Bpo c Chapter 25

7/29/2019 Bpo c Chapter 25

1/81

NO AC DS, PEPTPROTE NS, ENZYMES, ANDNUCLE

he chemistry of life is largely the chemistry of polyfunctional organic com-pounds. The functional groups usually are of types that interact rather stronglyas, for example, the hydroxyl and carbonyl functions of carbohydrates (Chap-ter 20). The interaction between amino and carboxyl functions of amino acidsfigures greatly in the present chapter. We will approach the very importantchemistry of amino acids and their derivatives in three stages. First, simplea-amino acids will be considered with emphasis on how the properties of aminefunctions and of acid functions are modified in molecules that possess bothgroups. Then we shall discuss some important properties of peptides andproteins, which are substances made up of amino acids linked together byamide bonds. Attention also will be given to the chemical problems presentedby enzymes, which are protein molecules able to act as efficient catalysts forspecific chemical reactions, and to the role of nucleic acids in protein synthesis.

25-1 TYPES OF BIOLOGICALLY IMPORTANT AMINO ACIDS25-1A Protein Amino Ac idsThe amino acids that occur naturally as constituents of proteins have an aminogroup (NH,) and a carboxylic acid group (C0,H) attached to the same


2/81

25-1A Protein Amino Acidscarbon. The y are cal led a-amino acids and have the general formula

They differ only in the nature of the R group on the a carbo n and , with fewexceptions, they are chiral molecules with the L configuration at the chirala carbon:l

L-amino acid(S-amino acid)

T h e struc tures and names of some particularly impo rtant a-amino acidsare shown in Table 25-1. You will notice that the names in common use foramino acids are not descriptive of their structural formulas; but at least theyhave the advantage of being shorter than the systematic names. T h e abbrevia-tions G ly, G lu, and so on, that a re listed in Table 25-1 a re particularly usefulin designating the sequences of amino acids in proteins and peptides, as willbecome evident later in the chapter.The nature of the substituent R varies considerably. In some aminoacids, R is a hydrocarbon group, whereas in others it possesses functionalgroups such as OH , S H , SCH,, CQ,H, o r NH,. Am ino acids that have amineo r oth er basic functions in the R group a re called basic amino acids (lysine andarginine), whereas those with acidic groups are called acidic amino acids(aspartic and glutamic acids). Three of the amino acids listed in Table 25-1(cysteine, cy stine , an d methionine) contain sulfur in -S H , -S-S-, an d-SCH , groups. Cy steine and cystine can be interconverted readily with awide variety of oxidizing and reducing agen ts according to the general reaction

[O I2 R S Hd SSR. This is an important process in the biochemistry of[ H Isulfur-containing peptides and proteins (Section 25-8A).The a-amino function of the common amino acids is primary -NH,in all except proline and hydroxyproline. Several of the amino acids havearo m atic R g roup s (phen ylalanine, tyrosine, tryptop han), w hile histidine andtryptophan have azarene R groups.

lA number of D-amino acids have been found to be constituents of peptides in the cellwalls of bacteria.


3/81

25 Amino Acids, Pept ides, Proteins, Enzymes, and Nucleic Acids


4/81

25-1A Protein Amino Acids


5/81

Table 25-1 (continued)Amino Acids Important as Constituents of Proteins

AbbreviationsName 3-letter 1 letter Structurea

histidine His H

phenylalaninec Phe

tyrosine TY

tryptophan

prol ine Pro

hydroxyprol ined Hyp

!?Q"For con venience only, the structures are represented as neutral nonpolar molecules. In real i ty, ionic and dip olar forms are present in aqueoussolution in amounts dependent on the p H (Section 25-2A). f0bWater solubi l i ty at isoelectric po int of the L isomer in g/100 g at 20C. The D,L mixtures are usua lly less soluble."Must be included in diet for maintenance of proper nitrogen equil ibrium in normal adult humans.dFound only in collagen.


6/81

25-1 B Nonprotein Amino Acids 1216

Exercise 25-1 Select the amino acids in Table 25-1 that have more than one chiralcenter and draw projection formulas for all the possible stereoisomers of each whichpossess the L configuration at the a carbon.

Exercise 25-2 Which of the amino acids in Table 25-1 are acidic amino acids andwhich basic amino acids? Which of the structures shown would have the most basicnitrogen ? The least basic amino nitrogen ? Give the reasons for your cho ices. (ReviewSection 23-7.)

25-1B Nonprote in Amino A c idsT h e most ab undan t amino acids ar e those that ar e protein constituents andthese are a lways a-amino ac ids. Ho wev er , there are many othe r amino ac idsthat oc cu r naturally in living sy stem s that ar e not con stituents of proteins, andare n ot a-a mino acids. Man y of these a re rare, but others are common and playimportant roles in cellular metabolism. For example, 3-aminopropanoic acidis a precu rsor in the biosynthesis of the vitamin, pantothenic acid,2

I/H 2 N C H 2 C M 2 C - O H 3-aminopropanoic acid (p-alanine)ew3 ow oI I / I~ O C H 2 - C - c - ~ pantothen~c c id

I IC H 3 Hand 4-aminobutanoic acid is involved in the transmission of nerve impulses.

0IH2NCH2CII[2Cw2C-ON[ 4-aminobutanoic acid (y-aminobutyr ic)Hom ocysteine3 and hom oserine a re among the important a-amino acids thatare not consti tuents of proteins. These substances are precursors in the bio-synthesis of methionine.

N H 2homocysteine

NH2homoserine

2Pantothenic ac id i s in turn a precursor for the synthes is of coenz ym e A, which isessential for the biosynthesis of fats and l ipids (Sect ions 18-8F a n d 30-5A).T h e prefix homo impl ies an addi tiona l carbon in the longest cha in .


7/81

1212 25 Amino A cids, P eptides, Proteins, Enzymes, and Nuc leic Ac ids25-2 THE ACID-BASE PROPERTIES OF a-AMINO ACIDSThe behavior of glycine is reasonably typical of that of the simple amino acids.Because glycine is neither a strong acid nor a strong base, we shall expect asolution of glycine in water to contain four species in rapid equilibrium. Theproportions of these species are expected to change with pH, the cationicconjugate acid being the predominant form at low pH and the anionic conjugatebase being favored at high pH:

con jugate ac idof glycine(dominant at p H 1)dipola r ion conjugate baseo r of glycinezwitterion (dominant at pH 12)

neutral glycine

Spectroscopic measurements show that the equilibrium between neutral gly-cine and the dipolar ion favors the dipolar ion by at least 100 to 1. This is to

0be expected because the H3N- group of the dipolar ion will stabilize the0-co,@ end while the - C O , ~ group will stabilize the H,N- end.

0The acid-ionization constant of H3NCH2C02W s 4.5 x 10-YpK, =2.34, Equation 25-I), which is about 25 times greater than K, for ethanoicacid. (Section 18-2). This is expected because of the electron-attracting

0 0electrostatic effect of the H3N- group. Ionization of the H,N- group of thedipolar ion ( K ,= 2.0 x 10-lo; pK =9.60; Equation 25-2) is oppositely affectedby the electrostatic effect of the -CO,@ group and is 10 times less than ofethanammonium ion (Section 23-7B). The manner in which the concentrationsof the charged glycine species change with pH is shown in Figure 25-1. Noticethat, between pH 3 and pH 8, almost all of the glycine is in the form of thedipolar ion. The pH at the center of this range, where the concentration of0 0H,NCH,CO,H is equal to the concentration of H,NCH,CO,, is called theisoelectric point, pl, and usually corresponds to the pH at which the aminoacid has minimum water solubility. Isoelectric points for the amino acids areshown in Table 25- 1. The isoelectric points are the average of the pK, valuesfor dissociation of the monocation and the dipolar ion forms of the amino acid.For glycine, pl = (2.34 + 9.60)/2.


8/81

25-2 The Acid-Base Propert ies of a-Amino Acids

concentrat ion,moles l i ter - '

0 0Figure 25-4 Con ce nt ra tio ns of H,NCH,CO,H, H,NCH,CO,~, and H,NCH,-CO,@ as a function of p H for a 0.1M solution of glycine in water

@pK a ' = pf-H$. loglo [ H ~ N C H ~ C O ~ ~ I9 -60[H,NCH~CO,@]

Exercise 25-3 How wo uld the general features of the plot of concentration of dipo larion and cha rged sp ecies versus p H for glycine (Figure 25 - 1 ) change for 6-amino-hexanoVicac id, wh ich has pK, values of 4.43 and 1 0 . 7 5 ? Giv e specia l attention to theposition of the isoelectric point and the width of the pH range over which the dipolarion is expected to be the most stable species present.Exercise 25-4 Use Equations 25-1 and 25-2 to show that the isoelectric point ofgly cin e is the average of the two pK, values for the ac id diss ociation of gly cin e.

T h e pH behavior of am ino acids with ei ther acidic or basic funct ionalgroups a t tached t o the s ide chains is more complicated than of s imple amino


9/81

4214 25 Amino Acids, Peptides, Proteins, Enzymes, and Nuc leic Acid sacids. For example, there are three acid dissociations starting with the di-conjugate acid of lysine:

diconjugateac i d of lysine conjugate baseof lysine

The pKa values for the side-chain functions of acidic and basic aminoacids are given in Table 25-1.0We already have mentioned how the H3N- group of the conjugateacid of glycine enhances the acid strength of the carboxyl group compared to

0 0ethanoic acid and how the -C02 group reduces the acidity of the H3N-group of the dipolar ion relative to ethanammonium ion. These effects willbe smaller the farther away the charged group is from the ionizable group. Asa result, one would predict that the carboxyl groups of aspartic acid wouldhave different pKa values, and indeed this is so:

C H ~I aspartic ac id


10/81

25-3 Physical and Spectroscopic Properties 11295Similarly; the side-chain ammonium group of lysine is less acidic than thatof the ammonium group close to the carboxyl group:

(CH2)4 conjugate acid of lysineI

Exercise 25-5 a. The equations for the acid-base equil ibria of lysine on p. 1214'show pos sible involvem ent of three forms of the mon ocation and three forms of the

neutral acid. Arrange the three forms of each set in expected order of stability. Giveyour reasoning.b. The conjugate acid of glutamic acid (Table 25-1) has three acid dissociat ionsteps with pK, values of 2.19, 4.25 and 9.67. Write equa tions for the equ ilibr ia inv olve dand assign pKa values to each. D o the same for arginine (Tab le 25-1) with pKa valuesof 2.17, 9.04 and 12.48. Ca lculate the isoelectr ic point for glutam ic ac id an d forarginine.

