130

GENERAL METHODOLOGY

[10]

have discussed in detail the approach used initially by Baker and subsequently in our laboratories to design an exo affinity label for a series of serine proteases. Other enzymes will present different problems and the relative ease of obtaining an exo affinity labeling reagent will be determined by the accessibility of the substrate to chemical modification. Regardless of the nature of the enzyme or substrate, the general considerations should be the same. Acknowledgments Supported by NIH Grants AI 11231, AM 17351, and CA 17376. Judith M. Andrews is an NIH Postdoctoral Fellow AI 100264. David H. Bing is an Established Investigator of the American Heart Association. The authors acknowledge the excellent secretarial assistance of Ms. Dale Levine and Ms. Rachelle Rosenbaum.

[10] H a l o k e t o n e s as A f f i n i t y L a b e l i n g R e a g e n t s ~ B y FRED C. HARTMAN General Considerations

One of the first affinity labels described was the chloromethylketone derived from N-tosyl-L-phenylalanine (TPCK), which provided convincing evidence for the catalytic involvement of an imidazole side chain in proteolytic enzymes. 2 This and other successes of Elliott Shaw s,4 and his colleagues stimulated many investigators to design ligandlike ahaloketones as probes of the active sites of many different enzymes and nonenzymic proteins, a-Haloketones remain one of the more popular chemical classes of affinity labels. Their principal advantages as affinity labels are that they are highly reactive and provide a means for introducing a radioactive marker, after the covalent modification has taken place, through reduction with sodium borotritride. Haloketones are more reactive as alkylating agents than are haloacetylated amines. Since haloketones are potentially reactive with most nucleophiles found in proteins, the chances are good that modification of some residue within the active site will occur, provided the reagent 1Research from the author's laboratory was sponsored by the U~S. Energy Research and Development Administration under contract with the Union Carbide Corporation. 2 G. Schoellmann and E. Shaw, Biochemistry 2, 252 (1963). E. Shaw, Physiol. Rev. 50, 244 (1970). 4E. Shaw in "The Enzymes" (P. D. Boyer, ed.), Vol. 1, p. 91. Academic Press, New York, 1970.

[10]

I-LkLOKETONE8

131

has an affinity for the active site. The opportunity to introduce conveniently and economically an isotopic label into the reagent moiety of the derivatized protein removes the necessity of synthesizing the labeled reagent. Haloketones also have some disadvantages. Their high reactivities can be a drawback in that the greater the reactivity, the greater the likelihood of undesired, nonselective modifications. It is possible to exert some control over reactivity by the choice of halogen; bromine is invariably a better leaving group than is chlorine (steric considerations aside), whereas fluorine is usually such a poor leaving group that it is of little use. Iodoketones are not always alkylating agents but instead can be oxidizing agents in which the reagent serves as a source of iodonium ion. 5 Haloketones (with the exception of the fiuoro compounds) are certainly too reactive for general use as chemotherapeutic agents. Another disadvantage of haloketones is that the investigator faces a unique problem in identifying the derivatized amino acids for each new reagent used successfully. This contrasts with the characterization of proteins modified with bromoacetylated compounds, in which, irrespective of the entire structure, the derivatized amino acids are converted to easily identified carboxymethyl amino acids upon acid hydrolysis2 ,7 A third disadvantage is that the synthesis of a desired haloketone frequently becomes a research problem in organic chemistry. Again, this contrasts with the preparation of bromoacetylated compounds, in which well-defined, simple procedures are applicable to many alcohols and aminesY Very recently a potential affinity label for the fl-adrenergic receptor in erythrocytes has been prepared by treating an amine with dibromoacetone to form the corresponding alkylated amine containing a reactive bromoketone group, s This provides a synthetic procedure that should be readily adaptable to a multitude of amines. Successful application of affinity labeling, with haloketones or any other class of reagent, typically requires four types of endeavors: (1) search for suitable reagents; (2) demonstration that inactivation results from an active-site-directed process; (3) characterization of the covalently modified protein; (4) determination of the relationship of the results to the mechanism of the enzyme-catalyzed reaction. In rare instances, suitable reagents can be obtained from commercial sources; more often newly designed reagents are synthesized after 5F. C. Hartman, Biochemistry 9, 1776 (1970). F. Naider, J. M. Becker, and M. Wilchek, Isr. J. Chem. 12, 441 (1974). M. Wilchek and D. Givol, this volume [11]. SD. Atlas, M. L. Steer, and A, Levitzki, Proc. Natl. Acad. Sci. U~%4. 73, 1921 (1976). See also this volume [69].

