many also possess additional weakly acidic functional groups such as OH, SH, guanidino, or imidazole groups. • The pKa values of all functional groups of an amino acid dictate its net charge at a given pH. pI is the pH at which an amino acid bears no net charge and thus does not move in a direct current electrical field. • Of the biochemical reactions of amino acids, the most important is the formation of peptide bonds. • The R groups of amino acids determine their unique biochemical functions. Amino acids are classified as basic, acidic, aromatic, aliphatic, or sulfur-containing based on the properties of their R groups. • Peptides are named for the number of amino acid residues present, and as derivatives of the carboxyl terminal residue. The primary structure of a peptide is its amino acid sequence, starting from the aminoterminal residue. • The partial double-bond character of the bond that links the carbonyl carbon and the nitrogen of a peptide renders four atoms of the peptide bond coplanar and restricts the number of possible peptide conformations. REFERENCES Doolittle RF: Reconstructing history with amino acid sequences. Protein Sci 1992;1:191. Kreil G: D-Amino acids in animal peptides. Annu Rev Biochem 1997;66:337. Nokihara K, Gerhardt J: Development of an improved automated gas-chromatographic chiral analysis system: application to non-natural amino acids and natural protein hydrolysates. Chirality 2001;13:431. Sanger F: Sequences, sequences, and sequences. Annu Rev Biochem 1988;57:1. Wilson NA et al: Aspartic acid 26 in reduced Escherichia coli thioredoxin has a pKa greater than 9. Biochemistry 1995;34:8931. ch03.qxd 2/13/2003 1:35 PM Page 20