Chemical studies of enzyme active sites.

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CHEMICAL STUDIES OF ENZYME

x904

Annu. Rev. Biochem. 1975.44:889-931. Downloaded from www.annualreviews.org by ILLINOIS STATE UNIVERSITY on 11/23/12. For personal use only.

ACTIVE SITES David S. Sigman Department of Biological Chemistry and Molecular Biology Institute, University of California at Los Angeles School of Medicine, Los Angeles, California 90024

Gregory M ooser Department of Biochemistry, University of Southern California School of Dentistry, Los Angeles, California 90033

CONTENTS INTRODUCTION. OXmOREDUCTASES .. Lactate Dehydrogenase and Lactate Oxidase. Monoamine Oxidase and a-Amino Acid Oxidase.

TRANSFERASES. Thymidylate Synthetase. Choline-O-Acelyltramferase and Acelyl-CoA: A ryl umine N-Acelyllransjerase.

f3-0xOQcyl-CoA Thiolase..

Creatine Kinase.. Aspartate Transcarbamylase ..... Aspartate Aminotransferase. HYDROLASES .. Serine Proteases..

Endopeptidases

Endopeptidases- Acid Proteases . Exopeptidases-Carboxypeptidases. Phospholipases and Lipases .

Nucleases-Ribonuclease A . N ucleases- Deoxyribonucleases .

LYASES. .... .. Pyridoxal 5-Phosphate-Dependent Decarboxylases . Tryptophan Synthetase. Non-Pyridoxal Phosphate-Dependent Decarboxylases . .

Fructose 1.6-Diphosphate Aldolase-Class I .. {i-Hydroxydecanoyl Thioesler Dehydrase.

ISOMERASES. Triose Phosphate Isomerase. Malldelate Racemase.

LIGASES Aminoacyl-tRNA Synthetase. CONCLUDING REMARKS. .

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890 891 891 892 895 895 897 899 900 901 902 905 905 907 909 912 914 915 916 916 917 917 918 920 922 922 924 924 924 925 889

890

SIGMAN & MOOSER


INTRODUCTION The primary focus of the present review will be the use of chemical modification of proteins as a technique for investigating enzyme mechanism. Although chemical modification has historically played a central role in understanding biological catalysis, the recent contributions of X-ray crystallography, magnetic resonance techniques, stereochemistry, and kinetics have tended to'dilute the reliance on chemical modification in the elucidation of enzymic mechanisms. However, in many cases, chemical modification still provides the best available evidence for defining the structure of essential intermediates or identifying catalytically important residues. In all cases, chemical modification can confirm suggestions originated by other techniques of mechanistic investigation. There are several different types of chemical modification experiments that have revealed the presence and/or function of reactive groups at the active sites of enzymes. They include the use of group-specific reagents, affinity labels, pseudo substrates, suicide substrates, the trapping of reactive intermediates, deletion of amino acid residues from the carboxyl or amino termini, and the chemical synthesis of either peptide fragments or entire proteins in which an amino acid of interest is replaced by another at a given position in the sequence. The intrinsic information content of these approaches varies. For example, the trapping of reactive intermediates probably provides the greatest insight into a catalytic mechanism in the absence of a large body of information regarding the mechanism of action of the target enzyme. Reactions of pseudo substrates and suicide substrates also possess high inherent information content. Pseudosubstrates react with their target enzyme via mechanisms with strong homologies to the enzyme-catalyzed reaction. Suicide substrates, which like pseudo substrates do not react readily with free amino acids, are transformed by the enzyme's catalytic site into highly reactive species which then modify active site amino acid residues. On the other hand, site-specific modifications with group-specific reagents or affinity labels often do not provide unambiguous information by themselves as to the mechanism of catalysis. Even though numerous examples of stoichiometric modi fications of active sites by group-specific reagents and affinity labels are available, in many cases it is still difficult to determine if the residue modified is involved in catalysis. The loss of biological activity is often not due to alteration of a vital catalytic group. It can be due to either the disruption of the conformation of the active site or steric hindrance resulting from the introduction of a bulky substituent. But in the context of an X-ray structure, for example, results from these approaches to the chemical modification of proteins can provide invaluable insights. Recent contributions of various chemical modifications to the elucidation or confirmation of the mechanism of action of different enzymes will be discussed below. By necessity, coyerage cannot be exhaustive. The most glaring area of omission is the many important and exciting contributions in the study of NAD/ NADP-dependent dehydrogenases. An attempt has been made to avoid treatment of subjects included in two recent excellent reviews of enzyme mechanisms in this

CHEMICAL STUDIES OF ENZYME ACTIVE SITES

891

series (1, 2). Many general and laboratory-oriented monographs and reviews on the chemical modification of proteins are available (3-20).

OXIDOREDUCTASES


Lactate Dehydrogenase and Lactate Oxidase

Several irreversible inhibitors of flavoenzymes have been recently studied in which the enzyme transforms a relatively unreactive molecule into a potenrinhibitor via a mechanism closely analogous to that occurring in the normal catalytic process. A full understanding of the mechanism of action of these suicide substrates on their target enzymes promises to reveal important features of these enzymes' catalytic mechanism and at the same time permit the design of extraordinarily specific inhibitors which might be of pharmacological value (21). Three different enzymes that catalyze the oxidation of lactate, bakers' yeast L-( + )-lactate dehydrogenase (cytochrome b2) (22), D-lactate dehydrogenase from Escherichia coli (23), and L-lactate oxidase from Mycobacterium smegmatis (24) are inhibited by a racemic mixture of 2-hydroxy-3-butynoate (I). Only the enantiomeric H- C = C

0

HO -

C C I

-

H

_

06

form of 1 that is consistent with the stereochemistry of the target enzyme serves as a specific inhibitor. Lactate oxidase from M. smegmatis. which catalyzes the reaction summarized in equation 1, is inactivated by 1 under either aerobic or OH I

e

R-C-C02 I

H

+

02

E-FMN

e

RCOO

+

CO2

+

H20

1.

anaerobic conditions. The flavin spectrum o f the modified holoenzyme is characteristic of the reduced form of the coenzyme. Confirmation of the flavin as the site of modification by I was provided by the demonstration that all the radioactivity incorporated into the holoenzyme by (4_ 3H) I was associated with the coenzyme after the flavin was resolved from th� apoprotein. Although the precise structure of the flavin adduct was not determined, the lack of any radioactivity incorporation into the coenzyme when enzyme was inactivated with (2_ 3H) I suggests that loss of the IX-proton precedes the formation of inactive enzyme. The absence of an exchangeable acetylenic proton in the flavin adduct suggests that the acetylenic linkage has been altered in the irreversibly inhibited product (24). An important feature of the reaction of I with lactate oxidase is that under anaerobic conditions a stoichiometric amount of I, based on the assumption that only the L form is reactive, is sufficient to completely inactivate the enzyme. However, under aerobic conditions, some oxygen consumption is observed prior

892

SIGMAN & MOOSER

to complete inactivation of the enzyme. Structure I is therefore both a substrate and an inhibitor of lactate oxidase. Since the amount of I consumed prior to the complete inactivation of the enzyme depends on the concentration of oxygen, the parti tioning of a common intermediate (see 2-lIb) between oxidation and irreversible inactivation is suggested. One possible kinetic scheme consistent with both reactions is indicated in equation 2. It includes an intermediate (2-III) implicit in a previous


E-FMN

+

9H

0

H-CEC-C-COO

H

Inactive

enzyme

0,

�

/ 8

__

OH 0 I " E-FMN-H-C5! c-c-c-oe

II

...

2-na

H .., 9H ? 6 E-FMN-C=C=C-C-O

®

'iT"

? ?

2. e

E-FMNH2-H-C;:C-C-C-O .- III

E-FMN

I

+

CO2

+

H 20

+

o "

H-C5C-C-0e

description (24). The isolatable flavin derivative obtained from lactate oxidase could arise from: 1. nucleophilic attack on the incipient allene (2-IIb) (or its fully protonated form in which another proton is added to C-4) derived from an enzyme-bound carbanion (2-IIa) (process A, equation 2) in analogy to the reaction of acetylenic inhibitors with [3-0H-decanoyl thioester dehydrase (25); or 2. rapid nucleophilic attack by reduced flavin at the acetylenic moiety of 2-keto-3-butynoic acid (process B, equation 2). This latter possibility would imply an as yet unreported spectro scopically identifiable transient intermediate in the inactivation reaction. A key question in the mechanism of action of flavoenzymes is whether or not electron transfer takes place via covalent intermediates (26). It is possible that some type of adduct exists in the overall process designated 2 in equation 2. If this is true, another pathway for the formation of inactive enzYIpe would be rearrangement of such a covalent adduct. No evidence is available to either support or exclude this possibility. The ability of lactate oxidase to dehydrohalogenate [3-chlorolactate to form pyruvate under anaerobic conditions and to form pyruvate and chloro pyruvate under aerobic conditions suggests the rate-limiting formation of a common carbanion intermediate (similar to 2-IIa in equation 2) in both processes, but does not exclude the existence of obligatory covalent intermediates for reduction of FMN to FMNH2 (27). Monoamine Oxidase and D-Amino Acid Oxidase

Both these flavoenzymes catalyze the oxidative deamination of amines (equation 3). The three readily identifiable partial reactions leading to the net reaction are 3. summarized in equations 4-6, where EnFI and EnFlH2 are the oxidized and reduced


893

forms of the flavin enzyme, respectively (28). The demonstration that the hydrolysis of the imine (equation 6) generated by oxidation (equation 4) takes place in solution R-CHz-NHz + EnFI .... R-CH=NH+EnFlHz

4.

EnFIHz+02 .... EnFI+ H202

5.


R-CH=NH+HzO-. RCHO+NH3

6.

is based on the production of equimolar amounts of lX-eH}-D- and L-methionine upon addition of eH}-NaBH4 to a reaction mixture composed of D-amino acid oxidase and D-methionine. Further evidence that the imino acid freely dissociates from the enzyme is that e-N-(l -carboxyethyl)-L-Iysine is generated by D-amino acid oxidase from o-alanine and L-Iysine in the presence of sodium borohydride at pH 8.3 (29). Apparently, IX-imino acids readily undergo transaldimination reactions with amines and the resulting Schiff bases are rapidly reduced by sodium boro h ydride. Previous workers have demonstrated that ('4 C) o-alanine is incorporated into o-amino acid oxidase as e-N(l-carboxyethyl)-L-Iysine in the presence of sodium borohydride (30), but in light of the facile transaldimination reaction, these results do not indicate the presence of an essential enzyme-bound Schiff base intermediate. In addition to the production of a rapidly dissociable IX-imino acid intermediate, formation of a carbanion, at least in the case of D-amino acid oxidase, seems essential prior to the reduction of the flavin (equation 4). In analogy to studies with lactate oxidase and p-chlorolactate, the most convincing evidence for this conclusion is the ability of the enzyme to catalyze elimination reactions using fi-chloro-L-alanine and IX-amino-fi-chlorobutyrate as substrates to yield pyruvate and a-ketobutyrate, respectively (3 1, 32). The spectra of tramients in the enzyme-catalyzed dehydrogena tion of a-amino-fi-chlorobutyrate resemble those of oxidized flavins. Since kinetic isotope studies reveal that their rate of formation depends on the cleavage of the IX-C�H bond, formation of the intermediate carbanion probably does not involve a flavin adduct. However, since a-amino-fi-chlorobutyrate is not oxidatively deaminated to fi-chloro-a-ketobutyrate, this does not exclude the formation of flavin adducts after carbanion formation. The D-amino acid oxidase-catalyzed oxidation of the carbanion of nitroethane has provided strong but not compelling evidence for adduct formation at N-5 (33). Several specific irreversible inhibitors of monoamine oxidase have been described whose mode of action depends on the prior generation of a reactive intermediate in an enzyme-catalyzed process. They can profitably be discussed in the context of the presumed mechanism of action of this enzyme, assuming its similarity to D-amino acid oxidase. 3-Bromoallylamine (II) is an effective inhibitor whose