25-3 PHYSICAL AND SPECTROSCOPIC PROPERTIES0 0The a-amino acids crystallize as the dipolar forms, H,N-CHR-CO,, and

the strong intermolecular electrical forces in the crystals lead to higher meltingpoints than those of simple amines or monocarboxylic acids (see Table 25-1).The melting points are so high that decomposition often occurs on melting.Th e solubility characteristics of amino acids in water are complex because ofthe acid-dissociation equilibria involved, but they are least soluble at theirisoelectric points. The dipolar structures of amino acids greatly reduce theirsolubility in nonpolar organic solvents compared to simple amines and car-boxylic acids.The infrared spectra of a-amino acids in the solid state or in solution donot show a carbonyl absorption band at 1720 cm-l characteristic of a carboxylgroup. Rather, they show a strong absorption near 1600 cm-I typical of thecarboxylate anion. The N-El stretch appears as a strong, broad band between3 100-2600 cm-l:


11/81

1216 25 Amino A cids, Pept ides, Proteins, Enzymes, and Nucleic Ac ids

Exercise 25-6 Indica te the approxim ate positions of C=O and N-H absorptions0 0 0 0you would expect in the infrared spectra of (a) CIH,NCH2C02H (b) H2NCH2C02Na.

Exercise 25-7 Sketch the nmr spectrum show ing the splitt ing pattern and che m icalshif ts you w ould a nt icipate for alanine dissolved in an excess of D 2 0.Do not neglectH-D exc hange (Sec tion 9-1 0E and 9-101).

Amino acids do not give any very useful ultraviolet absorption spectraunless they possess aromatic groups as in phenylalanine, tryptophan, andtyrosine. The absorption characteristics of these groups are more useful inmonitoring chemical and conformational changes in proteins than they are inthe simple amino acids.

It is not easy to obtain the mass spectra of amino acids because of theirlow volatility. However, a number of special techniques now make possibledetermination of the mass spectra of amino acids and also of peptides. Becausevery small amounts of sample are required, this is becoming a particularlyuseful method of amino acid and peptide analysis.

25-4 ANALYSIS OF AMINO ACIDS25-4A The Ninhydrin and Related TestsIn many kinds of research it is important to have simple and sensitive meansfor analysis of amino acids, particularly in small quantities. Detection of aminoacids can be achieved readily by the "ninhydrin color test," whereby an alco-holic solution of the triketone, "ninhydrin," is heated with an amino acid andproduces an intense blue-violet color. The sensitivity and reliability of this testis such that 0.1 micromole of amino acid gives a color intensity reproducibleto a few per cent, provided that a reducing agent such as stannous chloride ispresent to prevent oxidation of the colored salt by dissolved oxygen.

indane-l,2,3-tr ione "ninhydrin"


12/81

25-4 Analysis of Amino Ac ids 1217The color-forming reaction is interesting because most a-amino acids givethe same color irrespective of their s t r ~ c tu r e . ~he sequence of steps that leadsto the color is as follows:

0 RC=O + RCH-C02H -H20, m /C=N-CH-C02H -CO,I C\\ NH20 \o

/PR

rearr. >-H,O

blue/0 0

A new, very sensitive method of detection and analysis of amino acids, whichis useful down to the 10-I, mole (picomole) level, depends on the formationfrom RNH, and "fluorescamine," 1, of substances that are intensely fluorescentin ultraviolet light:

&rHs RNH,, &C6H5 fluorescent product\ C 0 2 H

01

4Proline and hydroxyproline are exceptions because neither has the necessary primaryNH, group needed for the reaction. However, these compounds do react with nin-hydrin to give yellow compounds, and these colors can be used to identify themsatisfactorily.


13/81

Y218 25 Amino Acids, Pept ides, Proteins, Enzymes, and Nucle ic Acids

Exercise 25-8 The reactions that lea d to the blue color p rodu ced between ninhydrinand a-amino acids are examples of reactions discussed previously in the context ofcarb onyl chemistry (see, for instan ce, Section 16-4C). Write mechanisms, basedinsofar as possible on analogy, for each of the steps involved in the ninhydrin test,using glyc ine as an ex ample. Would you expect am monia or m ethanamine to give theblue color? Explain.

25-4B Paper ChromatographyNinhydrin (or fluorescamine) is very useful in chromatographic methods forthe analysis of amino acids. One of these is paper chromatography, whereinamino acids are separated as the consequence of differences in their partitioncoefficients between water and an organic solvent. The aqueous phase is heldstationary in the pores of the paper because of strong interaction of the waterwith the hydroxyl functions of the cellulose. The differences in partition coeffi-cients show up as differences in rates of migration on the surface of moist (butnot wet) paper over which there is a slow flow of a water-saturated organicsolvent. We shall discuss one of several useful modes of operation. In thisexample, a drop of the solution to be analyzed is placed on the corner of a sheetof moist paper (often filter paper), which is then placed in an apparatus like thatof Figure 25-2, arranged so that the organic solvent can migrate upward bycapillarity across the paper, carrying the amino acids with it along one edge.The acids that have the greatest solubility in the organic solvent move mostrapidly and when the solvent reaches the top of the paper, the paper is removed,dried, then turned sidewise, and a different solvent allowed to migrate upward.This double migration process gives a better separation of the amino acids thana single migration and results in concentration of the different amino acids in

solvent front

Figure 25-2 Diagram of apparatus used to develop a pape r chromato-gram, Paper is suspended from its top edge within an airt ight container,here a glass box closed with a glass plate, having an atmosphere sat-urated with solvent vapor; the lower edge of paper dips into a troughcontaining the l iquid solvent.


14/81

25-4C Ion-Exchange Chromatography

methionine@ 0.4t ryptophan 2,4-dimethylazabenzene:

@ @ glycine 2,4,6-tr imethylazabenzene:arginine alanine @ water, 1 I : I4D@ 0.2aspart ic acid@lysine

I I I II 0.8 0.6 0.4 0.2

I 1startbenzenol-water, 5 : l

Figure 25-3 Ideal ized two-dimensional paper chromatogram of a mix-ture of am ino a cids . The horizontal and vertical scales represent thedistance of travel of a com ponen t of the mixture in a given solvent relativeto that of the solvent itself. This is known as the R, value an d is fair ly con-stant for a particular compound in a given solvent. A rough identif icationof the amino acids present in the mixture may therefore be made on thebas is of their R, values.

rather well-defined spots. These spots can be made visible by first drying andthen spraying the paper with ninhydrin solution. The final result is as shown inFigure 25-3 and usually is quite reproducible under a given set of conditions.The identities of the amino acids that produce the various spots are establishedby comparison with the behavior of known mixtures.

Analysis by thin-layer chromatography (see Section 9-2B) can be carried outin the same way as paper chromatography. The partitioning is now between asolid stationary phase (the coating on the plate) and the moving solvent front.

25-4C Ion-Exchange ChromatographyThe advent of ion-exchange chromatography has revolutionized the separationand analysis of amino acids as well as that of many inorganic substances. Asthe name implies, it involves the exchange of ions between a stationary and amoving phase. The stationary phase is an insoluble polymer (or resin) havingchains on which are located ionic functions such as sulfonate groups -SO,@ or

0quaternary ammonium groups, -NR,. The counterions to these groups, suchas Na@or C1@,are not bound to the resin and can be exchanged for other ionsin the mobile phase as the mobile phase travels through the resin. A commonapplication of this principle is in household water softeners, in which thecalcium and magnesium ions in ordinary "hard" water are replaced by sodium


15/81

25 Amino Acids, P eptides, froteins, Enzymes, and Nu cleic Ac ids

at pH 3_1_2volume (mi) of effluent --Figure 25-4 Part of amino-acid chromatogram obtained by the methodof automatic amino-acid analysis from a hydrolyzed sample of theenzyme ribonuclease. The component amino acids listed are present inthe ratio Asp:Thr:Ser:Glu:Pro:Gly:Ala = 15:10:15:12:4:3:12, as deter-min ed by peak intensity. The volume of effluent is a me asure of the reten-tion time of the amino acids on the column.

ions from the resin (Equation 25-3). The resulting "soft" water can be freed ofmetal ions, if desired, by exchanging the Na@ ons for protons (Equation 25-4):

In strongly acidic solutions (pH -0), the amine and carboxyl groups of anamino acid are completely protonated. This cationic form of the amino acidcan be exchanged with the cations associated with the sulfonate groups ofthe resin:

The process is reversible, and the amino acid cations can in turn be exchangedoff the columns. However, different amino acids have different affinities for theresin, and these are considerably influenced by the pH of the moving phase(eluent). The basic amino acids (arginine, lysine), which form cations mostreadily, are more strongly held by cation-exchange resins than are acidic aminoacids (asparatic and glutamic acids). There is a spectrum of affini ties of the otheramino acid cations for the resin between these extremes. Thus a mixture ofamino acids can be separated by ion-exchange chromatography by elution with


16/81

25-5 Reactions of Amino Acids 4223buffered aque ous solutions. T h e eff luent f rom the colum n is mixed with ninhy-drin solution and the intensity of the blue color is measured and plotted as afunction of t ime a t constan t flow rates (F igure 25-4). T h e identity of an aminoacid is determined by the volume of solvent required to elute the amino acidfrom t he column, and th e concentrat ion is determined from the intensity of thecolor developed.

Exercise 25-9 Explain why arginine elutes from an ion-exchange column using abuffer at p H 5-6, whereas glutamic ac id elutes at pH 3.Exercise 25-10 A cation-exchange resin can be prepared by radical-addit ionpolym erization of phe nylethene (styrene, Section 10-8) in the presence of about2-1 0% 1,4-diethenyl benzene (1,4-diviny lbenzene), H 2C=CH e C H = C H 2 ,follow ed by elec trop hilic sulfonation of the resulting polym er with H2S0,-SO, (seeSection 22-46), Explain how these reactions lead to a three-dimensional insolublepolymer with l inkages as shown below. Indicate the reaction mechanisms involved.

