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Some extremely toxic mushroom poisons, suchas amanitin, are also peptides, as are many antibiotics.Chapter 5 Amino Acids and PeptidesInsolublepolystyrenebeadCl—CH 2OR1,p" M'" n _(rijn(CH3)3C-O-C-N-CH-C-O -a-Amino ggroupprotectedppby *-butyloxycarbonylby £-butyloxycarbonylgroupl)HR1oAttachment of carboxyl-terminalamino acid to reactivegroup on resin.OI(CH 3 ) 3 C—O—C—N—CH—C—O—CH,HR2OO(5)Protecting group is removedby flushing withCF3COOH.®a-Amino group of aminoacid 1 attacks activatedcarboxy group of amino acid2 to form peptide bond.CF;,COOH(CH 3 ) 3 C—O-C-N—CH—C—OH©DicyclohexylcarbodiimideOR2IIAmino acid withprotecteda-amino group isactivated atcarboxyl groupby DCC.ONHIIIOR+II IIIICH 3 N—CH—C—O—CH2(CH3)3C—O-C-N—CH—C—O-CHHDicyclohexylureaR2oR1OOReactions ( ? ) to ( ? )repeated as necessaryII(CH 3 ) 3 C—O—C—N—CH—C—N—CH—C—O—CH,HHHF (5)R2OR1OI III II+CHH3N—CH—C—N—CH—C—CT + F—HA growing number of small peptides are proving to be importantcommercially as pharmaceutical reagents.
Unfortunately, they areoften present in exceedingly small amounts and hence are hard to purify. For these and other reasons, the chemical synthesis of peptideshas become one of the major technologies associated with biochemistry(Box 5-2).Completed peptide isdeprotected as inreaction (5) ; HF hydrolyzesester linkage betweenpeptide and resin.129130Part II Structure and CatalysisSummaryThe 20 amino acids commonly found as hydrolysisproducts of proteins contain an a-carboxyl group,an a-amino group, and a distinctive R group substituted on the a-carbon atom. The a-carbon atomof the amino acids (except glycine) is asymmetric,and thus amino acids can exist in at least twostereoisomeric forms. Only the L stereoisomers,which are related to the absolute configuration ofL-glyceraldehyde, are found in proteins. The aminoacids are classified on the basis of the polarity oftheir R groups.
The nonpolar, aliphatic class includes alanine, glycine, isoleucine, leucine, proline,and valine. Phenylalanine, tryptophan, and tyrosine have aromatic side chains and are also relatively hydrophobic. The polar, uncharged classincludes asparagine, cysteine, glutamine, methionine, serine, and threonine. The negativelycharged (acidic) amino acids are aspartate and glutamate; the positively charge (basic) ones are arginine, histidine, and lysine. There are also a largenumber of nonstandard amino acids that occur insome proteins (as a result of the modification ofstandard amino acids) or as free metabolites incells.Monoamino monocarboxylic amino acids arediprotic acids (+H3NCH(R)COOH) at low pH.
Asthe pH is raised to about 6, near the isoelectricpoint, the proton is lost from the carboxyl groupto form the dipolar or zwitterionic species+H3NCH(R)COCT, which is electrically neutral.Further increase in pH causes loss of the secondproton, to yield the ionic species H2NCH(R)COO~.Amino acids with ionizable R groups may exist inadditional ionic species, depending on the pH andthe pKa of the R group.
Thus amino acids vary intheir acid-base properties. Amino acids form colored derivatives with ninhydrin. Other colored orfluorescent derivatives are formed in reactions ofthe a-amino group of amino acids with fluorescamine, dansyl chloride, dabsyl chloride, andl-fluoro-2,4-dinitrobenzene. Complex mixtures ofamino acids can be separated and identified by ionexchange chromatography or HPLC.Amino acids can be joined covalently throughpeptide bonds to form peptides, which can also beformed by incomplete hydrolysis of polypeptides.The acid-base behavior and chemical reactions ofa peptide are functions of its amino-terminalamino group, its carboxyl-terminal carboxyl group,and its R groups.