II

894

SIGMAN

& MOOSER


7. suggested mode of action (equation 7) (34) parallels the postulated mechanism for the reduction of flavin by alcohols (26). In view of the unproven formation of covalent intermediates prior to production of the carbanion, the adduct suggested may not form. However, the central feature of the scheme, namely the enzyme catalyzed isomerization of the double bond to yield a potent alkylating agent capable of modifying a vicinal amino acid residue, is undoubtedly correct. One argument presented in support of the formation of a covalent adduct is that rapid dissociation of the inhibitor from the enzyme surface could lessen the likelihood of reaction. However, since the dissociation of products is a limiting step in catalysis by D-amino acid oxidase, covalent anchoring of the inhibitor may not be necessary for modification of the enzyme. One case where the anchoring of an enzyme-generated inhibitor is clearly un necessary for modification is the inhibition of monoamine oxidase by phenyl hydrazine (28). The enzyme reacts with phenylhydrazine under anaerobic conditions to yield a completely reduced flavin which, upon introduction of oxygen, is completely reoxidized with total restoration of activity. However, if oxygen is initially present, an irreversibly inhibited enzyme with reduced-type flavin spectrum, stable to autoxidation, is generated. Incubation with ( 1-14C)-phenylhydrazine yielded a protein where 1 .4 mol of inhibitor were incorporated per mole of flavin. Three lines of evidence suggest that phenyldiazene (III), the product formed upon

O

-N=N-H III

initial oxidation of phenylhydrazine, is the true inhibitor of monoamine oxidase. The most compelling is that III generated by in situ decarboxylation of phenylazo formate inhibits the enzyme to yield an inactive derivative that has the same flavin spectrum obtained from inhibition by phenylhydrazine. Secondly, the rates of oxidase inhibition by a series of p-substituted phenylhydrazines, yield a p value in a Hammett plot of - 1 .9. Phenylhydrazine reduction of the flavin is therefore the rate-determining step for inhibition. Finally, 2-phenylethylhydrazine, although a substrate, is a poor inhibitor because the diazene generated from it rapidly rearranges to yield phenylacetaldehyde hydrazone, which has independently been found to be an ineffective inhibitor.


895


Monoamine oxidase is also irreversibly inhibited by pargyline (N-benzyl-N methyl-2-propynylamine), an acetylenic compound presently used in antihyperten sive therapy. Pargyline inhibits monoamine oxidase in a 1: I stoichiometry and forms a stable flavin adduct (35, 36). Although the precise structure of the flavin adduct has not yet been determined, it is tempting to assume that monoamine oxidase catalyzes the formation of the carbanion of pargyline (IVa), which is reactive in its incipient allene form in analogy to the reaction of 2-0H-3-butynoate with lactate oxidase and lactate dehydrogenase from bakers' yeast and E. coli. The

IVa

IVb

inhibition of monoamine oxidase by pargyline provides indirect evidence that carbanion formation is as important for this flavoenzyme as it is in the others discussed.

TRANSFERASES Thymidylate Synthetase

The one carbon transfer from 5,1O-methylene tetrahydrofolate to deoxyuridine 5' phosphate to form thymidylic acid is catalyzed by thymidylate synthetase and is one of the slow steps in DNA synthesis (37). Since effective cancer chemotherapeutic agents such as 5-fluorouracil and 5-trifluoromethyl-2'-deoxyuridine inhibit thymidylate synthetase (38-43), the mechanism of inhibition of this enzyme by uracil analogs has been carefully studied. 5-Fluoro-2'-deoxyuridine 5'-phosphate (V) inhibition of thymidylate synthetase has been particularly intensively investigated. The enzymes most widely used in o

HN�F O� l. H

I(j)

o e 10 O-POC 2 Ie 0 o OH

v

these studies were isolated from an amethoptherin-resistant strain of Lactobacillus casei (44-47) or from T-2 bacteriophage-infected E. coli (48). All available evidence indicates that V is a suicide substrate which reacts with the enzyme via a mechanism closely analogous to the normal enzyme-catalyzed process. In the presence of 5,10methylene tetrahydrofolate, V forms an irreversible ternary complex composed of V, coenzyme, and enzyme. The absorption spectrum of the complex indicates that


896

SIGMAN & MOOSER

the 5,6 double bond in the pyrimidine is no longer present (44, 45, 47). The ternary complex is stable in guanidine hydrochloride (45, 48), sodium dodecylsulfate, and urea (46), and does not dissociate upon digestion with trypsin (47) or pronase (45). Therefore, a covalent bond between enzyme and V, which most likely arises from the nucleophilic addition of an amino acid residue to either the 5 or 6 position of V, must exist. The requirement of coenzyme for tight binding of V to the enzyme, coupled with the isolation of a single peptide containing both coenzyme and V (49), indicate that the coenzyme as well as the enzyme are covalently attached to V (47, 49). At present the precise structure of the adduct is unknown. However, since the 6 position of various uridine derivatives is very susceptible to nucleophilic attack (50-52), addition at this position is most likely responsible for the abolition of the double bond between C-S and C-6. Nucleophilic addition to C-6 should activate C-5 for electrophilic substitution. For example, model system studies have shown that 2',3'-O-isopropylidine uridine in alkaline solution exhibits a greatly enhanced rate of deuterium exchange at c-s in D20 relative to the corresponding S'-deoxy compound because of addition of the 5'-hydroxy group to the 6 position of the pyrimidine ring (53) (equation 8). The coenzyme, rather than a proton, must be the

H,/,N6 wlLNJ.O o

,

",\:JX

8.

electro phiIe capable of reacting with the nucleophilic center generated at C-5 as a consequence of the addition of active site amino acid residue to C-6 of V. At present, the form of the coenzyme that reacts is unclear, but the fluorescence and absorption spectra of the isolated peptide containing V and the coenzyme are characteristic of

CHEMICAL STUOIES

OF

ENZYME ACTIVE SITES

897


5-alkyltetrahydrofolates (49). However, before this spectral evidence is used as proof of the reactive form of the coenzyme, it should be noted that lO-methyltetrahydro folate, a potent competive inhibitor, facilitates the formation of a tight but non covalent complex with V, whereas S-methyltetrahydrofolate does not (47). In addition, the enzyme-10-methyltetrahydrofolate complex causes exchange of C-S protons of 2'-deoxyuridylate S'-phosphate (47). At present, the active site nucle op hilic amino acid that initially adds to C-6 is unknown. Even though the enzyme does possess an active site cysteine residue

whose modification by iodoacetamide is protected by V (47), the absence of a cysteine in the active site peptide containing covalently bound V excludes this amino acid as the enzyme-bound nucleophile that adds to C-6 (49). Since the only nucleophilic amino acid residues present in the isolated peptide are threonine and histidine (49), either a hydroxyl group or an imidazole residue adds to the C-6 position to initiate the formation of the irreversible complex. Details of the inhibi ti on of thymidyla te synthetase by V a re directly relevant to the mechanism of action of this enzyme. Apparently, the stability of the carbon fluorine bond effectively traps and causes accumulation of a complex of closely analogous structure to the steady-state intermediate. Although the general structure of the adduct or complex formed with V is likely represented by VI, the complete solution of its structure requires identification of the essential nucleophilic amino acid residue as well as the isomeric form of coenzyme bound to C-S of th e pyrimidine. HN

:+0

CH2- tetrohydrofolote

�N = O 0-�-OCH2 yO" 06 r OH e?

X (Thr, His, Alo, Leu, Pro2)

VI

Choline-O-Acetyltraniferase and Acetyl-CoA: Arylamine N-Acetyltransferase

Choline-O-acetyltransferase and acetyl-CoA : arylamine N-acetyltransferase, which catalyze the reactions summarized in equations 9 and 10, respectively, exhibit steady state kinetics and exchange patterns indicative of a covalent intermediate (54�57). choline + acetyl-CoA ...... acetylcholine + CoA

arylamine + acetyl-CoA ...... N-acetylarylamine + CoA

9.

10.

For both enzymes, the expected acetyl-enzyme intermediate has been isolated (56, 57). Because both enzymes are inhibited by sulfhydryl reagents (58, 59), the resistance of the isolated acetyl-enzyme intermediates to N-ethylmal eirnide in activation provides excellent evidence for a thioester structure of the essential intermediate (56, 60).


898

SIGMAN & MOOSER

The completc protcction of a reactive group by thc formation of a covalent intermediate permits the use of group-specific chemical modification reagents, such as iodoacetamide, to determine if the intermediate's formation or decomposition is rate determining under a given set of experimental conditions where all intermediates have achieved their steady-state concentrations. This technique, which is applicable to impure enzyme preparations, was originally devised in the study of papain (6 1 ) and has recently been applied to choline-O-acetyItransferase (60) and acetyl CoA ; arylamine-N-acetyItransferase (62). Studies on the latter enzyme were designed to confirm that, at saturating substrate concentrations, acylation is rate limiting with strongly basic aniline acceptors (e.g. p-toluidine), whereas deacylation is rate limiting with weakly basic anilines (e.g. p-cyanoaniline) (55). p-Nitrophenylacetate, which can be used as an acetyl donor, retards the rate of inactivation by iodoacetamide by 58% at concentrations as low as 1.7 x 10-8 M even though the observed Ks for this substrate in steady-state kinetic measurements is 8.6 x 1 0 - 3 M. Therefore, protection is not afforded by the reversible binding of substrate but must involve formation of the acetyl-enzyme intermediates as indicated in equation 1 1 . Addition of aniline acceptors, which by themselves do not protect o

!

-o-

E�SH +CH3� -O

0

!

N02:;;:>: E�S� �CH3 + H

a--Q-

N 02 1 1.

against iodoacetamide inactivation, decreases the extent of protection of the enzyme provided by p-nitrophenylacetate. The explanation for this loss of protection is that deacylation of the enzyme by aniline increases the concentration of the free enzyme, the form of the enzyme susceptible to inhibition. This observation suggests that the protected acetyl-enzyme is an intermediate in the enzymic reaction (62). As support for this qualitative conclusion, the ability of the various anilines to cause deprotection of the enzyme should show the same concentration dependence as the normal enzyme-catalyzed process. Therefore, when the second order rate constant for inactivation of the enzyme by iodoacetamide is measured as a function of aniline at a constant concentration of p-nitrophenylacetate, the concentration of aniline that provides 50% of the maximal deprotection should correspond to the apparent Km measured by steady-state kinetic techniques. Further, the extent of deprotection afforded by the various anilines at high concentrations should be a measure of the fraction of enzyme in the iodoacetamide-resistant acetyl-enzyme form under steady-state conditions. As a result, a low degree of deprotection indicates the accumulation of acetyl-enzyme and rate-limiting deacylation, whereas a large extent of deprotection indicates little acetyl-enzyme accumulation and rate limiting acylation. Since strongly basic amines effectively eliminate the p nitrophenylacetate protection towards iodoacetamide and weakly basic amines do not, the rate-limiting step for the enzyme reaction depends on amine structure in the manner predicted by steady-state kinetic measurements (55). The use of protection towards inactivation in the manner described above provides an additional way to investigate details of enzyme mechanisms using group-specific reagents.