Exercise 25-11 Co nsider a "ha rd" water co m pris ed of dilute MgCI,. Ion exchange0 0with resin-S03Na replaces Mg2@ i th Na@,and wi th res in -S03H, Nao is replaced

by He, thereby producing a dilute HCI solution. What kind of an ion-exchange resinwou ld you need to remove the CI@ rom the HC I solution and prod uce "de ionized "water? (Consider exchanging Clafor "OH.)

25-5 REACTIONS OF AMINO ACIDS25-5A Ester and A m ide FormationT o som e deg ree the reactions of amino acids are typical of isolated carboxylicacid and amine functions. Thus the carboxyl function can be esterified withan exc ess of an alcohol under acidic conditions, and the am ine function can be


17/81

1222 25 Amino Acid s, Pept ides, Proteins, Enzymes, and N ucleic Acidsacylated with acid chlorides or anhydrides under basic conditions:

The products, however, are not indefinitely stable because the functionalgroups can, and eventually will, react with each other. For example, in theacylation of glycine with ethanoic anhydride, the first-formed product maycyclize to the "azlactone" if the reaction is prolonged or excess anhydrideis used:

H CH 0\ / \ 2 /(CH3CO) 0 , N C (CH3CO),0H2NCH2C02H-H3C02H , _ O H C H 3 C 0 2 H


18/81

25-56 mino Acids with Aldehydes

Exercise 25-12 a. Draw the structure of the azlactone derived from L-phenylalanineand ethanoic anhydride.b. Wh ich of the hydrogens in this azlactone would you expect to be the most acid ic?Explain.c. Why do chiral azlactones derived from amino acids such as L-phenylalanineracemize easi ly on heating in ethanoic ac id in the presence of ethanoate ion?

25-5B Nitrous A cid React ionThe amine function of a-amino acids and esters reacts with nitrous acid in amanner similar to that described for primary amines (Section 23-10A). Thediazonium ion intermediate loses molecular nitrogen in the case of the acid,but the diazonium ester loses a proton and forms a relatively stable diazocompound known as ethyl diazoethanoate:

0 0I I HONO 0 I I -HOH2NCH2C-OC2H5 N=N-CH2-C-OC&5 >

ethyl .diazoethanoateThis diazo ester is formed because loss of N, from the diazonium ion resultsin formation of a quite unfavorable carbocation.

Exercise 25-13 Explain why glyc ine i tself, as the dipo lar ion, reacts with nitrous acidto eliminate nitrogen, whereas the ethyl ester of glycine forms ethyl diazoethanoate.

25-5C Amino A cids wi th Aldehydesa-Amino acids react with aldehydes to form decarboxylation and/or deamina-tion products. The reaction sequence is shown in Figure 25-5 and closelyresembles the ninhydrin reaction (Section 25-4A). In the first step the aminecondenses with the aldehyde to give an imine or Schiff base, 2. What happensnext depends on the relative rates of proton shift and decarboxylation of 2.


19/81

25 Amino Acids, Peptides, Proteins, Enzymes, an d N ucleic Acids

R ' C H O -H 20IR-CH-C02H

INproton \c HIs h y R' \

R-C-C02H 2IIN\

R-CHINproton 5shift(

+ R'C HO 'C HRfCH2NH2 R '6

Figure 25-5 Reactions of a-amino ac ids with an aldehyde, R'CHO. Theproducts are the result of decarboxylation and/or deamination; the frac-tion of the produc ts formed by eac h route is determine d by the ratio of therate of proton shift to the rate of decarboxylation of 2.

Proton shift produces a rearranged imine, 3, which can hydrolyze to the ketoacid 4. T h e keto acid is a deamination product. A lternatively, decarboxylationcan occur (see Section 18-4) and the resulting imine, 5, can either h ydrolyzeor rearrange by a proton shift to a new imine, 6. Hydrolysis of 5 or 6 gives analdehyde and an amine.There is an important biochemical counterpart of the deamination reaction thatutilizes pyridoxal phosphate, 7, as the aldehyde. Each step in the sequence iscatalyzed by a specific enzyme. The a-amino group of the amino acid combineswith 7 and is converted to a keto acid. The resulting pyridoxamine then reactsto form an imine with a different a-keto acid, resulting in formation of a newa-amino acid and regenerating 7. The overall process is shown in Equation 25-6and is called transamination. It is a key part of the process whereby amino acidsare metabolized.

pyridoxal phosphateRCHC02H+ RfCC02H < ' RCC02H+ R'CHC02H ( 25 -6 )I II I l Inzymes


20/81

25-6 Synthesis of a-Amino Acids 1225The biochemical process occurs with complete preservation of the L configura-tion at the a carbon. The same reactions can be carried out nonenzymaticallyusing p yridoxal phosphate, but they are nonstereospecific, require metal ionsas a catalyst, and give mixtures of products.

OHIO=P-0-c~~OH pyridoxal phosphate, 7IOHCH3

25-6 SYNTHESIS OF a-AMINO ACIDS

Many of the types of reactions that are useful for the preparation of aminoacids have been discussed previously in connection with separate synthesesof carboxylic acids (Chapter 18) and amino compounds (Chapter 23). Ex-amples include the S,2 displacement of halogen from a-halo acids by ammonia,

and the Strecker synthesis, which, in its first step, bears a close relationship tocyanohydrin formation (Section 16-4A):

0

cN Q - ~ - c = P JC H O NH, + HCN --+H@,H 2 0

NH2

Other general synthetic methods introduce the a-amino acid grouping,H2N-CH-C02H, by way of enolate anions. Two selected examples follow.INotice that in each a carbanion is generated and alkylated. Also the H,N-group is introduced as a protected amide or imide group.


21/81

1226 25 Amino Acids, Peptides, Proteins, Enzymes, and Nucleic Acids1 . phthalimidomalonic ester synthesis

heat H2NNH2-C02 >

2. N-formylaminomalonic ester synthesis

-IW-C Na0C2H5, CH2=CHCN

0 H,o@, -CO,I IH-C

The key step is the base-catalyzed addition of CH,=CHCN, which is aMichael addition (Section 18-9D).


22/81

25-7 Pep t ides and Proteins 1227With those amino acids that are very soluble in water, it usually is

necessary to isolate the product either by evaporation of an aqueous solutionor by precipitation induced by addition of an organic solvent like alcohol.Difficulty may be encountered in obtaining a pure product when inorganicsalts are coproducts of the synthesis. The best general method for removal ofinorganic salts involves passage of the solutions through columns of suitableion-exchange resins (Section 25-46).

The products of laboratory syntheses, starting with achiral reagents, areof course racemic a-amino acids. T o obtain the natural amino acids, the D,Lmixtures must be resolved (Section 19-3).

Exercise 25-14 Show how the fol lowing am ino acids may be prepare d from the indi-cated method and starting materials:a. glutamic acid from 2-oxopentanedioic acid (a-ketoglutaric acid) by the Streckermethodb. leucine from 2-methyl-I-propanol by the phthalimidomalonic ester synthesisc. aspart ic acid from ethyl chloroethanoate by the N-formylaminomalonic estersynthesisExercise 25-15 Suggest a synthetic route to proline from hexanedioic acid (adipicacid) that involves the transformations -C02H ---. NH2, and -CH 2C0 2H to-CH BrC 02H . Specify the reagents required to accom pl ish each step.

25-7 PEPTIDES AND PROTEINS25 -78 Classif icationAmino acids are the building blocks of the polyamide structures of peptidesand proteins. Each amino acid is linked to another by an amide (or peptide)bond formed between the NH, group of one and the 6 0 , H group of the other:

0 0 0 0I1C

INMCHCOH

I I> H,NCN-C-NHCHCOHI I I

In this manner a polymeric structure of repeating amide links is built into achain or ring. The amide groups are planar and configuration about the C-Nbond is usually, but not always, trans (Section 24-1). The pattern of covalent


23/81

9 228 25 Amino Acids , Pept ides, Proteins, Enzymes, and Nuc leic A cidsbonds in a peptide or protein is called its primary structure:

The distinction between a protein and a peptide is not completely clear.One arbitrary choice is to call proteins only those substances with molecularweights greater than 5000. The distinction might also be made in terms ofdifferences in physical properties, particularly hydration and conformation.Thus proteins, in contrast to peptides, have very long chains that are coiledand folded in particular ways, with water molecules filling the voids in the coilsand folds. Hydrogen bonding between the amide groups plays a decisive rolein holding the chains in juxtaposition to one another, in what is sometimescalled the secondary and tertiary structure.Wnder the influence of heat, or-ganic solvents, salts, and so on, protein molecules undergo changes, often ir-reversibly, called denaturation. The conformations of the chains and the degreeof hydration are thereby altered, with the result that solubility and ability tocrystallize decreases. Most importantly, the physiological properties of theprotein usually are destroyed permanently on denaturation. Therefore, if asynthesis of a protein is planned, it would be necessary to duplicate not onlythe amino-acid sequences but also the exact conformations of the chains andthe manner of hydration characteristic of the native protein. With peptides, thechemical and physiological properties of natural and synthetic materials usuallyare identical, provided the synthesis duplicates all of the structural and con-figurational elements. What this means is that a peptide automatically assumesthe secondary and tertiary structure characteristic of the native peptide oncrystallization or dissolution in solvents.

Representation of peptide structures of any length with conventionalstructural formulas is cumbersome. As a result, abbreviations are universallyused that employ three-letter symbols for the component amino acids. It isimportant that you know the conventions for these abbreviations. The twopossible dipeptides made up of one glycine and one alanine are

glycylala nine (Gly-Ala) alanylg lycine (Ala-Gly)Notice that in the conventions used for names and abbreviated formulas theamino acid with the free amino group (the N-terminal amino acid) always iswritten on the left. The amino acid with the free carboxyl group (the C-termi-

T h e di st inc tion be tween secondary and te r ti a ry s t ruc tu re i s no t sharp . Seconda rys tructure involves cons iderat ion of the in teract ions and spat ia l re la t ionships of theamino acids in the pept ide chains that are close together in the pr imary s t ructure ,whereas t e r t i a ry s t ruc tu re i s concerned wi th those tha t a re fur apart in the pr imarys tructure .


24/81

25-7B Determination of Amino-Acid Sequencesnal amino acid) always is written on the right. The dash between the three-letter abbreviations for the acids designates that they are linked together byan amide bond.