Peptides can be hydrolyzed toyield free amino acids. Some peptides occur free incells and tissues and have specific biological functions. These include some hormones and antibiotics, as well as other peptides with powerful biological activity.GeneralAmino AcidsCantor, C.R. & Schimmel, P.R. (1980) BiophysicalChemistry, Part I: The Conformation of BiologicalMacromolecules, W.H. Freeman and Company,San Francisco.Excellent textbook outlining the properties of biological macromolecules and their monomeric subunits.Corrigan, J.J. (1969) D-Amino acids in animals.Science 169, 142-148.Further ReadingCreighton, T.E.
(1984) Proteins: Structures andMolecular Properties, W.H. Freeman and Company, New York.Very useful general source.Dickerson, R.E. & Geis, I. (1983) Proteins: Structure, Function, and Evolution, 2nd edn, The Benjamin/Cummings Publishing Company, MenloPark, CA.Beautifully illustrated and interesting account.Meister, A. (1965) Biochemistry of the AminoAcids, 2nd edn, Vols.
1 and 2, Academic Press, Inc.,New York.Encyclopedic treatment of the properties, occurrence, and metabolism of amino acids.Montgomery, R. & Swenson, CA. (1976) Quantitative Problems in the Biochemical Sciences, 2ndedn, W.H. Freeman and Company, New York.Segel, I.H. (1976) Biochemical Calculations, 2ndedn, John Wiley & Sons, New York.Chapter 5 Amino Acids and PeptidesPeptidesHaschemeyer, R.H. & Haschemeyer, A.E.V.(1973) Proteins: A Guide to Study by Physical andChemical Methods, John Wiley & Sons, New York.131Smith, L.M. (1988) Automated synthesis and sequence analysis of biological macromolecules.Analyt.
Chem. 60, 381A-390A.Merrifield, B. (1986) Solid phase synthesis. Science 232, 341-347.Problems1. Absolute Configuration of Citrulline Is citrulline isolated from watermelons (shown below) aD- or L-amino acid? Explain.CH 2 (CH 2 ) 2 NH-C-NH 2H—C-NH 3Ocoo2. Relation between the Structures and ChemicalProperties of the Amino Acids The structures andchemical properties of the amino acids are crucialto understanding how proteins carry out their biological functions. The structures of the side chainsof 16 amino acids are given below.
Name the aminoacid that contains each structure and match the Rgroup with the most appropriate description of itsproperties, (a) to (m). Some of the descriptions maybe used more than once.(a) Small polar R group containing a hydroxylgroup; this amino acid is important in the activesite of some enzymes.(k) Forms disulfide cross-links between polypeptide chains; the pKa of its functional group is about10.(1) R group with pKa ~ 12, making it positivelycharged at physiological pH.(m) When this polar but uncharged R group is hydrolyzed, the amino acid is converted into anotheramino acid having a negatively charged R group atpH near 7.(1) —H(2) - C H 3.CH3(3) - C H\CH3(4)(5) - C H 2 O H(6) —CH2-V(7)—CH,(8) —CH,—CH 2CH\OH(b) Provides the least amount of steric hindrance.(c) R group has pKa « 10.5, making it positivelycharged at physiological pH.(d) Sulfur-containing R group; neutral at any pH.(e) Aromatic R group, hydrophobic in nature andneutral at any pH.(9) —CH2—C^(11) — C H 2 — C H 2 — S - C H 3(12) —CH 2 —SH(g) The only amino acid having an ionizing R groupwith a pKa near 7; it is an important group in theactive site of some enzymes.(13) - C H 2(i) R group has a pKa near 4 and thus is negativelycharged at pH 7.(j) An aromatic R group capable of forming hydrogen bonds; it has a pKa near 10.V\o-(f) Saturated hydrocarbon, important in hydrophobic interactions.(h) The only amino acid having a substituted aamino group; it influences protein folding by forcing a bend in the chain.(10) - C H 2 - C H 2 - ( /V -H(14) - C H 2 - C - N H 2OAH(15) -CH 2 —CH 2 -CH 2 —PC-NH2+H-NH(16) -CH 2 —CH 2 —CH 2 -CH 2 -NH 3Part II Structure and Catalysis3.