899

f3-0xoacyl-CoA Thiolases

p-Oxoacyl thiolases catalyze the thiolytic cleavage of p-ketoacyl-CoA esters (equation 1 2). Enzymes differ with respect to their specificity to fJ-ketoacyl-CoA

o

0

II

II

R-CHz-C-CHz-C-S-CoA+CoA

0

o


�

II

II

CH3-C-S-CoA + R-CHz-C-S-CoA

1 2.

substrates (63). For example, enzymes isolated from yeast (64) and rat liver (65) are specific for acetoacetyl-CoA, but an enzyme isolated from pig heart is able to cleave longer chain p-ketoacyl-CoA esters (66). Steady-state kinetics and exchange reactions indicate that the reaction proceeds by a two step mechanism as indicated in equations 13 and 14 (65, 67-(9), where the acyl-enzyme intermediate is assumed to be a thioester in view of the sensitivity of the enzyme to thiol reagents and the

o

II

0

II

0

0

II

II

RCH2-C-CH2-C-S-CoA +HSE� RCH 2�C-SE + CH3-C-S-CoA 13.

o

II

0

II

R-CH2- C-SE+HS-CoA�R-CH2-C-S-CoA+HSE

14.

isolation of an active site peptide following inactivation of the enzyme with iodoacetamide (67, 70). In analogy to the studies of acetyl-CoA-arylamine trans ferase, the effective protecti on of rat liver cytoplasmic acetoacetyl-CoA thiolase by acetoacetyl-CoA with respect to inhibition by iodoacetamide strongly supports the formation of a thioester intermediate (65). Acetyl-CoA provides some protection, but desulpho-CoA by itself is ineffective in preventing inhibition by iodoacetamide. The pig heart enzyme is effectively inhibited by 3-pentynoyl-, 3-butynoyl-, 4bromocrotonyl-, and 2-bromoacetyl-CoA but is not readily modified by 2-butynoyl or 4-pentynoyl-CoA (66). As might be expected from the protection of the enzyme by acetoacetyl-CoA towards iodoacetamide inhibition, this substrate prevents in activation by all the above acyl-CoA esters, whereas acetyl-CoA only prevents modification by 4-bromocrotonyl- and 2-bromoacetyl-CoA. Since the products of modification by these various reagents have not been identified, it is quite possible that the active site cysteine is not the sole amino acid modified. The protection by exogenous thiols, such as dithiothreitol, against modification by these reagents indicates that all affinity labels are capable of reacting with thiol groups but does not indicate that a cysteinyl residue is modified (66). The inhibition by the three acetylenic derivatives is perhaps of greatest interest because, under the experimental conditions used, they exhibit the least reactivity to free glutathione but are still very effective inhibitors. In view of the ineffectiv e

900

SIGMAN

&

MOOSER

inhibition of the enzyme by 2-butynoyl- or 4-pentynoyl-CoA, unassisted addition of an active site nucleophile to the acetylenic inhibitor appears unlikely. If a basic group is vicinal to the ()(-carbon and capable of removing an ()(-proton, in analogy to the proposed mechanism of inhibition of other enzymes by acetylenic derivatives, the modification of pig heart thiolase could proceed through an incipient allene anion (equation 1 5), where X, the attacking nucleophile, could be the active site cysteine. However, since the appropriate protein chemistry has not been performed, this conclusion must be regarded as tentative (66).


I

B , 0 H HsC- C5C-C- C-SCoA H I

I

I

X

BH

)

�

HSC- c��-c- SCoA e

H

15.

Yeast and pig heart thiolases are inhibited by incubation of acetoacetyl-CoA in the presence of sodium borohydride (64, 66). These results have been interpreted as evidence for an amino group at the active site of the enzyme, whose function may be to form a Schiff base with the f3-keto group of the substrate, which is then transformed to product in several steps. The product of the sodium borohydride inactivation with acetoacetyl-CoA has not been determined. In view of the ability of acetyl-CoA to substitute for acetoacetyl-CoA and the reduction by sodium borohydride of an enzyme-y-glutamyl-CoA intermediate of succinyl-CoA: aceto acetate coenzyme A transferase (71 ), we feel a reasonable explanation for the sodium borohydride inhibition observed for thiolase is the reduction of the thioester acetyl-enzyme intermediate of thiolase to form the corresponding inactive thio hemiacetal form of the enzyme. In the absence of further work, therefore, the presence of a Iysyl residue at the active site should not be assumed (63). In this context, it is pertinent to note the recent use of sodium borohydride to trap acylphosphate intermediates (72). Creatine Kinase

Creatine kinase has an active site cysteine that is readily modified by many sulfhydryf reagents (73). Substrate protection is generally possible with appropriate substrate combinations (74-77). By itself, these data are not sufficient to imply an essential catalytic role for the sulfhydryl, since the introduction of a new group can alter the conformation and accessibility of substrates at the active site. Indeed, isozymic forms of the enzyme from a number of mammalian and nonmammalian sources have shown half-site reactivity of the native dimer relative to alkylation, but not to inactivation (78-8 1 ). A recent approach to the solution of this persistent problem in the interpretation of chemical modification results, at least for the modification of essential sulfhydryl groups, has been the introduction of two new reagents, VII and VIII, which permit the introduction of small, uncharged, and nonhydrogen bonding alkane thio (RS-) groups (82, 83) according to equations 16 and 1 7. The disulfides formed by


E-SH+R'-S02-S-R -> E-S--S-R + R'S02H

901

16.

VII o

II

E-SH + R'-O-C-S-SR -+ E-S--S-R + OCS + R'OH

17.


VIII modification with VII and VIII can bc fully reactivated by treatment with mercaptoethanol or dithiothreitol. The advantage of temporary masking of cysteinyl residues is that it allows the modification of other amino acid residues by group specific reagents which may have some partial reactivity with cystcinyl residues (3). Treatment of creatine kinases with VII (R R' CH3) results in modification of one cysteine residue per active site. Significantly, the modified enzyme retains 18% activity which cannot be decreased by treatment with iodoacetamide at con centrations sufficient to cause complete inactivation of the native enzyme (83). Complete activity can be restored upon incubation with mercaptoethanol. There fore, despite the fact that 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) can cause complete inactivation of the enzyme, the reactive cysteine of creatine kinase has no apparent essential catalytic role. A single e-amino group of lysine at the active site of creatine kinase is modified upon treatment with dansyl chloride (84). The magnetic resonance techniques of internuclear double resonance spectroscopy (INDOR) and the intermolecular nuclear Overhauser effect (NOE) were used to monitor the interaction of formate in both the native and dansylated enzymes (85), since previous magnetic resonance studies had shown that formate as well as nitrate bind tightly to an enzyme creatine-ADP complex in a position consistent with that of the transferring phos phoryl group (86, 87). A pos sible role for the protonated lysine residue would be to bind formate in the inactive complex presumed to be analogous to the transition state. A large NOE for formate was observed only in the presence of both creatine and ADP and indicated that the structure comparable to the transition state is achieved only in the presence of the full complement of substrate or substrate analogs. However, the disappearance of the NOE for formate in the modified enzyme, coupled with a chemical shift appropriate for the c-CHz group of lysine in the native enzyme as measured with the INDOR technique, suggests that formate and, by inference, the phosphoryl group interact with the c-amino group of lysine (85). Site-specific chemical modification, in conjunction with magnetic resonance tech niques, therefore provides insights not accessible with either technique alone. =

=

Aspartate Transcarbamylase

Aspartate transcarbamylase from E. coli. which catalyzes the synthesis of N carbamylaspartate from carbamylphosphate and aspartate, contains 12 peptide chains which may be dissociated into two trimeric cataly tic (C) subunits and three

dimeric regulatory(R) subunits. The C subunits can be isolated free of the R subunits and have full catalytic activi ty but do not respond to allosteric effector s (88). Each


902

SIGMAN & MOOSER

of the three peptides in the C subunit has a single cysteine residue that is sensitive to a limited number of sulfhydryl modification reagents. N-Ethylmaleimide, iodoacetic acid, and some organomercurials react slowly or not at all (89, 90). However, certain negatively charged reagents, induding DTNB, p-hydroxymercuri benzoate (89, 91, 92), 2-chloromercuri-4-nitrophenolate (93, 94), and potassium permanganate (90), react with the free sulfhydryl with concomitant inactivation. As might be expected, negatively charged reversible inhibitors of the enzyme, phosphate, and the aspartate analog, succinate, protect the enzyme against in activation by these sulfhydryl reagents. Despite the inactivation of the enzyme accompanying its modification, in analogy with the studies on creatine kinase discussed above, this cysteine residue is not directly involved in catalysis. This conclusion is based on an important and general method involving DTNB first used on aspartate transcarbamylase (89). The mixed disulfide composed of 5-thio-2-nitrobenzoate and cysteine, formed from DTNB and an enzymic sulfhydryl group, is susceptible to nucleophilic attack by cyanide, sulfite, and 2-mercaptoethanol or any organic thiol. Because 5-thio-2-nitrobenzoate is an effective leaving group, the nucleophiles will displace 5-thio-2-nitrobenzoate from the cysteine residue of the enzyme to yield either S-cyano, S-sulfo, or mixed disulfides. For aspartate transcarbamylase, the S-sulfo or S-2-mercaptoethanol derivatives are inactive, but the S-cyano derivative is fully active. The results with the small and uncharged S-cyano group clearly indicate no essential catalytic function for the sulfhydryl group modified (89). Pyridoxal 5-phosphate, but not pyridoxal, is an effective reversible inhibitor of aspartate transcarbamylase, which competes with carbamylphosphate (95). It forms a Schiff base with an active site lysine residue that has a characteristic absorption maximum at 430 nm. Three moles of pyridoxal 5-phosphate bind per trimer of catalytic subunit. Consistent with its observed behavior as a competitive inhibitor, the absorption maximum is lost upon addition of carbamylphosphate or the effective reversible inhibitor (N-phosphonacetyl)-L-aspartate. The efficacy of pyridoxal 5phosphate as a modification reagent, as opposed to pyridoxal, indicates that the phosphate moiety must interact specifically with the enzyme and that the Schiff base contributes only a part of the favorable free energy of binding. Many of the enzymes that are potently inhibited by pyridoxal 5-phosphate catalyze reactions involving phosphorylated substrates (3). Of particular interest for aspartate transcarbamylase is the ability of the Schiff base of pyridoxal phosphate to serve as a specific photosensitizing agent which, upon irradiation, yields an inactive enzyme derivative in which two histidine residues are destroyed per active site. These results demonstrate that a lysine residue and two histidine residues are located at the active center. Since photo-inactivated enzyme does not regain activity upon dissociation of pyridoxal 5-phosphate, at least one of the histidine residues oxidized probably has a direct role in catalysis (95). Aspartate Aminotransferase

The basic kinetic scheme of aspartate aminotransferase, a pyridoxal phosphate dependent enzyme, is summarized in equation 1 8 (96, 97). Aspartate aminotransferase

903


H

A-b-cooe H 0 II I e R-C-C-O I

'NH,

18-1

I N "

[3

18.