Exercise 25-16 The structure of the hormonal peptide oxytocin is abbreviated toI~ y s - ~ y r - l i e - G l n - ~ s n - c i s - p r o - ~ e u - G I Y N H , . Draw its ful l co valent structure

25-78 Determination of Am ino-Acid SequencesThe general procedure for determining the primary structure of a peptide orprotein consists of three main steps. First, the number and kind of amino-acidunits in the primary structure must be determined. Second, the amino acids atthe ends of the chains are identified, and third, the sequence of the componentamino acids in the chains is determined.The amino-acid composition usually is obtained by complete acid hy-drolysis of the peptide into its component amino acids and analysis of themixture by ion-exchange chromatography (Section 25-4C). This procedure iscomplicated by the fact that tryptophan is destroyed under acidic conditions.Also, asparagine and glutamine are converted to aspartic and glutamic acids,respectively.Determination of the N-terminal acid in the peptide can be made bytreatment of the peptide with 2,4-dinitrofluorobenzene, a substance veryreactive in nucleophilic displacements with amines but not amides (see Sec-tion 14-6B). The product is an N-2,4-dinitropheny derivative of the peptidewhich, after hydrolysis of the amide linkages, produces an N-2,4-dinitrophenyl-amino acid:

O2N - ! - C H 2 - C 0 2 H + amino acidsN-2,4-dinitrophenylglyc~ne(low solubil ity in acid solutions)


25/81

4 230 25 Amino Acids, Pept ides, Proteins, Enzymes, and Nuc leic Ac idsThese amino-acid derivatives can be separated from the ordinary amino acidsresulting from hydrolysis of the peptide because the low basicity of the 2,4-dinitrophenyl-substituted nitrogen (Section 23-7C) greatly reduces the solu-bility of the compound in acid solution and alters its chromatographic behavior.The main disadvantage to the method is that the entire peptide must be de-stroyed in order to identify the one N-terminal acid.A related and more sensitive method makes a sulfonamide of theterminal NH, group with a reagent called "dansyl chloride." As with 2,4-dinitrofluorobenzene, the peptide must be destroyed by hydrolysis to releasethe N--sulfonated amino acid, which can be identified spectroscopically inmicrogram amounts:

dansyl chlor ide

q/ + amino acidsS 0 2 N H C H C 0 2 HIli

sulfonated N-terminal acid

A powerful method of sequencing a peptide from the N-terminal end isthe Edrnan degradation in which phenyl isothiocyanate, C,W,N=C=S,reacts selectively with the terminal amino acid under mildly basic conditions.If the reaction mixture is then acidified, the terminal amino acid is cleavedfrom the peptide as a cyclic thiohydantoin, 8:


26/81

25-7B Determ ination of Am ino-Acid Sequences

a thiohydantoin 8

The advantage of the Edman procedure is that the residual peptide after onedegradation now has a new N-terminal amino acid that can react further withphenyl isothiocyanate. In practice, it is possible to carry out sequential Edmandegradations by an automated procedure that identifies each of the amino acidsin sequence from the N-terminus as a thiohydantoin. Figure 25-6 illustrateshow the procedure works. If the N-terminal nitrogen is not a free amino group,but for example is an ethanoylamide, CH,CONH--, the Edman degradationdoes not proceed.

There are simple reagents that react selectively with the carboxyl termi-nus of a peptide, but they have not proved as generally useful for analysis ofthe C-terminal amino acids as has the enzyme cnrboxypept idnse A . This en-zyme catalyzes the hydrolysis of the peptide bond connecting the amino acidwith the terminal carboxyl groups to the rest of the peptide. Thus the aminoacids at the carboxyl end will be removed one by one through the action of theenzyme. Provided that appropriate corrections are made for different rates ofhydrolysis of peptide bonds for different amino acids at the carboxyl end ofthe peptide, the sequence of up to five or six amino acids in the peptide canbe deduced from the order of their release by carboxypeptidase. Thus a se-quence such as peptide-Ser-Leu-Tyr could be established by observing thatcarboxypeptidase releases amino acids from the peptide in the order Tyr,Leu, Ser:

carboxypeptidasepep tide-Ser-Leu-T yr1-12 0 > peptide-Ser-Leu + Tyr

-+ eptide-Ser + Leu + Tyr -+ peptide + Ser + Leu + TyrDetermining the amino-acid sequences of large peptides and proteins is verydifficult. Although the Edman degradation and even carboxypeptidase can beused to completely sequence small peptides, they cannot be applied successfullyto peptide chains with several hundred amino acid units. Success has beenobtained with long peptide chains by employing reagents, often enzymes, toselectively cleave certain peptide bonds. In this way the chain can be brokendown into several smaller peptides that can be separated and sequenced. Theproblem then is to determine the sequence of these small peptides in theoriginal structure. To do this, alternative procedures for selective cleavagesare carried out that produce different sets of smaller peptides. It is not usuallynecessary to sequence completely all of the peptide sets. The overall amino-acid composition and the respective end groups of each peptide may suffice to


27/81

25 Amino Acids, P eptides, Proteins, Enzymes, and Nu cleic A cids

rest of peptide chain

S\\C-NHI \,N ,CH-CH, + residual peptideC6H5 IIFigure 25-6 Result of a series of Edman degradations on an N-terminalGly-Tyr-His-Ala-peptide

show overlapping sequences from which the complete amino-acid sequencelogically can be deduced.The best way to show you bow the overlap method of peptide sequencingworks is by a specific example. In this example, we will illustrate the use ofthe two most commonly used enzymes for selective peptide cleavage. Oneis trypsin, a proteolytic enzyme of the pancreas (MW 24,000) that selectivelycatalyzes the hydrolysis of the peptide bonds of basic amino acids, lysine and


28/81

25-7B Determinat ion of Amino-Acid Sequencesarginine. Cleavage occurs on the carboxyl side of lysine or arginine:

M Lys-GIy- >H2O - ys + Gly-trypsinArg-Gly- ~0 > r\A Arg + Gly-

Chyrnotiypsin is a proteolytic enzyme of the pancreas (MW 24,500) that cata-lyzes the hydrolysis of peptide bonds to the aromatic amino acids, tyrosine,tryptophan, and phenylalanine, more rapidly than to other amino acids. Cleav-age occurs on the carboxyl side of the aromatic amino acid:

chymotrypsinF-"-.. Phe-Ala H2.0

>w he + AlaOur example is the sequencing of a peptide (P) derived from partial hydrolysis

of a protein which, on complete acid hydrolysis, gave Ala, 3 Gly, Glu, His,3 Lys, Phe, Tyr, 2 Val, and one molar equivalent of ammonia.

1. Treatment of the peptide (P) with carboxypeptidase released alanine, andwith 2,4-dinitrofluorobenzene followed by hydrolysis gave the 2,4-dinitrophenylderivative of valine. These results establish the N-terminus as valine and theC-terminus as alanine. The known structural elements now areP =Val Ala

2. Partial hydrolysis of the peptide (P) with trypsin gave a hexapeptide, atetrapeptide, a dipeptide, and one molar equivalent of lysine. The peptides,which we will designate respectively as M, N, and 0 , were sequenced by Edmandegradation and found to have structures:Gly- Ala 0Val-Tyr-Glu-Lys NVal-Gly-Phe-Gly-His-Lys M

With this information, four possible structures can be written for the originalpeptide P that are consistent with the known end groups and the fact that trypsincleaves the peptide P on the cnrboxyl side of the lysine unit. Thus

3. Partial hydrolysis of the peptide P using chymotrypsin as catalyst gavethree peptides, X, Y , and Z. These were not sequenced, but their amino-acidcomposition was determined:Gly, Phe, Val XGly, His, Lys, Tyr, Val YAla, Glu, Gly, 2 Lys Z


29/81

25 Amino Acids, Peptides, Proteins, Enzymes, and Nucleic AcidsThis information can be used to decide which of the alternative structures

deduced above is correct. Chymotrypsin cleaves the peptide on the carboxylside of the phenyialanine and tyrosine units. Only peptide M contains Phe,and if we compare M with the compositior~s f X, Y, and Z, we see that onlyX and Y overlap with M. Peptide Z contains the only Ala unit and must be theC-terminus. If we put together these pieces to get a peptide, P' (which differsfrom P by not having the nitrogen corresponding to the ammonia formed oncomplete hydrolysis) then P' must have the structure X-Y-Z:

MI 7

P' = Val-Gly-~he;Gl~-His-~ys- yr- laThis may not be completely clear, and it will be well to consider the logic insome detail. Peptides M and N both have N-terminal valines, and one of themmust be the N-terminal unit. Peptide M overlaps with X and Y, and because Xand Y are produced by a cleavage on the carboxyl side of Phe, the X and Yunits have to be connected in the order X-Y. Because the other Val is in Y,the N-terminus must be M. This narrows the possibilities to

There are two Lys units in Z, and this means that only the sequence M-N-Lys-0 is consistent with the sequence X-Y-Z, as shown:

The final piece of the puzzle is the placement of the mole of ammonia re-leased from the original peptide on acid hydrolysis. The ammonia comes froma primary amide function:

The amide group cannot be at the C-terminus because the peptide would thenbe inert to carboxypeptidase. The only other possible place is on the side-chaincarboxyl of glutamic acid. The complete structure may be written asP = Val-Gly-Phe-Gly-His-Lys-Val-Tyr-Glu-Lys-Lys-Gly-Ala

INH,

Exercise 25-17 What problem s m igh t be encountered in using the 2,4-dinitrof luoro-benzene method for determination of en d group s on Gly-Lys-Ala? Exp lain.