Relationship between the Titration Curve andthe Acid-Base Properties of Glycine A 100 mL solution of 0.1 M glycine at pH 1.72 was titrated with2 M NaOH solution. During the titration, the pHwas monitored and the results were plotted in thegraph shown. The key points in the titration aredesignated I to V on the graph. For each of thestatements below, identify the appropriate keypoint in the titration and justify your choice.(n) At what point do the structures of the predominant species consist of a 50:50 mixture of+H3N—CH2—COOH and + H 3 N—CH 2 —COO?(o) What point corresponds to the isoelectricpoint?(p) At what point is the average net charge onglycine -£?(q) What point represents the end of the titration?(r) If one wanted to use glycine as an efficientbuffer, which points would represent the worst pHregions for buffering power?(s) At what point in the titration is the predominant species H2N—CH2—COO?4.
How Much Alanine Is Present as the CompletelyUncharged Species ? At a pH equal to the isoelectric point, the net charge on alanine is zero. Twostructures can be drawn that have a net charge ofzero (zwitterionic and uncharged forms), but thepredominant form of alanine at its pi is zwitterionic.pHCH3oHo-CH3H2N—C—CZwitterionic0.51.01.5OH~ (equivalents)2.0(a) At what point will glycine be present predominantly as the species +H3N—CH2—COOH?(b) At what point is the average net charge ofglycine +£?(c) At what point is the amino group of half ofthe molecules ionized?(d) At what point is the pH equal to the pKa ofthe carboxyl group?(e) At what point is the pH equal to the ipKa ofthe protonated amino group?(f) At what points does glycine have its maximum buffering capacity?(g) At what point is the average net charge zero?(h) At what point has the carboxyl group beencompletely titrated (first equivalence point)?(i) At what point are half of the carboxyl groupsionized?(j) At what point is glycine completely titrated(second equivalence point)?(k) At what point is the structure of the predominant species +H3N—CH2—COO"?(1) At what point do the structures of the predominant species correspond to a 50:50 mixture of+H3N—CH2—COO" and H 2 N—CH 2 —COO?(m) At what point is the average net charge ofglycine -1?HOHUncharged(a) Explain why the form of alanine at its pi iszwitterionic rather than completely uncharged.(b) Estimate the fraction of alanine present atits pi as the completely uncharged form.
Justifyyour assumptions.5. Ionization State of Amino Acids Each ionizablegroup of an amino acid can exist in one of twostates, charged or neutral. The electric charge onthe functional group is determined by the relationship between its pKa and the pH of the solution.This relationship is described by the HendersonHasselbalch equation.(a) Histidine has three ionizable functionalgroups.
Write the relevant equilibrium equationsfor its three ionizations and assign the proper ipKafor each ionization. Draw the structure of histidinein each ionization state. What is the net charge onthe histidine molecule in each ionization state?(b) Draw the structures of the predominant ionization state of histidine at pH 1, 4, 8, and 12. Notethat the ionization state can be approximated bytreating each ionizable group independently.(c) What is the net charge of histidine at pH 1, 4,8, and 12? For each pH, will histidine migrate toward the anode ( + ) or cathode (-) when placed inan electric field?6. Preparation of a Glycine Buffer Glycine is commonly used as a buffer. Preparation of a 0.1 M glycine buffer starts with 0.1 M solutions of glycinehydrochloride (HOOC—CH 2 -NH 3 + C1) and gly-Chapter 5 Amino Acids and Peptidescine ( OOC—CH2—NH3 ), two commercially available forms of glycine.
What volumes of these twosolutions must be mixed to prepare 1 L of 0.1 M glycine buffer having a pH of 3.2? (Hint: See Box 4-2)7. Separation of Amino Acids by Ion-ExchangeChromatography Mixtures of amino acids areanalyzed by first separating the mixture into itscomponents through ion-exchange chromatography. On a cation-exchange resin containing sulfonate groups (see Fig. 5-12), the amino acids flowdown the column at different rates because of twofactors that retard their movement: (1) ionic attraction between the —SO3 residues on the columnand positively charged functional groups on theamino acids and (2) hydrophobic interaction between amino acid side chains and the strongly hydrophobic backbone of the polystyrene resin.
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