18- II


R ...

c II

",cooe

N

18- V

18 - I V

18- III

in mammalian tissue exists in at least two isozymic forms which differ in cellular location, kinetics, physiochemical, and immunochemical properties (98). One isozyme is of cytoplasmic origin and the other of mitochondrial origin. Both the mitochondrial and cytoplasmic enzymes are relatively insensitive to iodoacetate, iodoacetamide, a-bromopropionate, a-bromobutyrate, and bromosuccinate (99). However, J3-bromopropionate selectively inhibits the mitochondrial enzyme and not the cytoplasmic enzyme. The site of modification is the E-amino group of the lysine involved in coenzyme binding (100). An active site sulfhydryl in the pig heart cytoplasmic aspartate aminotransferase can be modified with concomitant inactivation by tetranitromethane, N ethylmaleimide, and DTNB ( 10 1-105). However, this residue does not appear to be critical for activity. The S-cyano derivative, prepared as described above for aspartate transcarbamylase (89), had 60% activity ( 106). The bulky or charged derivatives prepared with glutathione, sulfite, mercaptoethanol, or methanethiol had less than 20% activity (106). Experiments with aspartate aminotransferase have revealed that conformational changes during the course of catalysis can be reflected by changes in the reactivity of an amino acid residue to a group-specific reagent. The reactivity of the active site cysteine residue discussed above toward DTNB and other reagents is lowest in the free holoenzyme but increases slightly in the presence of a-methylaspartate and erythro-J3-hydroxyaspartate. The aldimine ( 1 8-11) accumulates with the former amino acid (107), whereas the quinoid intermediate ( 1 8-I1I) predominates with the latter (108, 109). Reactivity is enhanced two orders of magnitude in the presence of the substrate pair, glutamate and oc-ketoglutarate, after the equilibrium con centrations of all covalent intermediates are established. Therefore, the active site sulfhydryl, which plays no direct catalytic role, is most reactive in the ketimine form (l8-IV) to modification reagents such as DTNB (106, 1 10, 1 1 1 ).


904

SIGMAN & MOOSER

Bifunctional compounds such as bromopyruvate (1 12, 1 13), L-serine-O-sulfate (114), fJ-chloroalanine ( 1 1 5, 1 16), and fJ-bromoalanine (1 12) are potent irreversible inhibitors of the cytoplasmic and mi tochondrial forms of aspartate aminotransferase. The amino acid derivatives react with the pyridoxal form of the enzyme, whereas bromopyruvate is reactive with only the pyridoxamine form. These modification reagents also serve as substrates and yield pyruvate as product. Tn addition, transamination reactions have also been reported for ,B-chloroalanine ( 1 1 5) and L-serine-O-sulfate ( 1 14). The amino acids at the active site modified by ,B-chloroalanine and bromopyruvate are different. ,B-Chloroalanine modifies a lysine residue which can be isolated from the inactivated enzyme following sodium borohydride reduction as the lysine derivative, IX. A cysteine residue is modified by bromopyruvate (1 13) although

coo-

I

H3+N�CH

I

(CH 2)4

I

H

I

N�CH2�C�COO-

I

I

H

NH

I

CH2

I

� �N� IX because the appropriate protein chemistry has not been performed, it is not known if this is the same residue that is rapidly modified by DTNB in the presence of ex ketoglutarate and glutamate. The sequence of reactions presented in equation 1 9 summarizes the reaction o f bromo pyruvate with the enzyme. Although two pathways (A and B) could account for the modification of the cysteine residue, the identical maximal rates for pyruvate formation and the inactivation reaction indicate a common intermediate for both processes and suggest that pathway B is responsible for the alkylation of the sulfhydryl group. The sequence of steps most likely responsible for the inactivation reaction by fJ-chloroalanine is summarized in equation 20, where pathway B probably leads to the alkylation of the lysine residue. The lysine residue modified may be involved initially in the Schiff base linkage to the coenzyme in the holoenzyme and serve as a general base catalyst for the labilization of the ex-hydrogen of the substrate during the course of the reaction (97).


e a,CHZ -C-COO " SH H N Z

e a,CHZ -C-COO H N Z I CH

�

SH

CHz

I

-

o N

H

905

N

o N

+

H

19 -10

19-111


��____________________�.,____________________-J'

/

inactive enzyme 9 H1C:C-coo

I

CH I

c -coo

� I

H ZN

SH o

H ZC:C-COO SH

3

a

z

-

+

NH;

19.

e

I

N •

CH

o N

H

+

19-11

H 0 I

II

e

X-CH2-C-C-O I

N

" CH

o

N 20-1

J'

20.

inactive

enzyme

t,ansaminatlon

pyruvate

HYDROLASES Endopeptidases- Serine Proteases Chemical modification has played a central role in establishing the mechanism of action of chymotrypsin, trypsin, and elastase from the points of view of identifying

906

SIGMAN & MOOSER

reactive groups and establishing the existence of essential intermediates. The use of diisopropylfluorophosphate and phenylmethane sulfonyl fluoride to modify an essential serine residue, the use of haloketone affinity labels to alkylate active site histidine residues, and the application of pseudosubstrates such as p-nitrophenyl acetate and cinnamoylimidazole to establish the acyl-enzyme mechanism in equation 2 1 have been extensively reviewed (1 1 7-121). However, it is pertinent to note that 0

o

II

k,

II


E�OH + R�C---'-X � EOH . R--C - X L,

0 k, -+

II

k,

E��C�R ---+ E�OH + X-

H,O

21.

+ o

II

R�C�Othe experimental approaches largely pioneered in the studies of serine proteases have been useful in such diverse areas as classifying glycosidases ( 1 22) and lipases ( 1 23) as to basic kinetic scheme and in devising sensitive fluorimetric techniques to titrate the active site normality of acetylcholinesterase ( 1 24, 1 2 5). Several new reactive groups have been introduced which exhi bit high reactivity toward serine proteases. Alkylisocyanates react rapidly and irreversibly in a 1: 1 stoichiometry with the serine residue of chymotrypsin and elastase (1 26). The carbamyl derivatives formed are sufficiently stable to permit their isolation using standard techniques of protein chemistry (127). In view of the reactivity of alkyl isocyanates with other nucleophilic amino acid residues (e.g. cysteine) ( 1 28, 1 29), the site of inactivation of alkylisocyanates cannot be assumed in the absence of appropriate confirmatory experiments. Certain aldehydes, arsonates, and boronates have recently been demonstrated to be potent specific inhibitors of serine proteases ( 1 30- 1 34). The inactive complexes formed can be reactivated by dialysis even though it is likely that the active site serine residue adds to the electrophilic cen ters of the various inhibitors. The probable reason for the potent inhibition is that the adducts generated resemble the tetrahedral intermediate through which both the formation and hydrolysis of the acyl-enzyme proceed. Because X-ray crystallographic analysis of subtilisin and chymotrypsin have revealed a hydrogen bonding network of appropriate geometry to bind a tetrahedral intermediate (or a transition state closely resembling the tetrahedral intermediate), the inhibitors are probably interacting with the enzyme in a manner closely analogous to a true substrate (135). The importance of tetrahedral structures in inhibiting serine proteases is emphasized by the discovery that several naturally occurring peptides terminating in arginylaldehyde are potent inhibitors of trypsin and plasmin ( 1 36). Further, in the bovine trypsin-pancreatic trypsin inhibitor complex, a tetrahedral adduct is formed by the addition of the reactive serine of the enzyme to a carbonyl group of a specific lysine residue of the inhibitor ( 137). Another use of pseudosubstrates and specific inhibitors of serine proteases has been to study the zymogens of pancreatic proteolytic enzymes. X-ray crystallographic



907

results have indicated that a surprisingly minor degree of reorganization in tertiary structure accompanies the transformation of chymotrypsinogen to active chymo trypsin (138). The spatial arrangement of the essential charge relay system in chymotrypsin composed of a hydrogen bonding network between the side chains of a serine, histidine, and aspartic acid residues (139, 140) is effectively intact in the zymogen (138). Nuclear magnetic resonance studies have confirmed the presence of charge relay system in the zymogen ( 1 4 1 ). One of the few pronounced and readily interpretable structural changes in the generation of enzyme activity from chymo trypsinogen is the movement of the side chain of a methionyl residue (Met-I92) from a deeply buried position in the zymogen to the surface of the enzyme, where it serves as a flexible lid of the substrate binding cavity partially created by this movement. Studies on the reactivity of the zymogen with active site titrants and substrates are consistent with the absence of effective binding sites on the zymogen. A measurable reaction of chymotrypsinogen and trypsinogen with diisopropylfluoro phosphate, even though the rate constants are 103 to 104 less than their respective enzymes, shows that a reactive serine residue pre-exists in the zymogen (142). Further, chymotrypsinogen catalyzes the hydrolysis of p-nitrophenyl guanidinobenzoate 106 to 107 times less rapidly than the enzyme, despite the fact that the deacylation of the acyl zymogen is -it that of the enzyme (143). The significantly lower acylation rate but roughly comparable deacylation rate indicate that the ineffective catalytic properties of the zymogen are due primarily to an inefficient or poorly developed binding site rather than to a distorted catalytic site. There are at least three further lines of evidence supporting the pre-existence of a competent active site in trypsinogen and chymotrypsinogen (144). First, acetyltryp sinogen, which cannot be activated by either trypsin or acetyltrypsin, causes measurable activation of chymotrypsinogen, even though the rate is 105 less than that of trypsin (145). Second, specific oxidation of the Met- 192 of chymotrypsinogen to methionine sulfoxide (146) by hydrogen peroxide yields a zymogen derivative that is phosphorylated by diisopropylfluorophosphate two times more rapidly and acylated by p-nitrophenyl-p-guanidinobenzoate eight times more rapidly than the native zymogen (147). The deacylation rate for the acyl zymogen formed from the latter titrant is the same for the native and modified zymogen. These results suggest that the oxidation of Met-I92 has induced an alteration in the position of the methionine similar to that which occurs in the zymogen-to-enzyme conversion. The binding of the pseudosubstrates, and hence their reactivity, are facilitated ( 147). Finally, methane sulfonyl fluoride, which because of its relatively small size does not exhibit high binding specificity, reacts with trypsinogen only 40 times less rapidly than it does with the enzyme (148). Endopeptidases-Acid Proteases

The unusually low pH optimum of pepsin has long indicated that this enzyme has a structure fundamentally different from the serine proteases and that carboxylate groups are probably directly involved in catalysis (149). Determination of the amino add sequence of a peptide from the carboxyl terminus of both porcine and human