30/81

25-7B Determinat ion of Amino-Acid SequencesExercise 25-18 The tripeptide, eisenine, has only one free carboxyl group, doesnot react with 2,4-dinitrof luorobenzene, and on complete hydrolysis yields 2 molesof L-glutamic acid, 1 mole of L-alanine, and 1 mole of ammonia. Alanine is indicatedto be the C-terminal amino acid. Write a structure for eisenine that is in accord withthe above facts.Exercise 25-19 * Eledo isin is a pe ptid e isolated from the salivary glands of eledone,a Mediterranean eight-armed cephalopod. The peptide is a powerful hypotensiveagent. Ded uce a pos sible structure from the fol lowing information: (1) Com pletehydrolysis gives equal amounts of ammonia, Ala, Asp, Glu, Gly, Ile, Leu, Lys, Met,Phe, Pro, and Ser. (2) No free amino N-terminal group or free carboxyl C-terminalgroup can be detected. (3) Chymotrypsin hydrolysis forms two peptides, L and M.Their composit ions areL = Aia, As p, Glu, Lys, Phe, Pro, Ser (unsequenced)

M = I le-Gly-Leu-MetNH, (sequenced)(At this point you should be able to ded uce the sequence of f ive amino acids at theC-terminus of eledoisin.) (4) Trypsin hydrolysis gives two pep tides, P and Q, with theindicated composit ions:P = Glu, L ys, Pro, SerQ = Ala, Asp , Gly, Ile, Leu, Met, Phe(At this point, you can ded uce two possible sequences for Q.) (5) Trypsin hydrolysisof L gives a pe ptide of com posit ion Ala, Asp, Phe which, with 2,4-dinitrof luorobenzene,gives the 2,4-dinitrophenyl derivative of asp art ic ac id. (6) Partial acid hydrolysis ofeledo isin gives several dipe ptid es , among them Ser-Lys and Pro-Ser.Exercise 25-20 A hexapeptide was subjected to the transformations diagramm edbelow. (The com mas between the amino acids indicate the sequence is unknown orunspecif ied.) Ded uce the structure of the hexapeptide.

hexapeptide HCI Arg + Gly + Lys + Pro + Trp + Val + NH,I I boil for 24 hrcarboxypeptidase + no reaction

(Arg-Trp) + (Gly, Lys, Pro, Val)

'0 no reaction(Gly-Lys) + (Pro-Val) + (Lys-Pro) + NH,


31/81

25 Amino Acid s, Peptides, Proteins, Enzymes, and Nuc leic Ac idsUsing procedures such as those outlined in this section more than 100 proteinshave been sequenced. This is an impressive accomplishment considering thecomplexity and size of many of these molecules (see, for example, Table 25-3).It has been little more than two decades since the first amino acid sequence of aprotein was reported by F. Sanger, who determined the primary structure ofinsulin (1953). This work remains a landmark in the history of chemistrybecause it established for the first time that proteins have definite primarystructures in the same way that other organic molecules do. Up until that time,the concept of definite primary structures for proteins was openly questioned.Sanger developed the method of analysis for N-terminal amino acids using 2,4-dinitrofluorobenzene and received a Nobel Prize in 1958 for his success indetermining the amino-acid sequence of insulin.

25-7C Me thods for Forming Pep tide BondsT h e problems involved in pe pt ide synthe ses a re of much pract ical im por tanceand hav e received con sidera ble atten tion. T h e major diff iculty in putt ing to-geth er a chain of say 100 am ino acids in a particular or de r is on e of overallyield. A t least 100 separa te s ynth etic s tep s would be required and , if th e yieldin ea ch s te p were equal to n X 100% , the overall yield would be (nloO 100%).If the yield in each step were 90%, the overall yield would be only 0.003%.Ob vious ly, a practical laborato ry sy nthes is of a peptide chain m ust be a highlyeff icient process. T h e ex traord inary abili ty of l iving cells to achieve synth esesof this na ture, no t of just on e but of a wide variety of such su bstan ces, istruly impressive.Several metho ds for the format ion of amide bonds have been d iscussedin Sect ions 18-7A and 24-3A. The most general react ion is shown below, inwhich X i s so me reac t ive leaving group (see Table 24-1) :

X N HR'W hen applied to coupling t w o different am ino acids, diff iculty is to be exp ecte dbecause these same react ions can l ink two amino acids in a to ta l o f fourdifferent ways. Thus if we star ted with a mixture of glycine and alanine, wecould generate four d ipeptides , Gly-Ala , Ala-Gly , Gl y-G ly , and Ala-Ala.T o avoid unw anted coupl ing react ions a pro tect ing group is subs t i tu tedon the amino funct ion of the acid that is to act as the acyla ting agent . Fur the r -more, al l of the amino, hydroxyl, and thiol functions that may be acylated tog ive undes ired p roducts usual ly m ust be pro tected . Fo r ins tance, to synthes ize


32/81

25-7C Methods for Fo rming Peptide BondsCly-Ala free of other possible dipeptides, we would have to protect the aminogroup of glycine and the carboxyl group of alanine:

n INHCH2-C-NHCH-C-peptide) 1bond CH3

In general, peptide synthesis is a sequence of steps involving (a) protection offunctional groups, (b) conversion of the carboxyl group to a more reactivegroup, (c) coupling, and (d) removal of the protecting group, as shown inFigure 25-7.

Some methods of protecting amine and hydroxyl functions were dis-cussed previously in Sections 23-13 and 15-9, respectively. A summary ofsome commonly used protecting groups for NH,, OH, SH, and C 02Hfunc-tions is in Table 25-2, together with the conditions by which the protectinggroups may be removed. The best protecting groups for NIP, functions arephenylmethoxycarbonyl (benzyloxycarbonyl) and tert-butoxycarbonyl. Bothgroups can be removed by treatment with acid, although the tert-butoxycar-bony1 group is more reactive. The phenylmethoxycarbonyl group can be re-moved by reduction with either hydrogen over a metal catalyst or with sodiumin liquid ammonia. This method is most useful when, in the removal step, itis necessary to avoid treatment with acid:

II / HCl(CH3)3C-0-C+NHCHRC02H ----+tert- butoxycarbony j (CH3),@=CH2 + C 0, + H,NCHRCO,H

0 / I H c l CGH5CH2Cl C0 ,I/ /C6H5CH2-0-C:+NHCHRCO,H + H,NCHRCO,HIphenylmeth- /oxycarbonyl I Pt > CGH,CH3 +CO,or Na, NM,(l)

In most cases, formation of the ethyl ester provides a satisfactory protectinggroup for the carboxyl function.


33/81

25 Amino Acids, Peptides, Proteins, Enzymes, and Nucleic Acids

Irotection of

amino groupINHCHC-OHIformation of

-C-X

protection ofcarboxyl group

INHCH-C-X I I+ H2NCH-CI I

-HX coupling1protecting 1 1 11 protecting-NHCHC-NHCHC-group I I group

removal ofprotecting groups

Figure 25-7 Sequence of reactions for forming a peptide bond from twodifferent amino acids. The same type of procedure can be used to makepeptide bonds between two peptides or between an amino acid anda peptide.

Conv ersion of th e carboxyl group to a mo re reactive group and couplingare key s teps in peptide synthesis . The coupling reaction must occur readilyan d quantitatively, an d with a minimum of racemization of th e chiral cen ter sin the molecule. This last criterion is the Achilles ' heel of many possiblecoupling sequences. The importance of nonracemization can best be appre-ciated by a n example. C onsider synthesis of a tr ipeptide from three protectedB AL-amino acids, A, B, an d C , in two sequential coupling s teps , C -+ B-C -+A-B-C. Su pp ose tha t th e coupling yield is quantitative, bu t the re is 20%formation of the D isomer in the acylating com pon ent in each coupling s tep.


34/81

25-76 Methods for Forming Peptide Bonds

I,003 , 7?"g E I gjElu_mL & a ,I O f Z Z

II l o "o=o O"00 q , z 10, I c 5 yO n ,f2 1 . 5w -

(I)Q$ I0 -X0+-3I)

Ca,-0+PQ

cnm w-cv r j- -a r -

(I)a ,3E 1.? 10Q)4-

s :a '-2 If i :3 l


35/81

4 240 25 Amino Acids, Peptides, Proteins, Enzymes, and Nucleic AcidsThen the tripeptide will consist of a mixture of four diastereomers, only 64%of which will be the desired L,L,Ldiastereomer (Equation 25-7):

This is clearly unacceptable, especially for longer-chain peptides. Nine cou-pling steps with 20% of the wrong isomer formed in each would give only 13%of the decapeptide with the correct stereochemistry.

Exercise 25-21 How could an opt icaj ly pure N-acylamino acid racemize and leadto racem ic N-a cylpep t ides as the result of a pept ide coup l ing react ion wherein thecarboxyl group of the amino acid was converted to an anhydride group? (ReviewSection 25-5A.)Exercise 25-22 Suppose there is 1% formation in each step of the wrong isomer ofthe acylating com ponent in an otherwise quantitative 100-step pe ptide synthes is.What is the yield of the desired polypeptide isomer?

The most frequently used carboxyl derivatives in amide coupling areazides, RCO-N,, mixed anhydrides, RCO- 0-CORr ,and esters of moder-ately acidic phenols, RCO-OAr (see Table 24-1). It also is possible to couplefree acid with an amine group using a diimide, R-N=C=N-R, mostfrequently N,N -dicyclohexylcarbodiimide.

The diimide reagent may be thought of as a dehydrating agent. The "elementsof water" eliminated in the coupling are consumed by the diimide to form a


36/81

25-76 Methods for Forming Pept ide Bondssubstituted urea. T h e overall reaction is

-IIR-C-OH + H 2 N R 1+

I

This reaction takes place because diimides, -N=C=N-, have reactive\cumulated double-bond systems like those of ketenes, C=C=O; isocy-/anates, -N=C=O; and isothiocyanates, -N=C=S; and are susceptible

to nucleophilic attack at the central carbon. In the first step of the diimide-coupling reaction, the carboxyl function adds to the imide to give an acyl inter-mediate, 9. This intermediate is an activated carboxyl derivative RCO-Xand is much more reactive toward an amino function than is the parent acid.The second step therefore is the aminolysis of 9 to give the coupled productand N, N I-dicyclohexylurea:

C:]

Afte r com pletion of a coupling reaction, and before anoth er amino acidcan b e added to the N-terminu s, it is necessary to remo ve the protecting group.Th is must b e do ne by selective reactions that do not destroy the peptide bondsor side-chain protecting groups. This part of peptide synthesis is discussed inSection 23-1 3, and som e reactions useful fo r removal of the N-terminal pro-tecting group s are summ arized in Tab le 25-2.


37/81

4242 25 Amino Acids, P ept ides, Proteins, Enzymes, an d N ucle ic Ac ids

A cha in S SI IGly-lle-Val-G lu-GI n-Cy-Cy-Ala-Se r-Val-Cy-Ser-Leu-Tyr-Gln-Leu-G lu-Asn-Tyr-Cy-AsnI I

B cha i n s sI IPhe-Val-Asn-Gln-His-Leu-Cy-Gly-Ser- His- Leu -Val-G lu- Ala- Leu-Tyr-Leu-Va l-Cy 1

Figure 25-8 Amino-ac id seque nce in beef insul in. The A chain of 21amino-acid residues is l inked to the B chain of 30 residues by way oftwo d isul f ide b r idges.