908

SIGMAN & MOOSER

pepsin has supported the former view (1 50, 1 51 ). Of particular interest is the apparent common evolutionary origin of calf renin and hog pepsin as suggested by the high degree of homology of their carboxyl-terminal sequences ( 1 52). The chemical modification of pepsin with a series of reagents has supported the presence of carboxylate residues, in particular aspartyl residues, at the active site. Reagents that either have been proven to or are likely to esterify active site carboxylate residues include p-bromophenacyl bromide ( 1 53, 1 54), 1,2-epoxy3(p-nitrophenoxy)-propane (1 55, 1 56), L-l-diazo-4-phenyl-3-tosylamido butanone-2 ( 1 57), diazoacetyl-D, L-norleucine methyl ester ( 1 58), diazoacetyl-L-phenylalanine methyl ester ( 1 59), a-diazo-p-bromoacetophenone ( 160), and I-diazo-4-phenyl-2butanone ( 1 61). All the modification reagents bearing diazo linkages require cupric ion for rapid stoichiometric modification of pepsin. Few unambiguous conclusions can be drawn from these chemical modification experiments other than the certain existence of aspartyl residues at the active center of the enzyme. These studies indicate the inherent limitations of chemical modifica tion experiments when the derivatization reaction does not bear a basic similarity to the enzyme-catalyzed process. Two moles of 1,2-epoxy-3(p-nitrophenoxy} propane are incorporated per mole of pepsin (1 55). This stoichiometry is not altered even if thc enzyme is pretreated with p-bromophenacyl bromide or diazoacetyl-D, L-norleucine methyl ester (1 55). One mole of 1,2-epoxy-3(p-nitrophenoxy)-propane reacts with an aspartyl residue, while the second mole reacts with a methionine residue. The amino acid sequences around the reactive methionine and aspartate are indicated in sequences l' and II', respectively, where the residue modified is Phe-Glu-Gly-Met-Asp-Val-Pro-Thr-Ser-Ser-Gly I' Ile-Phe-Asp-Thr-Gly-Ser-Ser-Asn II' underlined (1 56, 1 62, 1 63). The aspartyl residue modified by I-diazo-4-phenyl-2butanone (161), diazoacetyl-L-phenylalanine methyl ester ( 1 59), and apparently a diazo-p-bromoacetophenone ( 1 63) is located in sequence III'. The carboxylate group modified by diazoacetyl-D, L-norleucine methyl ester has not yet been reported nor Ile-Val-Asp-Thr-Gly-Thr-Ser-Leu III' has that modified by L- l -diazo-4-phenyl-3-tosylamido butanone-2 ( 1 57). The aspartyl residue modified by a-bromo-p-bromoacetophenone is not in sequences II' and III' because it does not inhibit the incorporation of either 1 ,2-epoxy-3(p-nitrophenoxy} propane or a-diazo-p-bromoacetophenone into the enzyme ( 1 55, 1 60). Most likely this residue is not essential for activity in any case, since enzyme modified by a-bromo-p-bromoacetophenone still retains substantial activity toward denatured hemoglobin ( 1 60). In addition, gastriesin, which is homologous in structure to pepsin,



909

is not inhibited by ()(-bromo-p-bromoacetophenone, although it is inhibited by ()( diazo-p-bromoacetophenone and diazoacetyl-o,L-norleucine methyl ester (164). The assignment of a catalytic function to the aspartyl residue in either sequence II' or III' has been complicated by the report that pepsin modified by either ()(-diazo-p-bromoacetophenone or 1 ,2-epoxy-3(p-nitrophenoxy)-propane retains its sulfite esterase activity but not its peptidase activity using hemoglobin as its substrate ( 1 63). However, there is evidence that a single active site is responsible for both activities. The initial report on the sulfite esterase activity of pepsin demonstrated that both the peptidase and sulfite esterase activity were lost upon modification with diazoacetyl-o, L-norleucine methyl ester (1 65). Furthermore, the Ki calculated for the inhibition of sulfite ester hydrolysis by N-carbobenzoxY-L-phenylalanyl-L tyrosine is the same as the Km calculated using the dipeptide as a substrate ( 1 65). If, as seems likely, diazoacetyl-o,L-norleucine methyl ester reacts with the aspartyl residue in either sequence II' or III' and the mechanisms of sulfite ester and peptide hydrolysis are the same, there is no unequivocal evidence that an aspartyl residue participates directly in bond making and breaking at the active site. Inhibition could be due to steric effects resulting from the introduction of a bulky substituent in the active site. On the other hand, sulfite ester and peptide hydrolysis may proceed by different mechanisms. Identification of the acidic residue esterified by diazoacetyl-n, L-norleucine methyl ester would help resolve this question, as would a detailed analysis of the peptide products and catalytic activities of pepsin inhibited by the bifunctional reagents, 1, 1-bis-diazoacetyl-2-phenylethane (X) or o, L-l diazoacetyl- l -bromo-2-phenylethane (XI) ( 1 66). Esterification of active site carboxyo II

NzC H - C -

o II

� H - C H Nz

2]2 x

Br 0 I

II

C Hz - C - C - C H Nz

O �

XI

lates, using reagents that introduce small substituents, such as trimethyloxonium borate, might be an alternative approach ( 1 67). Just as diisopropylfluorophosphate has been a useful reagent to classify enzymes as serine proteases, it appears that diazoacetyl-o, L-norleucine methyl ester might be valuable to determine if proteases are homologous to pepsin. In addition to pepsin from a variety of sources, enzymes inhibited in a cupric ion-dependent reaction by diazoacetyl-o,L-norleucine include renin from mouse submaxillary glands (1 68), penicillopepsin from Penicilliumjanthinillum (169), and human gastricsin (164). Exopeptidases- Carbox ypeptidases

Bovine carboxypeptidase A, a zinc metalloenzyme, has been extensively studied by chemical modification and X-ray crystallography (e.g. 170, 1 71 ). The X-ray structure of the carboxypeptidase A-glycyl-tyrosyl complex has revealed that the carbonyl


910

SIGMAN & MOOSER

group of the peptide coordinates to the active site zinc ion, and two amino acid residues, Glu-270 and Tyr-248, are close enough to the scissile peptide bond to be catalytically important. The positive charge of the guanidinium group of Arg-145 interacts with the terminal carboxylate ion and is responsible for the specificity of carboxypeptidase as an exopeptidase. The role of the zinc ion is to polarize the carbonyl group and enhance its susceptibility to nucleophilic attack. The glutamate residue either attacks the coordinated peptide carbonyl group to form an anhydride intermediate which rapidly h ydrolyzes or serves as a general base catalyst for the nucleophilic attack of water on the peptide carbonyl group. The presumed role of the tyrosine is to serve as a general acid and donate a proton to the nitrogen of the susceptible peptide bond ( 170, 1 72). Although affinity labeling experiments have confirmed that Glu-270 is at the active site, they cannot, by their intrinsic nature, resolve the ambiguity with respect to its precise catalytic function. Both the esterase and peptidase activities of carboxy peptidase A are inhibited by incubation with N-bromoacetyl-N-methyl-L phenylalanine (XII) ( 173). Two moles of XII are incorporated per mole of enzyme, CH2Br

I

C=O

I

CH 3-N 0

0 , -

t �

CH2- - -O -

I

H XII

but in the presence of D-phenylalanine, a competitive inhibitor of the enzyme, only one mole of XII is incorporated and there is no loss of activity. Therefore, there are two loci of XII attachment on the enzyme, one at the active site and one at a site that does not affect enzymic activity ( 173). An analysis of the products reveals that the alkylation of Glu-270 to form an ester is responsible for the loss of activity ( 1 74). The second mole of XII reacts with the IJ(-amino group of the N terminal asparagine (0.5 residue/mol) and the imidazole moiety of a histidine residue (0.2 residue/mol) in the N-terminal portion of the molecule (1 74). The rate of enzyme inactivation by XII depends on an ionizable group of pKa 7.0 which reacts in the deprotonated form. Because the inhibition of carboxypeptidase by Woodward's Reagent K shows a com;Jarable pH dependence ( 1 7 5, 1 76) and an ionizable group of approximately this value is important in peptidase activity (177, 1 78), Glu-270 is most likely the residue responsible for this ionization despite the divergence of its pKa from that of a normal carboxylic acid. Both XII and IJ(-N-bromoacetyl-o-arginine inhibit carboxypeptidase B (179, 180). As might be expected from the similarity of these two enzymes, the glutamate residue of carboxypeptidase B esterified is located in a peptide that is homologous to the active site peptide of carboxypeptidase A ( 1 8 1 ).



911

The chemical modification of tyrosine residues has provided interesting details with respect to carboxypeptidase A-catalyzed hydrolyses. Acetylation of the enzyme with acetic anhydride (182) or acetylimidazole ( 183) abolishes peptidase activity using carbobenzoxyglycyl phenylalanine as substrate but enhances esterase activity using hippuryl-DL-phenyllactate as substrate. The interpretation of these findings is somewhat tentative since the enzyme is susceptible to substrate and product inhibition (1 84-186), and the products of the modification have not been determined, although the acetylation of Tyr-248 is assumed to be primarily responsible for the observed kinetic effects. However, the results indicate that a free hydroxyl group on Tyr-248 may not be absolutely required for ester hydrolysis, even though in all cases examined it is essential for peptidase hydrolysis (172). Experiments involving the modification of Tyr-248 in the crystalline state with the diazonium salt of p-arsanilic acid have questioned whether substrate-induced conformational changes deduced from X-ray crystallography occur in solution (1 87, 188). These changes involve the movement of the phenolic hydroxyl group of Tyr-248 by roughly 12 A when the substrate (glycyltyrosine) binds to the free enzyme. These structural changes appear to be triggered by the formation of a salt link between the terminal carboxylate ion of the substrate and the guanidinium group of Arg-145. Initially, the hydroxyl group of the tyrosine is 17 A from the zinc ion, but after binding of the substrate, it is located near the nitrogen of the scissile peptide bond (189). The tyrosine derivative formed by modification with the diazonium salt, XIII,

is yellow in its protonated form and red in its phenolate form. The free phenolate has an absorption maximum at 485 nm but, coordinated to zinc ion, it has an absorption maximum at 540 nm (190). The ex, P. and }' forms of monoarsanilazo tyrosine-248 carboxypeptidase A in crystalline forms distinct from that used in the X-ray studies are yellow at pH 8.2 but red Pmax 510 nm) in solution at the same pH (190). Circular dichroism and absorption spectra of modified carboxypeptidase with and without the active site zinc ion indicate that the red color in solution at pH 8.2 is due to the coordination of the active site zinc ion by monoarsanil=


912

SIGMAN & MOOSER

azotyrosine-248. These studies indicate that the position ofTyr-248 in this crystalline form is different from that in solution (190). This conclusion is supported by the pKa difference of the modified tyrosine of nitrotyrosyl-248 carboxypeptidase in the crystalline state and in solution (1 91). However, the monoarsanilazotyrosine-248 derivative of carboxypeptidase A, (the form used in the X-ray studies) is red in solution at pH 8.2 and red in the crystal structure used in the X-ray analysis at pH 8.2 (192). Another important difference between the crystals used in the X-ray studies and the other crystalline form is that the former hydrolyzes carbobenzoxyglycyl-L-tyrosine 1 00 times more rapidly than the latter (192). At pH 7.5 and 4°C, the conditions used in the X-ray investiga tion, the absorption spectra of the modified enzyme retain a residual absorption maximum at 510 nm ( 1 92) both in solution and in the crystal form used in X-ray studies. These results suggest that some fraction of the carboxypeptidase A, molecules in both the crystalline and solution states exists in a conformation in which Tyr-248 coordinates to the zinc ion. Re-examination of the electron density map of un modified carboxypeptidase A, reveals that roughly 1 5-25% of the molecules do exist in a conformation consistent with the tyrosine hydroxyl group coordinated to the active site zinc ion, although in the majority of enzyme molecules, the hydroxyl group is 17 A from the zinc ion (189). The spectra of arsanilazotyrosine248 carboxypeptidase therefore accurately reflect conformational states of the un modified enzyme, and Tyr-248 of carboxypeptidase A has greater conformational flexibility than had been previously noted. Since substrates and inhibitors such as glycyltyrosine and L-phenylalanine destroy the red color characteristic of chelation of zinc ion by arsanilazotyrosine, the direct coordination of the metal ion by the carbonyl group of the peptide deduced from the X-ray studies is confirmed. The crucial problem to resolve is whether the pattern of induced conformational changes deduced from the X-ray studies is an obligatory pathway in solution to achieve the interactions essential for catalysis or whether alternative pathways, beginning with the conformation of the enzyme in which the tyrosine is coordinated to the zinc ion, also exist. This question is complicated by the finding that the rJ., p, and y forms of the enzyme, which are very similar in their catalytic properties, possess different ratios of the two conformational states ( 1 90). The kinetics of displacement of the modified tyrosine from the zinc ion by substrates and inhibitors should help determine the kinetic competence of the con formational forms of the em:yme. Carboxypeptidases from non mammalian sources, such as yeast and cotyledons of germinating cotton seedlings, do not appear to be homologous to the pancreatic enzyme (193-196). Both are inhibited by DFP and are therefore serine proteases. However, the sequence of the active site peptide containing the reactive serine of the yeast enzyme does not correspond to that of the serine proteascs (194). Phospholip ases and Lip ases