In spite of the large number of independent steps involved in the syn-thesis of even small peptides, each with its attendant problems of yield, racemi-zation, and selectivity, remarkable success has been achieved in the synthesisof large peptides and certain of the smaller proteins. The synthesis of insulin(Figure 25-8) with its 51 amino acid units and 3-disulfide bridges has beenachieved by several investigators. Several important hormonal peptides,namely glutathione, oxytocin, vasopressin, and thyrotropic hormone (seeFigure 25-9) have been synthesized. A major accomplishment has been thesynthesis of an enzyme with ribonuclease activity reported independently bytwo groups of investigators, led by R . Hirschman (Merck) and R. B. Merrifield(Rockefeller University). This enzyme is one of the simpler proteins, havinga linear structure of 124 amino-acid residues. It is like a peptide, not a pro-tein, in that it assumes the appropriate secondary and tertiary structure withoutbiochemical intervention (Section 25-7A). As a specific example of the strategyinvolved in peptide synthesis, the stepwise synthesis of oxytocin is outlinedin Figure 25-10, using the abbreviated notation in common usage.

25-7D Solid-Phase Peptide SynthesisThe overall yield in a multistep synthesis of a peptide of even modest size isvery poor unless each step can be carried out very efficiently. An elegantmodification of classical peptide synthesis has been developed by R . B. Merri-field, which offers improved yields by minimizing manipulative losses thatnormally attend each step of a multistage synthesis. The key innovation is toanchor the C-terminal amino acid to an insoluble support, and then add amino-acid units by the methods used for solution syntheses. After the desired se-quence of amino acids has been achieved, the peptide can be cleaved from thesupport and recovered from solution. All the reactions involved in the synthesismust, of course, be brought to essentially 100% completion so that a homo-geneous product can be obtained. The advantage of having the peptide anchoredto a solid support is that laborious purification steps are virtually eliminated;solid material is purified simply by washing and filtering without transferringthe material from one container to another. The method has become knownas solid-phase peptide synthesis. More of the details of the solid-phase synthesisfollow.


38/81

25-7D Solid-Phase Peptide Synthesis 1243SH Glutathione (GSH) is widely distr ibuted in cel lI t issue. I ts biological function is not completelyC0,H 0 ,I ' 3 2 0 1II / I II / understood but i t is thought to be a coenzymeH2NCHCH2CH2C ; NHCH-C ; NHCH2C02H for a Cannizzaro-type reaction interconverting

GSH watery-glutamyl [ cysteinyl-g lycine glyoxal a nd lactic acid , CH,COCHOd

I CH,CH(OH)CO,H. GSH is very easily oxid ize d byy-G u I CY G ~ Y - Hglutathione air to the disul f ide, 2GSHw GSSG. Noticealso that the pe ptide bon d to the glutamyl residue

is to C5, not to C1.

C ~ - A S ~ - G ' I ~IPro-Arg-Gly-NH,vasopressin

--I---O=C O CH2CONH2 0 (CHz),CONHzI I l l I I II ITY SCH,CHNH-C-CHNH+C-CHNHS I Asn ' G lnI ( ' ) 9 0II I

I I IVJSCH,CHC----~-- NHCHC--NHCHC

N H Tyr CH, I le ,C sn-Phe-His-Pro

His+ is-ProIf each coupling step proceeds in 80% yield, which of the two routes would give thehighest overall yield?Exercise 25-25 lndicate the steps that would be necessary to attach each of theamino acids l isted to the N-terminus of a peptide chain. Assume that any side-chainfunctions in the pe ptid e are suitably protected , but do not assume that the amino ac idswil l couple with the peptide without suitable protection of their functional groups.a. lysine b. aspart ic acid c. cystine d. serineExercise 25-26 Show how each of the following substances may be synthesizedstart ing with the individual amino acids. lndicate the reagents needed in each step.a . glutam ylglycine (Glu-Gly) b. Tyr-Ala-Val


43/81

1248 25 Amino Acids, Pept ides, Proteins, Enzymes, and Nuc leic Ac ids25-7E Separation of Pe ptides an d Proteins

In many problems of peptide sequencing and peptide synthesis it is necessary tobe able to separate mixtures of peptides and proteins. The principal methodsused for this purpose depend on acid-base properties or on molecular sizes andshapes.

Ultracentrifugation is widely used for the purification, separation, and molec-ular-weight determination of proteins. A centrifugal field, up to 500,000 timesthat of gravity, is applied to the solution, and molecules move downward in thefield according to their mass and size.

Large molecules also can be separated by gel filtration (or gel chromatog-raphy), wherein small molecules are separated from large ones by passing asolution over a gel that has pores of a size that the small molecules can penetrateinto and be trapped. Molecules larger than the pore size are carried on withthe solvent. This form of chromatographic separation is based on "sieving"rather than on chemical affinity. A wide range of gels with different pore sizesis available, and it is possible to fractionate molecules with molecular weightsranging from 700 to 200,000. The molecular weight of a protein can be esti-mated by the sizes of the pores that it will, or will not, penetrate. The chemicalstructure of a gel of this type is described in Exercise 25-27.

The acid-base properties, and hence ionic character, of peptides and proteinsalso can be used to achieve separations. Ion-exchange chromatography, similarto that described for amino acids (Section 25-4C), is an important separationmethod. Another method based on acid-base character and molecular sizedepends on differential rates of migration of the ionized forms of a protein inan electric field (electrophoresis). Proteins, like amino acids, have isoelectricpoints, which are the pH values at which the molecules have no net charge. Atall other pH values there will be some degree of net ionic charge. Becausedifferent proteins have different ionic properties, they frequently can be sepa-rated by electrophoresis in buffered solutions. Another method, which is usedfor the separation and purification of enzymes, is affinity chromatography,which was described briefly in Section 9-2B.

Exercise 25-27* A resin known as Sephadex that is useful in ge l filtration is prepare dfrom a polysaccharide that is cross-linked into a three-dimensional matrix with

0/ \"epichlorohydrin," CH,-CH-CH,CI . The degree of cross-linking determines thepore size of the gel. Write equations, specifying the con ditions as clos ely as poss ible,for reactions whereby a glucose unit of one polysaccharide chain co uld b e l inke d tothe glucose of another chain through an epichlorohydrin molecule.Exercise 25-28 Hemoglobin, the protein responsible for carrying oxygen from thelungs to the body t issues, contains 0.355% iron. Hydrolysis of 100 g of hemoglobingives 1.48 g of tryptophan. Calculate the minimum molecular weight of hemoglobinthat is consistent with these results.


44/81

25-8 Structure an d F unction of Proteins 8249

25-8 STRUCTURE AND FUNCTION OF PROTEINSThe biological functions of proteins are extremely diverse. Some act ashormones that regulate various metabolic processes. An example is insulin,which regulates blood-sugar levels. Enzymes act as catalysts for biologicalreactions, and other proteins serve as biological structural materials-forexample, collagen and elastin in connective tissue and keratin in hair. Iron-containing proteins (hemoglobin and myoglobin in mammals) and copper-containing proteins (hemocyanins in shellfish) transport molecular oxygen.Some blood proteins form antibodies, which provide resistance to disease,while the so-called nucleoproteins are important constituents of the genesthat supply and transmit genetic information in cell division. Motion by meansof muscle contraction and the generation and transmission of nerve impulsesalso involve proteins.How can a group of compounds, made from a common basis set ofamino acids, be so remarkably heterogeneous and exhibit such varied yetspecific functions? Clearly, the primary structure and the presence or absenceof special functional groups, metals, and so on, are of paramount importance.Of complementary importance are the t h r e e - d i m e n s i o n a l s t r u c t u r e s of proteins,which are dictated not just by the primary structure but by the way the pri-mary structure is put together biochemically. The polypeptide chains areseldom, if ever, fully extended, but are coiled and folded into more or lessstable conformations. As a result, amino-acid side chains in distant positionsin the linear sequence are brought into close proximity, and this juxtapositionoften is crucial for the protein to fillfill its specific biological function.

25-8A Three -Dim ensional Structure of ProteinsThe elucidation of the detailed s h a p e of protein molecules-in fact, the spatiallocations of the individual atoms in a protein-is accomplished primarily byx-ray crystallography. The three-dimensional structures of more than twentyproteins have now been established by this technique. The importance of x-raycrystallography to structural and biological chemistry has been recognized inthe award of six Nobel Prizes in this area.6 A number of important proteinsand their properties are listed in Table 25-3.

T h e fol lowing Nobel laurea tes rece ived the ir awards for contributions to the use ofx-ray crystallography fo r s tru cture determination: 19 14, M ax von Lau e (phy sics) ,diffraction of x rays in cry stals; 191 5, William Bragg and L aw ren ce Bragg (physics),s tudy of crystal s tructure by means of x rays ; 1 954 , Linus Pauling (chemistry), s tudy ofs t ruc ture of prote ins ; 1962, Max Perutz and John Kend rew (chemis t ry) , s t ruc tures ofmyoglobin and hemog lobin; 196 2, Fran cis C rick, Jam es Watson, and M aurice Wilkins(physiology and m edicine), double helix of D N A ; 1964, Do roth y Hodgkin (chemistry),determ ination of str uctu re of vitamin B,, a nd penicillin by x-ray method s. Sh e laterdetermined the three-dimensional s tructure of insulin.


45/81

Table 25-3A Few Important Proteins of Known Structure

Name Approx. Amino Disulfide Prosthetic lsoelectricMW acids bonds group point Occurrence Function

insulinri bonucleasemyoglobinhemoglobin

cytochrome c

lysozyme

17,800 153 - heme64,500 a-141" - heme 6.7P-146

a-chymotrypsin 24,500 241

carboxypeptidase A 34,600 307

pancreaspancreasmusclered bloodcorpusclesall cells

egg white

pancreas

pancreas

regulation of bloodsugar levelsenzyme that hydrolyzesRNArespiratory protein; storesO2 in muscle tissuerespiratory protein; trans-ports 0, from lungs; trans-ports CO, to lungsrespiratory protein;electron carrier foroxidative phosphorylationcenzyme that breaks downthe cell walls of bacteria byhydrolysis of P(1 -+ 4)glycoside linkagesdigestive enzyme; hydro-lyzes ester and peptidebondsdigestive enzyme; hydro-lyzes carboxyterminalpeptide bond in proteins

"Hemoglobin has four subunits, two a chains and two P chains, each with a heme group.bCytochrome c has two cysteine units covalently bonded to the two ethenyl side chains of the heme group:protein-SH + CH2=CH-heme ---+ protein-S-CH2CH,-heme"Oxidative phosphorylation is the process in which ATP is formed as electrons are transferred (by way of the cytochromes) from NADH orFADH, to 0,. For example, ' 120 , + NADH + H@- 20+ NAD@, nd the energy from this process is used to synthesize ATP (see Section20-10).


46/81

25-8 Structure and Function of proteins

-0 ydrogenbonds

Figure 25-11 Peptid.e chain of a protein coiled to form a right-handedalpha helix. Configuration of the helix is maintained by hydrogen bonds,shown as vertical dotted (or solid) l ines. The helix on the left shows thedetailed atom structure of the peptide chain. The helix on the right is aschematic representation without structural detail.

An especially favorable conformation of a polypeptide chain that wasoriginally deduced by Pauling and Corey is the alpha helix (Figure 25-1 1).The principal feature of the a helix is the coiling of the polypeptide chain in\ /such a way as to form hydrogen bonds of the type N-II . . .O=C between/ \

amide N-H and amide carbonyl groups that are four amino-acid units apart.The coiling is possible because the chain can twist about the C,-C and C,-Nsingle bonds of most amino acid units, as shown in Figure 25-12.There are several other points to notice about the a helix shown inFigure 25-1 1. The amide groups are planar and normally retain the stabletrans configuration in the helical structure; bond lengths and bond angles are


47/81

25 Amino Acids, Peptides, Proteins, Enzymes, and Nucleic Acids

Figure 25-12 Ball-and-stick model of a pept ide unit showing the co-planarity of the CNCC atoms of the amide linkage, here in the trans con-figuration, and the possibility of rotation about the C-C,, and N-C, bonds.

\ /normal, and the N H . . - O = C hydrogen bonds ar e nearly linear. How eve r,/ \the hydrogen bonds are n ot q uite parallel to the long axis of the coil, so the reare 3.6 rather than 4 amino-acid units per helical turn, and the spacing be-tween turns is about 5.4 A. T h e a helix in proteins has right-handed turns likea right-hand screw thread.T h e a mino acids of the side chains lie outside the coil of the a helix andare in c lose proximity to th e side chains three and four amino-acid units apart.Becau se of this proximity, steric hindrance b etween larger side cha ins can b esufficient to reduce the stability of the normal a helix. When such hindranceoccu rs, there is a d iscontinuity in the helical structure , and the peptide ch ainmay assum e mo re random arrangem ents about the C-C, and N-C, bonds(see Figure 25-12), thereby allowing the molecule to fold back on itself andform new hydrogen bonds. The helical structure apparently is always inter-rupted at proline or hydroxyproline residues because the C,-N bonds ofthese am ino acids ar e not free to rotate (they are incorporated in five-memberedrings) and also because the proline and hydroxyproline amide nitrogens haveno hydrogens to participate in hydrogen bonding to carbonyl groups.Pauling and Corey recognized a second stable conformation of poly-peptide chains- he e xten ded ch ain o r p-pleated shee t (Figure 25- 13). In thisconformation the chains are fully extended with trans amide configurations.In this arrangement the distance is maximized between adjacent amino-acid

\ /side chains. Hydrogen bonding of the type N-H. . O =C is now between/ \


48/81

25-8 Structure and Function of Proteins

tiH 0 H R H H , ,I II I/ N \ ~ / c \ ~ / C \ ~ / N \ ~ / C \ ~ / ' C \ ~ / N \/$ I 11 ' x , I 11H R H 0 H R H 0

Figure 25-13 Hyd rogen -bond ed structure of si lk f ibroin. Notic e that thepe ptid es run in different directions in alternate cha ins. This structure iscal led an ant iparal le l p-pleated sheet.

chains rather than between amino acids in a single chain (as in the a helix).Th is type of s t ruc ture is not a s common as the a helix a nd is foun d extensive lyonly in silk fibroin. However, a number of proteins with a single polypeptidechain ca n form s ho rt sections of "antiparallel" p-pleated s hee ts by foldingback on them selves, a s i llustrated in Figure 25-14.Another very important factor in protein archi tecture is the disulf ide-S-S- l ink. Remote parts of the polypeptide chain can be held close to-gether through the oxidat ive coupling of two cysteine thiol groups to form adisulfide bridge:


49/81

25 Amino Acids, Peptides, Proteins, Enzymes, and Nuc leic Ac ids

Figure 25-14 Diagrammatic representat ion of the coi l ing of a proteinchain showing areas of (a) a helix, (b) p-pleate d sheet, and (c) randomcoi l ing

Su ch -S-S- bridges grea tly restrict th e num ber of con form ations availa bleto a protein and ar e of fundam ental importance in determining the shap e of aprotein, and hence, i ts biological activity. Lysozyme, which can be isolatedfrom hen egg-white, provides an excel lent example. This substance is anenzyme that catalyses hydrolysis of the glycoside links in polysaccharideco m po ne nts of bacterial cell walls. It is a relatively small prote in of 129 am inoacid units in a single polypeptide chain that is cross-linked by four disulfidebridges (Figure 25- 15). It b eco m es inac tive if the disulfide bridges a re cleavedo r combined in oth er combinations than th e ones shown.The disulfide bridges in some proteins are between different peptidechains. Insulin, for instance, h as tw o interchain a s well as on e intrachain S-Sbridges (Fig ure 25-8).

25-8B Myoglobin and Hemoglob inSome idea of the complexity of protein conformations can be gained fromthe s tructure of myoglobin. This protein is responsible for the s torage and


50/81

25-8 S tructure and Fun ction of P roteins

Figure 25-15 Lysozyme from hen egg-white showing the amino-acidsequence (primary structure) and the four intrachain disulfide bridges.[Adapted from D. C. Phill ips, Sci. Amer. 5, 215 (1966).]


51/81

25 Amino Acids, Peptides, Proteins, Enzymes, and Nu cleic Ac ids

Figure 25-16 A model of myoglobin to show the way in which the poly-peptide chain is coi led and folded. The shaded sections correspond toregions in which the chain is coi led into an a helix. Each fold, and theregions near the C-terminus an d the N-terminu s, represent discon tinuitiesin the h elical structure. The position of the heme group is represented bythe disclike shape.

transport of molecular oxygen in the muscle tissue of mammals. It is a compactmolecule of 153 amino-acid units in a chain that is extensively coiled as ana helix. There are eight regions of discontinuity in the helical structure, andin these regions the chain folds on itself as shown in Figure 25-16. Four ofthe eight nonhelical regions occur at proline residues; the reason for the dis-continuity at the other regions is not entirely clear. With the exception of twohistidine units, the interior regions of myoglobin accommodate only the non-polar side chains; the interior, therefore, is mostly hydrocarbonlike and re-pellent to water and other polar molecules. In contrast, the polar side chainsare on the exterior of the protein.

A number of proteins, including myoglobin, possess one or more nonpeptidecomponents associated with specific sites on the polypeptide chain. Thesecomponents are called prosthetic groups and are essential to the biologicalactivity. When the prosthetic group is removed, the residual protein is referredto as an apoprotein.

In myoglobin the prosthetic group is a molecule of heme. The heme groupbelongs to a class of interesting compounds called metatlloporphyrins, whichare metal complexes of a highly conjugated ring system composed of fourazacyciopentadiene (pyrrole) rings linked by -CH= bridges between the2 and 5 positions. The parent compound is known as porphin. Porphyrins have


52/81

25-8B Myoglobin and Hemoglobin 1257highly stabilized electronic excited states and absorb visible light. As a resultthey usually are brightly colored compounds (e.g., chlorophyll, Figure 20-6).

porphin metalloporphyrinM = Fe, Cu , Mg, Zn, Cr, and other metals

The porphyrin of heme is known as protoporphyrin IX, and the associatedmetal is iron [as Fe(I1) or Fe(III)]. You will notice that the porphyrin ringcarries methyl, ethenyl, and propanoic acid side chains:

protoporphyrin IX

HCH2=CH CH,

heme

A major effort on the part of several eminent chemists in the early part of thecentury led to the elucidation of the structure of heme. The German chemistHans Fischer successfully synthesized heme in 1929, a feat for which, in 1930,


53/81

25 Am ino Ac ids, Pep t ides, Proteins, Enzymes, and Nu cleic A cidshe received the Nobel Prize in chemistry. [Some years earlier (19 15), RichardWillstatter received a Nobel Prize for structural studies of chlorophyll andplant pigments.]A very important question is, how does the particular combination of pro-

tein and iron-porphyrin allow myoglobin to reversibly bind molecular oxygen?The answer to this question is not known in all its details, but it is well estab-lished that Fe(I1)-porphyrins will complex readily and reversibly with oxygen.There are two additional coordination sites around the iron in heme besidesthe four ring nitrogens. These are indicated below as the general ligands L:

The disclike heme molecule fits into a cleft in the protein structure and isbound to it through one of the L coordination sites to a histidine nitrogen. Theremaining coordination site on the other side of the ring is occupied by molecu-lar oxygen. In the absence of the coordination by histidine, the porphyrin ironwould be oxidized rapidly to the ferric state, which does not bind oxygen.

A number of model compounds have been synthesized which have Fe(I1)-porphyrin rings carrying a side chain with histidine arranged to be able tocoordinate with the metal on one side. Several of these substances showpromise as oxygen carriers with properties similar to myoglobin.