Enzymes that act on Iipid-water in terfaces, such as phospho lipase A 2 and triglyceride lipase, are difficult to study because of the complex physiochemical structure of their substrates. Although magnetic resonance techniques have helped define the



913

micellar structures composed o f Triton X- I OO and dipalmitoyl phosphatidylcholine for phospholipase A2 from the venom of Naja naja (197). comparable studies have not been performed for pseudo substrates and chemical modification reagents. Nevertheless, the efficacy of certain reagents does appear to depend on their physical state. For example, p-bromophenacyl bromide alkylates a single histidine residue in both porcine pancreatic phospholipase A2 and its zymogen, prophospholipase, at the same rate. The enzyme is inactivated and the potential activity of the zymogen is destroyed (1 98). The modification of the enzyme by p-bromophenacyl bromide is retarded by D-dihexanoyllecithin at a constant concentration of alkylating agent until the critical micellar concentration is reached. At this point, p-bromophenacyl bromide can be incorporated into micelles of D-dihexanoyllecithin, and the rate of enzyme inhibition is enchanced. For the zymogen, increasing concentration of D dihexanoyllecithin inhibits alkylation with the phenacyl bromide until the critical micellar concentration is reached. After this point, the rate of modification of pro phospholipase is constant and independent of the phospholipid concentration ( 198). The reactivity of the enzyme and zymogen toward p-bromophenacyl bromide parallels their reactivity toward substrates. Substrates in micelles are more effectively hydrolyzed by the enzyme than the substrate in its monomeric form. On the other hand, the zymogen is active toward the monomeric substrate but is inactive toward the substrate in micellar form (199). In analogy to the studies on the pancreatic proteolytic enzymes discussed above, the zymogen apparently possesses a competent catalytil: site but an ineffective binding site for its native substrate. As a result, it is not modified by p-bromophenacyl bromide in micelles. The enhanced reactivity of the p-bromophenacyl bromide in micelles towards the enzyme is probably due partially to the increased local concentration of the alkylating agent at the active site when it is incorporated into micelles. In addition, micelles may induce con formational changes in the enzyme which enhance the histidine's nucleophilicity. The catalytic function of the modified histidine residue in the porcine pancreatic enzyme is at prcscnt unknown. However since calcium ion, which is essential for the catalytic activity (:JOO), protects against p-bromophenacyl bromide inactivation, the metal ion probably binds at or near the reactive histidine. Recent studies on the phospholipase A2 from the venom of Crotalus adamanteus, which also requires calcium ion (201), have suggested a possible role for the histidine residue in the porcine enzyme. The venom enzyme is inactivated upon modification of a lysine residue by ethoxyformic anhydride (202). A possible catalytic role for this lysine residue and, by analogy, the histidine residue in the pancreatic enzyme is suggested by the primary amine-catalyzed methanolysis of phosphatidylcholine to lysopho phatidylcholine and the methyl esters of the fatty acids originally present in thc lecithin (203). Since the reaction exhibits a kinetic isotope effect in both CH30D and CD30D, the primary amine probably serves as a general base catalyst. The general base-catalyzed methanolysis is further accelerated by calcium ion. In analogy to other metal ion-catalyzed transesterification reactions (204, 205), the calcium in


914

SIGMAN & MOOSER

the model reaction for phospholipase probably forms a reactive complex where the metal ion, besides serving as a template for reactants, facilitates the deprotonation of methanol by lowering its pKa. The deprotonation of the methanol is further assisted by the amine serving as a general base catalyst. In the pancreatic enzyme, the general base catalyst could be the histidine alkylated by p-bromophenacyl bromide ; in the snake venom enzyme, it could be the lysine acylated by ethoxyformic anhydride. Although phospholipases do not possess reactive serine residues, lipases from a variety of sources appear to react with serine esterase pseudosubstrates. For example, an extracellular lipase from Corynebacterium acnes is inhibited by p-nitrophenyl acetate and diisopropylfluorophosphate (206). In addition, pancreatic lipase is inhibited by diethyl-p-nitrophenylphosphate (207) and hydrolyzes p-nitrophenyl acetate ( 1 23). In analogy with inhibition studies on phospholipase, the reactions of these pseudo substrates are dependent on their physiochemical state and proceed more rapidly when incorporated into micelles. Nucleases-Ribonuclease A

As methods for the chemical synthesis of peptides improve, a direct way to investigate the role of an amino acid in catalysis will be the chemical synthesis of modified enzymes in which the residue of interest is replaced by an amino acid of different structure and properties. P resently, the most practical way to achieve this goal is to first develop procedures to cleave native enzymes into well-defined peptide products which, though inactive individually, can be added together to restore enzymic activity by noncovalent association of the separated fragments. Since the synthesis of the smaller peptides is now practical (208), systematic substitution and permuta tion of residues in these peptides through chemical synthesis are feasible. The pioneering studies in this area, which were based on the cleavage of pancreatic ribonuclease A by subtilisin into a 20 unit and 104 unit peptide, have been recently reviewed (209), as has work on staphylococcal nuclease cleaved by trypsin (210-2 13). Recent studies on bovine pancreatic ribonuclease A have involved complementa tion of inactive enzyme in which residues from the carboxyl terminus have been removed by limited proteolytic digestion and enzymic activity regenerated by addition of appropriate carboxyl-terminal peptides (214). One question investigated was whether Phe- 1 20 plays an essential role in binding substrates. X-ray studies had revealed the close association of the aromatic rings of Phe- 1 20 and the pyrimidine substrate. To evaluate the significance of this interaction, the regeneration of enzyme activity in ribonuclease deficient in amino acid residues 1 19-124 by synthetic tetradecapeptides corresponding to residues 1 1 1-124 was examined (21 5). In particular, tetradecapeptides were synthesized in which the phenylalanine at position 120 was substituted with isoleucine, leucine, and tryptophan. Although the peptide containing tryptophan yielded only a trace amount of activity upon addition to the large fragment, the peptides containing isoleucine and leucine yielded roughly 1 5 % activity. The peptide containing phenylalanine regenerated 98% activity. The lower activity in the peptide containing amino acids other than phenylalanine at position 120 is largely due to reduced binding of the two fragments. However, since the Km values for the active product formed from the phenylalanine,



915

isoleucine, and leucine containing peptides were all equal within experimental error, these results demonstrate that the interaction of the phenyl ring of phenylalanine with the pyrimidine ring of the substrate is not essential for binding (21 5). The enzymically active products generated by complementation of fragments apparently have multiple conformations. When native ribonuclease A is alkylated by iodoacetate at pH 5.5, two inactive protein products are formed. The major product (87%) is carboxymethylated at His- 1 1 9, whereas the minor product is carboxymethylated at His- 1 2 (21 6). When ribonuclease A lacking residues 1 21-125 is complemented with the tetradecapeptide corresponding to residues 1 1 1-1 24, the essential catalytic function of His-1 19 can be contributed by the histidine residue either from the abbreviated protein or from the peptide. To determine which histidine residue is at the catalytic site, this enzymically active complex was alkylated by iodoacetic acid. Since the histidine residue at 1 19 is modified in both components (4 : 1, ratio favoring the protein), the complemented enzyme does not possess a unique conformation and the histidine residue can be provided by either the peptide or the protein. When ribonuclease lacking residues 12{)-124 was alkylated in the presence of the same tetradecapcptide, His- 1 19 in both components was modified to the same extent (217). Nucleases-Deox yribonucleases

The mechanisms of action of pancreatic ribonuclease and deoxyribonuclease must be significantly different because deoxyribonuclease cannot form an intermediate comparable to the cyclic 2',3' cyclic phosphate intermediate of ribonuclease (209). However, a related covalent enzyme phosphodiester intermediate may be involved in the deoxyribonuclease-catalyzed reaction if the hydroxyl group of a threonine, serine, or tyrosine replaces the 2'-OH group of a ribonucleotide as a nucleophile. Although no covalent intermediates have been detected in the staphylococcal

nuclease-catalyzed hydrolysis of deoxythymidine 3-phosphate-5-p-nitrophenyl phosphate (218), the rapid inactivation of pancreatic DNase by methane sulfonyl chloride at pH 7.0 and 5.0 has revealed a highly reactive serine residue at the active site of this enzyme (219). Evidence for a serine residue as the site of sulfonylation is 1. the alkaline lability of the 35S-methane sulfonyl label in the modified enzyme, and 2. the generation of S-aminoethylcysteine upon incubation of the modified enzyme with f3-mercaptoethylamine (equation 22). Both these prop erties are shared by chymotrypsin, which has been specifically sulfonylated at its reactive serine (220, 221).

0

o H

II

I

I

. . . C-C-CHz-O-S -CH3 + H S-CH z CH z

0

NH .....

CH 3

NH z

-

I

I

0 H

o -

I

S

II

0

-

o

-

II I

+ . · C-C-CH z-S CH zCH zNH z

I

NH

22.


916

SIGMAN & MOOSER

Although the active serine of DNase reacts roughly 3500 times more rapidly than would be expected for a primary alcohol under comparable conditions, these data merely suggest but certainly do not prove an essential catalytic role for the serine. The generality of reactive serine residues in deoxyribonucleases is uncertain because porcine spleen deoxyribonuclease is not inhibited by diisopropylfluoro phosphate (222). However, these two enzymes have fundamentally di fferent prop erties. Pancreatic DNase produces 5'-phosphorylated ends, requires a divalent metal ion, and has optimal activity at neutral pH. However, the spleen deoxyribo nuclease yields 3'-phosphorylated products, has no apparent metal ion requirement, and is optimally active at acidic pHs (223). Both enzymes are inhibited by iodoacetate (222-224). In each case, the inactivation is due to the alkylation of a histidine residue to yield 3-carboxymethyl histidine. Because the complete sequence of the 257 residue pancreatic enzyme is known (225, 226), it is possible to designate His- 1 3 1 as the reactive residue. Although no definitive proof of the catalytic function of the histidine residue is available, in analogy to ribonuclease, it may serve as a general base catalyst. In line with the differing metal ion requirements of the two enzymes, a divalent metal ion is essential for the inhibition of pancreatic DNase by iodoacetate but not for inactivation of the spleen enzyme (223). Inhibition of the pancreatic enzyme in the presence of cupric ion with a series of halo acetates revealed that chloroacetate was the best

alkylating agent followed by the bromo and iodo derivatives (227). Since this reactivi ty sequence corresponds to the stability of the various cupric halide complexes and not the normal leaving group tendencies of the halide (227), the cupric ion probably serves as an electrophilic catalyst by coordinating the halide le.aving group in the transition state of the alkylation reaction. If correct, this conclusion implies that the binding site of the metal ion is at or near the reactive histidine. Since metal ions protect the enzyme against inactivation of the enzyme by methyl sulfonyl chloride (219), the reactive serine and histidine residues may be adjacent to each other at the active site.

LYASES PyridoxaI 5-Phosphate-Dependent Decarboxylases

Although important mechanistic features of pyridoxal 5-phosphate-dependent de carboxylases remain to be determined, the general structure of reactive intermediates and their sequence of formation are well understood (96). The sodium bo�ohydride reduction of Schiff bases formed between pyridoxal 5-phosphate and lysine residues pioneered in the study of phosphorylase (228), has played a key role in elucidating the active site chemistry of all pyridoxal 5-phosphate-dependent enzymes. For example, the demonstration of a Schiff base or aid imine linkage between a lysine residue and the coenzyme, prior to addition of substrate, has indicated that the first step in the catalytic reaction involves a transaldimination reaction in which the amino group of the substrate displaces the s-amino group of the lysine from the pyridoxal phosphate (see equation 1 8). Sodium borohydride reduction of the holoenzyme has permitted the isolation



917

of pyridoxyl peptides from the active sites of these pyridoxal 5-phosphate-dependent enzymes (229, 230). Striking sequence homologies in the peptides from E. coli l ysine and arginine decarboxylases suggest a common evolutionary origin of these enzymes despite the fact that the two proteins are sufficiently distinct to show no immunological crossreactivity (230). A basic residue, either histidine or lysine, is frequently adjacent to the pyridoxyllysine residue in many pyridoxal phosphate dependent enzymes. No conclusive statement can be made as to the role, if any, of these residues, but molecular models indicate that interaction with the phosphate moiety of pyridoxal 5-phosphate is possible. Alternatively, if the neighboring basic group is charged, it may lower the pKa and therefore increase the reactivity of the active site lysine (229). f3-Chloroalanine is both a substrate and an irreversible inhibitor of L-aspartate-f3decarboxylase from Alcaligenes faeca:is (231). In analogy to aspartate transaminase ( 1 1 5, 1 1 6), the decarboxylase also catalyzes the transformation of f3-chloroalanine into pyruvate. Since fi-chloro-L-alanine does not alkylate the apoenzyme or the 4' deoxypyridoxine 5-phosphate-enzyme complex (231), the catalytic and inactivation reaction most likely proceeds through intermediates comparable to those sum marized in equation 20 for the reaction of p-chloroalanine with aspartate trans aminase. All the data are consistent with a glutamate residue as the sole site of modification. The catalytic function of this glutamate is unclear, although its location within the active site suggests that it could serve as the donor of the proton that replaces the ,B-carboxyl group (232). Tryptophan Synthetase

The use of pyridoxal 5-phosphate to effect the photosensitized oxidation of a histidine residue proximal to it in the three-dimensional structure of the f3-subunit of E. coli tryptophan synthetase has recently been described (233-235). Analysis of the peptide products indicates that the modified histidine residue either immediately precedes the pyridoxyllysine in the primary sequence or is five residues away in the direction of the N terminus. The suggested catalytic function of the histidine is to serve as a general base catalyst to assist the labilization of the a-carbon proton, although it is possible that the lysine displaced from the coenzyme by substrate in some cases might also serve in this catalytic role (229). Pyridoxal 5-phosphate-induced photoxidation of amino acids may be a general and useful technique to probe the environment in close proximity to lysine residues capable of forming Schiff bases with the coenzyme. As noted elsewhere in this review, this technique has been successfully applied to aspartate transcarbamylase and aldolase. Pyridoxal phos phate is not the only cofactor capable of serving as a photosensitizing agent. Protoporphyrin has been used in the photoxidation of apoperoxidase (236). Non-Pyridoxal Phosphate-Dependent Decarboxylases

Sodium borohydride has played an important role in elucidating the active site chemistry of decarboxylases lacking pyridoxal phosphate. For example, it has been used to trap a covalent Schiff base intermediate formed in decarboxylation reactions catalyzed by acetoacetate decarboxylase from Clostridium acetobutylicum (237). Since


918

SIGMAN

&

MOOSER

the inactive enzyme formed upon reduction contains an isopropyllysine residue, the ketimine of acetone and not of acetoacetate is reduced (238). These experiments suggest that electrostatic effects may play an important role in the ability of sodium borohydride to trap imine intermediates. Reduction of the acetoacetate imine may be inhibited by repulsive electrostatic interactions between the negatively charged carboxylate and borohydride. In agreement with this conclusion, acetonylsulfonate, an effective inhibitor, is reduced onto the enzyme only one fifth as rapidly as acetone (239), and acetonylphosphonate cannot be reduced onto the enzyme by borohydride (240). Removal of one negative charge from the phosphonate by forming the monomethyl ester yields a derivative that is readily reduced onto the enzyme even though it is a poorer reversible inhibitor (240). For acetoacetate decarboxylase, the addition of cyanide ion to the enzymic imine shows a greater sensitivity to electrostatic repulsion than borohydride because it fails to facilitate the binding of acetonylphosphonate and acetonylsulfonate to the enzyme (240). Phosphatidylserine decarboxylase from E. coli is inhibited by sodium borohydride but apparently is not a pyridoxal phosphate-dependent enzyme (241 ). This enzyme may be similar to histidine decarboxylase from Lactobacillus, in which a pyruvylphenylalanyl residue at the N terminus provides the carbonyl cofactor necessary for Schiff base formation (242, 243). The use of borohydride to trap inter mediates has therefore helped to identify a new type of cofactor. Fructose 1 ,6-Diphosphate Aldolase- Class I

There are two types of fructose 1 ,6-diphosphate aldolases (244). Class I aldolases form Schiff base intermediates and are therefore inhibited by sodium borohydride. Class II aldolases require metal ions, are not inhibited by borohydride, and proceed via ene-diolate intermediates similar to those observed in triosephosphate isomerase (244). The inhibition of class II but not class I aldolases by phospho glycolohydroxamate (XV), a stable analog of the proposed ene-diolate intermediate (XIV), provides further evidence for this distinction (245). Class I aldolases are H

OH " / C " c

/ \ 6

C H2

\

0

0

e e I 0 - p- 0 II

OH I N " C

/ \ 6 0

C H2

\

0 I e 0 - P- 0

6

0

0

XIV

XV

generally found in animals and higher plants, whereas class II aldolases are found in bacteria and molds (244). Recent studies have indicated that this distinction is not absolute (246). The sequence of intermediates formed in class I aldolase-catalyzed reactions is summarized in equation 23 (244). Unambiguous evidence for the formation of Schiff


H Z -0 P 03- N H Z IC = O E � H2 OH +

I


I

H z -0 P 03CI = O HOC-H H�-OH NH2- E I

I

R

t

o

r�� OP 03-

C=N � HZ OH 23-1 I

-

R

23-

IV

j ®

E

rz�� OP 03 C=N-E HOC-H HC-OH I

H

919

+

r�� OP03C=N-E HO-eCH 23 I

-II

J

23.

r�-OP03C-N-E CH OH " I

23 - III

base intermediates includes l . the trapping by borohydride of 23-J (247-249) and 23-IV (249) ; 2. rates of fructose 1,6-diphosphate carbonyl 180 exchange which exceed the rate of the cleavage reaction (250) ; and 3. formation of inactive aminonitrile enzyme derivatives by addition of cyanide ion to 23-1 (251 ). Examination of the reactive properties of the essential active site lysine residue of rabbit muscle aldolase, using a competitive labeling technique involving eH)-acetic anhydride (252), has demonstrated that this residue is less readily acetylated than a normal surface lysine in the pH range 7-12 (253). Its apparent pKa is greater than 1 1 .5 even though the pH optimum of class I aldolase is 6. 5-8.0. Since the deprotonated form of lysine is essential for Schiff base formation, a basic group of the enzyme must facilitate the formation of 23-1 or 23-IV after initial formation of the non covalent enzyme-substrate complex (253). The pKa of the lysine residue of aldolase contrasts with that of the essential lysine residue at the active site of acetoacetate decarboxylase. 2,4-Dinitrophenyl propionate acylates this group, causing inactivation. The pH rate profile for this reaction is governed by an ionizing group of pKa 5.9, which is reactive in its basic form. Although alternative explanations are possible, the observed pKa is most likely that of the active site lysine residue which is acylated (254). These studies indicate that imine formation at the active sites of aldolase and acetoacetate de carboxylase takes place by substantially different mechanisms. Pyridoxal 5-phosphate inhibits rabbit muscle aldolase reversibly (255). Reduction of the inactive complex with sodium borohydride and isolation of the peptide products reveal that the lysine modified is distinct from that involved in forming the essential Schiff base intermediates. In view of the ability of the reduced pyridoxal phosphate derivative of aldolase to form a Schiff base with dihydroxyacetone


920

SIGMAN

&

MOOSER

phosphate, the lysine residue of aldolase which reacts with pyridoxal phosphate may serve as a binding site for the 6-phosphate of fructose 1,6-diphosphate (255). Pyridoxal phosphate has proven to be a remarkably specific affinity label for the binding site of phosphorylated substrates (3, 256). The Schiff base formed between pyridoxal phosphate and either rabbit muscle or spinach aldolase can serve as a photosensitizer for the oxidation of histidine residues below pH 8.5 (257) and for the oxidation of a tyrosine residue above this pH (258). The enzyme derivative in which histidine is oxidized retains its ability to form a Schiff base with dihydroxyacetone phosphate. In addition, the modified enzyme retains transaldolase activity. Aldolase alkylated at a histidine two residues from the C-terminal tyrosine by N-bromoacetylethanolamine (259, 260) and altered by carboxypeptidase A treatment with concomitant release of the C-terminal tyrosine (261 , 262) exhibits similar partial reactions as the photo-oxidized enzymes. These and other results (263) indicate that the C-terminal region of the molecules plays some role in the chemistry of the carbanion intermediate (23-II). The histidine and tyrosine in the C-terminal region of the rabbit muscle enzyme may serve con formational rather than direct catalytic roles, since liver aldolase is not affected by carboxypeptidase treatment nor is the C-terminal tyrosine residue modified by photo-oxidation of the enzyme-pyridoxal phosphate complex (258).

/3-Hydroxydecanoyl

Thioester Dehydrase

fJ-Hydroxydecanoyl thioester dehydrase permits the synthesis of long chain un saturated fatty acids in bacteria by an anaerobic pathway (25). The enzyme from E. coli catalyzes the dehydration of fJ-hydroxydecanoyl thioesters to trans-rx-fJ decenoyl and cis-fJ-y-decenoyl thioesters as well as the direct interconversion between the two unsaturated fatty acids. At equilibrium, the relative concentrations of the fJ-hydroxydecanoyl, rx-fJ-decenoyl, and fJ-y-decenoyl thioester derivatives are 70 : 27 : 3. Kinetic and isotopic studies have indicated that all dehydrase reactions proceed through an enzyme-a,fJ-decenoyl intermediate (equation 24) (25).

� fJ,y (free) /3-0H (free) � (a-f3)-Enzyme � a,fJ (free)

24.