Hemoglobin is related to myoglobin in both its structure and function. Itreversibly binds molecular oxygen which it transports in the red corpusclesof blood rather than in muscle tissue. However, hemoglobin is made up of fourpolypeptide chains, in contrast to myoglobin which has only one chain. Two ofthe hemoglobin chains are of one kind with 143 amino acid residues, called thea! chains, and two are of another kind with 146 amino acids, called the ,8 chains.Each chain, or s~~buni t ,ontains one heme group identical with the heme inmyoglobin. The subunits are held in the hemoglobin by noncovalent interactionsand provide four hemes and hence four binding sites for molecular oxygen. Thea! and p hemes have different affinities for oxygen but function in a cooperativeway to increase oxygen availability to the cells.

arrangeme nt of the four cha ins andfour heme group s o f hemog lob in


54/81

25-8C Quaternary Structures of Proteins 4 259In spi te of the fac t tha t the a and ,8 chains of hemoglobin a re nonidentica l

with the myoglobin chain, the three-dimensional s t ructures of al l three chainsare st r ikingly similar; myoglobins and hemoglobins differ s l ight ly in aminoacid composi t ion, depending on the spec ies , but the prote in shape remainsessent ial ly the same.

25-8C Quaternary Structures of ProteinsMany factors contribute to the three-dimensional structures of proteins. Wealready have mentioned hydrogen bonding between am ide grou ps, locationand character of prosthetic groups, and disulfide bonds. Other important in-fluences include electrostatic interactions between ionic groups (-NH3@,-COZe), hydrogen-bonding involving side-chain substitue nts (-CH,O H),and nonbonded interactions. Except for the disulfide linkages, most of theseinteractions ar e weak c om pare d to covalent bond strengths, and the conforma-tions of m any prote ins ca n be altered rathe r easily. In fac t, several have confor-mations tha t clearly a re in dynam ic equilibrium un der physiological conditions.Such structural flexibility may be necessary for the protein to be functional,but if the conformation is altered irreversibly- that is, if it is denatured - tsbiological activity usually is destroyed.In many cases there are important interactions between protein mole-cules that may lead to highly organized structures such as the pleated sheetof silk fibroin (Figure 25-1 3) o r the coiling of a helices, a s found in a-ke ratins,the fibrous proteins of hair, horn, and muscles (Figure 25- 17 ) . This sort oforganization of protein molecules is called quaternary structure and is an im-portant feature of many proteins that associate into dimers, tetramers, andso on. Th e tetrame ric structure of hemoglobin is an important example.

Figure 25-17 Representation of the quaternary structure of a-keratinshowing (a) three a-hel ical polypept ide strands coi led into a rope and(b) eleven units of the three-stranded rope arrange d to form one micro-f ibr i l


55/81

11260 25 Amino Acid s, Pept ides, Proteins, Enzymes, and Nu cleic A cidsENZYMES

Virtually all biochemical reactions are catalyzed by proteins called enzymes.The catalytic power and specificity of enzymes is extraordinarily high. Thereactions that they catalyze are generally enhanced in rate many orders ofmagnitude, often as much as lo7, over the nonen:zymatic process. Conse-quently enzymatic reactions may occur under much milder conditions thancomparable laboratory reactions. For example, the simple hydrolysis of anamide proceeds at a practical rate only on heating the amide in either stronglyacidic or strongly basic aqueous solution, and even then reaction may not becomplete for several hours. In contrast, hydrolysis of amide or peptide bondscatalyzed by typical proteolytic enzymes, such as trypsin, chymotrypsin, orcarboxypeptidase A, occurs rapidly at physiological temperatures and physio-logical pH.7 It is one of the remarkable attributes of many enzymes that theycatalyze reactions that otherwise would require strongly acidic or basic con-ditions. Enzymes are strictly catalysts, however, and affect only the rate ofreaction, not the position of equilibrium; they lower the energy of the transi-tion state, not the energies of the reactants or products (see Figure 4-4).

Many enzymes appear to be tailor-made for one specific reaction in-volving only one reactant, which is called the substrate. Others can functionmore generally with different reactants (substrates). But there is no such thingas a universal enzyme that does all things for all substrates. However, nothingseems to be left to chance; even the equilibration of carbon dioxide with wateris achieved with the aid of an enzyme known as carbonic an hydra~e .~learly,the scope of enzyme chemistry is enormous, yet the structure and function ofrelatively few enzymes are understood in any detail. We can give here only abrief discussion of the mechanisms of enzyme action-first some generalprinciples then some specific examples.

25-9A Aspe cts of the Mec han ism s of Enzyme ReactionsAn enzyme usually catalyzes a single chemical operation at a very specificposition, which means that only a small part of the enzyme is intimately in-volved. The region of the enzyme structure where key reactions occur as the

7The slowness with which amide bonds are hydrolyzed in the presence of either strongacids or strong bases, and their susceptibility to hydrolysis under the influence of en-zymes, clearly is a key advantage in the biological functioning of peptides. Amidehydrolysis in neutral solution has a favorable, but not large, equilibrium constant.Therefore it does not take a great deal of biochemical energy either to form or tohydrolyze peptide bonds. The resistance to ordinary hydrolysis provides needed sta-bility for proteins, and yet when it is necessary to break down the peptide bonds ofproteins, as in digestion, this can be done smoothly and efficiently with the aid of theproteolytic enzymes.8Many enzymes are named by adding the suffix -rise to a word, or words, descriptiveof the type of enzymatic activity. Thus, esternses hydrolyze esters, proteinnses hydro-lyze proteins, reductnses achieve reductions, and synthetases achieve syntheses ofpolypeptide chains, nucleic acid chains, and other molecules.


56/81

25-9A Aspects of the Mechanisms of Enzyme Reactions

lockandkey

induced f i t

Figure 25-18 Illustration of the lock-and-key concept of enzyme-sub-strate interaction (top) and of the induced-fit theory, whereby the enzymemolds to the substrate through conformational changes (bottom)

result of association of the substrate with the enzyme is called the active site.The initial association of the enzyme (E) and the substrate (S) is formation ofan enzyme-substrate complex (ES):

Complexation could occur in many different ways, but for the intimate com-plexation required for catalysis, the enzyme must have, or must be able toassume, a shape complementary to that of the substrate. Originally, it wasbelieved that the substrate fitted the enzyme somewhat like a key in a lock;this concept has been modified in recent years to the induced-fit theory,whereby the enzyme can adapt to fit the substrate by undergoing conforma-tional changes (Figure 25-18). Alternatively, the substrate may be similarlyinduced to fit the enzyme. The complementarity is three-dimensional, an im-portant factor in determining the specificity of enzymes to the structure andstereochemical configuration of the substrates.

Detailed structures for the active sites of enzymes are difficult to obtainand have been worked out only for a few enzymes that have been studiedextensively by both chemical and x-ray methods. Very revealing informationhas been obtained by x-ray diffraction studies of complexes between theenzyme and nonsubstrates, which are molecules similar to actual substratesand complex with the enzyme at the active site, but do not react further. Thesesubstances often inhibit reaction of the normal substrate by associatingstrongly with the enzyme at the active site and not moving onward to products.The x-ray studies of enzymes complexed with nonsubstrates show that theactive site generally is a cleft or cavity in the folded structure of the enzymethat is largely hydrophobic in character. The enzyme-substrate complex can


57/81

1262 25 Am ino Ac ids, Pept ides, Proteins, Enzymes, and Nu cleic Ac idsbe inferred to be held together largely by van der Waals attractive forces be-tween like groups (Section 12-3C), hydrogen-bonding, and by electrostaticattraction between ionic or polar groups. To achieve a stereospecific catalyzedreaction, there must be at least three points of such interactions to align prop-erly the substrate within the cavity of the enzyme.

The reaction of the ES complex may convert the substrate to product(P) directly, and simultaneously free the enzyme (E) to react with more ofthe substrate:

However, the reaction between enzyme and substrate often is much morecomplex. In many cases, the substrate becomes covalently bound to theenzyme. Then, in a subsequent step, or steps, the enzyme-bound substrate(ES') reacts to give products and regenerate the active enzyme (E):

25-9B Carboxypept idase AThe considerable detail to which we now can understand enzyme catalysis iswell illustrated by what is known about the action of carboxypeptidase A. Thisenzyme (Section 25-7B and Table 25-3) is one of the digestive enzymes of thepancreas that specifically hydrolyze peptide bonds at the C-terminal end. Boththe amino-acid sequence and the three-dimensional structure of carboxy-peptidase A are known. The enzyme is a single chain of 307 amino-acid resi-dues. The chain has regions where it is associated as an a helix and otherswhere it is associated as a 6-pleated sheet. The prosthetic group is a zinc ionbound to three specific amino acids and one water molecule near the surfaceof the molecule. The amino acids bound to zinc are His 69, His 196, and Glu 72;the numbering refers to the position of the amino acid along the chain, with theamino acid at the N-terminus being number 1. The zinc ion is essential for theactivity of the enzyme and is implicated, therefore, as part of the active site.

X-ray studies%f carboxypeptidase complexed with glycyltyrosine (with whichit reacts only slowly) provide a detailed description of the active site, which isshown schematically in Figure 25-19a and is explained below.

1. The tyrosine carboxylate group of the substrate is associated by electro-static attraction with the positively charged side chain of arginine 145 (W) :

V.N . Lipscomb, Accounts of Chemical Research 3, 8 1 (1970);E. T. Kaiser and B. L.Kaiser, ibid. 5, 219 (1972). Lipscomb received the 1976 Nobel Prize in chemistry forstructural work on boranes.


58/81

25-9B Carboxypept idase A

Figure 25-19 Steps in a possible mechanism of carboxypept idaseaction. (a) The substrate is shown complexed to the enzyme surfacethrough X, Y, Z, and W ; X is a nonpolar pocket; Y is a hydrogen bond,possibly f rom OH of Tyr 248; Z is the prosthetic group, Zn; and W is an@ionic intera ction with =NH2 of Arg 145. The C-terminal am ide bo nd of thesubstrate is held close to the catalytic site, which is the carboxyl of Glu270. (b) A tetrahedral intermediate could be formed by attack of Glu 270carboxylate anion at the amide carbonyl of the substrate. (c) Cleavage ofthe tetrahedral interme diate of (b) releases the C-terminal am ino ac id andforms an acy l-enzym e interme diate. (d) The residue of the substrate chainis released from the enzyme by hydrolysis of the acyl-enzyme inter-med iate. These dra win gs are defic ien t in that they try to reprodu ce a three-dimensional situation in two dimensions. The third dimension is espe-cial ly important in understanding the stereospecif icity of the enzyme.

2. The tyrosine side chain of the substrate associates with a nonpolar pocketin the enzyme (X).

3. Hydrogen bonding possibly occurs between the substrate tyrosine amideunshared pair and the side-chain HO groups of the enzyme tyrosine

Documents

Bpo c Chapter 25