The enzyme is inhibited by alkylation of an active site histidine and the nitration of a tyrosine by tetranitromethane (264). More importantly, all the activities of the enzyme are irreversibly and stoichiometrically inhibited by the N-acetylcysteamine (NAC) thioester of 3-decynoyl acid (XVI) at concentrations less than 1O� 7 M (265). The site of inactivation is a histidine residue because an acid hydrolysate of the

o

I

H

I

0

II

CH3-(CH2)5�C=C�CH2�C�S---C -- H2�CH2�N�C�CH3

XVI modified enzyme contains one less histidine than that of the native enzyme (264). Three structural properties of the 3-decynoyl-NAC contribute to its inhibitory



921

ability. First, a thioester linkage is essential. 3-Decynoic acid and its methyl ester are ineffective inhibitors (265). Second, the carbon chain of ten, which exactly corresponds to the substrate specificity of the enzyme, is important. Although comparable derivatives of nine and eleven carbons are potent inhibitors, derivatives with eight and twelve carbons are substantially weaker inhibitors. Finally, and most significantly, the triple bond must be positioned between the fJ and y carbons. The N-acetyicysteamine thioesters of 2-decynoic and 4-undecynoic acids are in effective inhibitors. The mechanism of enzyme inhibition by 3-decynoyl-NAC proceeds via an enzyme-generated allenic derivative according to equations 25-27 (266). The process summarized in equations 26 and 27 is analogous to the enzyme-catalyzed iso merization of cis-3-decenoyl-NAC to trans-2-decenoyl-NAC and is consistent with

H

I

H 0

I

II

� E-CH 3 (CH2)S-C=C=C-C-NAC

H

I

H

I

26.

0

II

E-CH3 (CH2ls-C=C=C-C-NAC -+ E' (inactive)

27.

the requirement for a fJ-y triple bond in the inhibitor. Direct proof for the inactivation scheme involved the demonstration that 2,3-decadienyl-NAC (XVII) (shown in its H

I

H 0

I

I

CH 3 (CH2)s-C=C=C-C-SNAC XVII complexed form in equation 27) inhibits the enzyme more rapidly than 3-decynoyl NAC at equal concentrations (266). The residue modified is a histidine at the active site. Further, whereas a-dideutero-3-decynoyl-NAC reacted slower than its hydrogen analog, the inactivation of the enzyme by a-deutero-2,3-decadienoyl-NAC exhibited no isotope effect in its inactivation reaction with the enzyme. Therefore, the slow step in the inactivation by acetylenic inhibitors is the removal of an a-proton to form the enzyme-bound allene (equation 26). A comparable process is not required for inhibition by XVII. The absolute requirement of thioester derivatives for acetylenic but not dienic acids is explicable in these terms, since thioesters are


922

SIGMAN

&

MOOSER

known to facilitate labilization of IJ(-protons. In addition, the thioester may be essential in binding the acetylenic inhibitor in the proper orientation for proton abstraction from the IJ(-carbon (266). Although the dienic acids have high intrinsic reactivity, the enzyme is most potently inhibited by dienic acids of ten carbons. This correspondence to the enzyme specificity, also observed for inhibition by acetylenic derivatives, indicates that inhibition by the dienic acids proceeds via reversible binding followed by covalent bond formation (267). Comparison of the various C- l O dienic acid deriva tives shows that the effectiveness of inhibition decreases in the series thioester, oxygen ester, free acid, and amide. This pattern of reactivity would be expected if a Michael-type addition reaction (equation 28) was responsible for covalent bond

H

H

H

I � �

28.

N

�

I HN

H

I I R - C = C- C - C -X

1 1 R - C = C = C - C-X II o

N

\)

R

R

formation, since electrophilicity would be greatest for those derivatives where the resonance form, O-C X, is least important. Although the precise structure of the histidine adduct has not been determined, model studies have demonstrated that the derivative with the /3-y double bond (28-1) is more likely than the 1J(-/3 isomer (267). It is not yet known which nitrogen in the imidazole nucleus of histidine serves as the nucleophile. However, these chemical modification experiments un ambiguously demonstrate the essential role of a histidine residue as a general base catalyst at the active site of fl-hydroxydecanoyl thioester dehydrase. -

=

ISOMERASES Triose Phosphate Isomerase

The mechanism of triose phosphate isomerase involves proton abstraction from dihydroxyacetone phosphate by a basic group at the active site to form an ene diolate intermediate (29-1) which, in turn, is reprotonated to yield 3-phospho glyceraldehyde (equation 29) (268, 269). The proton abstracted readily exchanges

E

-

B:

H I H-C -OH

I

C=O

o

H I c = o

H I

+

E - BH

C - OH

II

e

c-o

I

C H20

o

� - Oe �e

29 - I

E - B;

0

HC-OH

I

II e C H20 P - 0

Ie o

29.


923

with solvent and very little, if any, is added back to the ene-diolate (270). The effective inhibition of the enzyme by phosphoglycollate (271 ) and its hydroxamic acid (245) supports the presence of an ene-diolate intermediate. Active site-directed inhibitors have implicated the y-carboxyl group of a glutamate residue as the participating base in the reaction. Halohydroxyacetone phosphates (XVIII) (272-277) and glycidol phosphate (XIX) (278-28 1 ) inhibit the enzyme by alkylating a single glutamate residue. The isolation of homologous active site


H zC

XCHz-C-CHz-OP-O -

I

=

C--CHz-OP-O "H I 0-

--

0x

II

/ �

I

I

0

o

0

o

Cl, Br, I XIX

XVIII

peptides containing the reactive glutamate residue from rabbit muscle (275, 279), chicken muscle (273, 282), human erythrocyte (272), and yeast (274) triose phosphate isomerase underscores the essential role of glutamate in the catalytic mechanism. Analysis of the reaction products formed by the inactivation of chicken muscle triose phosphate isomerase by bromohydroxyacetone phosphate revealed that this affinity label can serve as a crosslinking reagent at the active site of this enzyme (273). After triose phosphate isomerase is inactivated by bromohydroxyacetone 3 2 p-phosphate, the inactive enzyme product loses its label upon overnight dialysis without regain of enzymic activity. Inactive enzyme prepared from 1 4 C-labeled inhibitor does not lose its radioactivity under comparable conditions. Reduction by sodium borohydride prevents the loss of the 3 Zp label. Isolation of a labeled hexapeptide from inactive enzyme that had been reduced by borohydride showed that glutamate is the site of alkylation by the affinity label. The sequence of the active site peptide is Ala-Tyr-Glu-Pro-Val-Trp. When the same

-Br

dH

o e "

o "

,C, O - POCH 2 CH 2

�e

b

1�O

�

30.

924

SIGMAN

&

MOOSER


peptide was isolated from inactive enzyme that had not been reduced with boro hydride, the adjacent tyrosine was modified. A migration of affinity label from the initial site of alkylation, the glutamate residue, to the neighboring tyrosine residue as indicated in equation 30 is therefore consistent with all experimental results. Sodium borohydride reduction limits the intramolecular migration since the phos phate in the glycerol derivative is less susceptible to nucleophilic attack than the phosphate on the IX-carbon of an acetone derivative. These results indicate that the phenolic side chain of the tyrosine residue is adjacent to the y-carboxylate of the glutamate in the tertiary structure of the enzyme. They also illustrate the complexity of product analysis using multifunctional affinity labels. Mandelate Racemase

Mandelate racemase from Pseudomonas putida is irreversibly and stoichiometrically inhibited by D, L-a-phenylglycidate (XX), a structural analog of mandelate (XXI) (283). The release of fJ-phenylglyceric acid from the modified enzyme provides tentative support for the modification of an aspartate or glutamate residue (283). Acidic residues seem to be the preferred, but not the exclusive, site of attack by affinity labels containing epoxides (3). OH

I rnrCH t=o

o xx

I

OH

XXI

Both the inactivation of the enzyme by XX and the enzyme-catalyzed racemization of mandelate require a tightly bound magnesium ion (284). If one assumes that magnesium ion is coordinated by the oxygen of XX and XXI, internally consistent mechanisms for both processes can be offered (283, 284). In the inactivation reaction with XX, the function of the metal ion would be to polarize the epoxide in order to make it more susceptible for nucleophilic attack by the active site aspartate or glutamate (283). In the enzyme-catalyzed reaction, the coordination by the oxygen would facilitate the formation of the probable carbanion intermediate by making the a-proton more acidic (284).

LIGASES Aminoacyl-tRNA Synthetase

Enzymes that utilize macromolecular substrates, such as the aminoacyl-tRNA synthetases, present unique problems in the study of their active site chemistry by chemical modification. Clearly, the synthesis of affinity labels using tRNA as the carrier ligand will be limited by the available techniques of nucleic acid chemistry.



925

Two approaches that have been successful in the selective modification of aminoacyl tRNA synthetase include photo-induced covalent bond formation of the enzyme tRNA complex and acylation of the a-amino group of aminoacyl-tRNA with reactive groups. The photo-induced formation of a covalent nucleic acid-enzyme complex was first reported with DNA and RNA polymerase from E. coli using alternating dA"dT copolymers (285). Irradiation of tRNA lyr in the presence of E. coli tyrosyl tRNA, followed by selective nuclease digestion, demonstrated that covalent bonds with the enzyme were formed with the dihydrouridine arm, the anticodon, and the extra loop of tRNAtyr (286). p-Nitrophenyl-carbamylmethionyl-tRNA has been used to modify methionyl tRNA synthetases from E. coli (287). A single Iysyl residue at the active site was modified and the sequence of the active site peptide containing it was determined. Similarly, N-bromoacetyl-isoleucyl-tRNA inhibits isoleucyl-tRNA synthetase from E. coli (288, 289). N-Bromoacetyl-isoleucine is not an effective inhibitor. Irreversible inhibition of the enzyme could be blocked with tRNAile but not by tRNAphe. The amino acid residue modified has not yet been identified.

CONCLUDING REMARKS The studies cited in this review illustrate different ways that chemical modification can be used to study enzyme mechanisms. The increasing use of suicide substrates merits particular attention. The various suicide substrates discussed above for lactate oxidase, monoamine oxidase, thymidylate synthetase, aspartate transaminase, aspartate fJ-decarboxylase, and fJ-hydroxydecanoyl thioester dehydrase have revealed and/or confirmed details of the active site chemistry of these enzymes in a satisfying manner. Because of their inherent specificity, the design of suicide substrates may represent the best strategy in developing pharmacologically useful enzyme inhibitors. Sodium borohydride and pyridoxal 5-phosphate have emerged as two reagents of impressive generality. In addition to the extensive use of sodium borohydride in reducing imines, recent work has shown that this reducing agent can also be used to trap thioester and acylphosphate intermediates. Pyridoxal 5-phosphate has exhibited remarkable specificity for lysine residues at the binding sites for phos phorylated substrates of many enzymes. The ability of pyridoxal 5-phosphate in its Schiff base linkage with several of these enzymes to cause the photosensitized oxidation of neighboring amino acid residues has greatly extended its utility. The accurate interpretation of the results of chemical modification experiments by group-specific and affinity labeling reagents remains a ceJ?tral problem. However, examination of modified enzymes by magnetic resonance and X-ray crystallographic techniques has permitted in some cases precise structural explanations for the loss of biological activity caused by these reagents. The introduction of new reagents and procedures to assess the importance of steric effects associated with modification of a given amino residue has also facilitated the interpretation of chemical modifica tion experiments. Parallel chemical modification studies on the same enzyme from

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different biological sources also help to evaluate whether a reactive amino acid is of fundamental importance to the catalytic process. Perhaps, in the future, the chemical synthesis of modified enzymes will replace chemical modification experiments as a method to identify which amino acid residues play functional roles in catalysis. However, certain types of chemical modification experimen ts will still be essential to define the precise nature of these catalytic functions.


ACKNOWLEDGMENTS

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