132

GENERAL METHODOLOGY

[10]

thoughtful deliberations on the known structural requirements for substrafe binding and on the most appropriate placement, with respect to available mechanistic information, of the reactive group within the reagent. Many of the criteria for ascertaining whether a reagent is acting as a true affinity label are universally applicable and are described in a previous chapter of this volume2 Methods for determining the kinds of amino acid residues modified depend somewhat upon the type of reagent used; the introduction of a carbonyl group into a protein provides a handle which can facilitate the identification of the labeled residue, as will be seen later in this chapter. The degree to which data from affinity labeling will increase understanding of the enzymatic mechanism cannot be predicted a priori. In some cases nothing more is obtained than an indication of a particular residue's presence at the substrate binding site, whereas in other cases detailed information is obtained concerning a residue's role in a precisely defined catalytic step and its geometric relationship to other active-site residues. Most of the methodologies used and problems encountered with haloketones as affinity labels are covered by considerations of bromopyruvate, haloacetol phosphates, and haloketone derivatives of pyridine nucleotides. Similar information is gained from numerous elegant studies in which halomethylketone derivatives of amino acids and peptides have been used as affinity labels for proteases2 ,4,1° These investigations are excluded from the present article, since they are considered elsewhere in this volume. 1°

Selected Examples Bromopyruvate Detailed investigations by H. Paul Meloche and his associates of the reaction of bromopyruvate with 2-keto-3-deoxyglucomate-6-phosphate (kdGtP) aldolase exemplify the wealth of information that can be gleaned from affinity labeling. Although this particular aldolase is not ubiquitous (in most studies the enzyme from Pseudomonas putida,was used), it typifies a large number of aldolases, isomerases, and fl-decarboxylases that activate a hydrogen atom (as a proton) a to the substrate carbonyl to generate an intermediate carbanion or enol. When Meloche initiated his studies, the kdGtP aldolase mechanism could be depicted as shown in oF. Wold, this volume [1]. ~oj. C. Powers, this volume [16].

[10]

HA.LOKETONES

133 o

H20 ~ :

(CH2)4-~H r~=C

C=O

H2)4- ~IH3

/

+

H- C - H

o

BH H2)4 - N"~-'~! - H

HC=O H-C-OH I .OH2C- OP--. 0 0 o

0 II C-OI C=O I

Enzyme

H20

(CH2)4 - NH = C t CH2

/

'BHI

J

CH2 +

II

@

I

HC-OH f HC- OH | .,OH2C- O ~ O-

C-OI

/"

k

I

.0

H2C - OP~0

|

]I

O

FIG.

1. Possible mechanism for 2-keto-3-deoxygluconate-6-phosphate aldolase.

Fig. I. Schiff-base formation was detected by reduction with borohydride, 11 and the presence of a basic group (B1) that abstracts a proton 1, E. Grazi, H. Meloche, G. Martinez, W. A. Wood, and B. L. Horecker, Biochem. Biophys. Res. Commun. I0, 4 (1963).

134

GENERAL METHODOLOGY

[10]

from C3 was assumed from the demonstration that the enzyme catalyzes the exchange of tritium from water into the methyl group of pyruvate. 12 Melochels,14 hypothesized that placement of a good leaving group at C3 would form an electrophilic agent capable of alkylating the unidentified basic group (B1). Thus, on the basis of the probable mechanism, bromopyruvate appeared to be an ideal reagent for the selective modification of an active-site residue with a known catalytic function. Bromopyruvate is a rare example of a reagent that not only appeared ideal from mechanistic considerations, but also was readily available. The compound can now be purchased from commercial sources. 1-[1~C] Bromopyruvate can be obtained by direct bromination of commercial [~4C]pyruvate. ~4 [SH]Bromopyruvate has also been prepared from randomly tritiated pyruvate obtained by the action of kdGtP aldolase on pyruvate in the presence of T20. ~ Bromopyruvate is assayed by its conversion at pH 8.0 to hydroxypyruvate, which is quantitated either enzymically with lactate dehydrogenase or chemically as the semicarbazone. ~2,14 Inactivation of kdGtP aldolase by bromopyruvate is clearly an activesite-directed process. 1~ Inactivation is first order with respect to the remaining native enzyme, and a rate-saturation effect is observed as the concentration of bromopyruvate is increased. Pyruvate protects against inactivation, and kinetic experiments in the presence of pyruvate show that reagent and substrate compete for the same site. The aldehyde substrate, D-glyceraldehyde 3-phosphate, affords no protection, indicating that only the pyruvate domain within the active site is alkylated. With enzyme that had prior treatment with nonradioactive bromopyruvate in the presence of pyruvate so that nonselective sites were blocked, the extent of specific incorporation as determined with [~C]bromopyruvate is close to 1 mole of reagent per mole of catalytic subunit inactivated. These observations meet the usual criteria indicative of affinity labeling. To determine the kinds of residues modified, the aldolase inactivated with 1-[~4C] bromopyruvate was first reduced, in the presence of a protein denaturant, with sodium borohydride to convert the ketone group of the incorporated pyruvyl moiety to a hydroxyl group. This was necessitated by the decarboxylation of the pyruvyl moiety (and therefore loss of the 14C label) during hydrolysis of the protein. ~6 Borohydride reduction of covalently incorporated ketones has become a widely practiced procedure. Not only does such reduction confer stability on the protein-reagent linkage and other labile groups a to the ~2H. P. is YI. P. ~4If. P. ~ 1=I. P. ~eH. P.

Meloche and W. A. Wood, J. Biol. Chem. 239, 3511 (1964). Meloche, Biochem. Biophys. Res. Commun. 18, 277 (1965). Meloche, Biochemistry 6, 2273 (1967). Meloche, M. A. Luczak, and J. M. Wurster, J. Biol. Chem. 247, 4186 (1972). Meloche, Biochemistry 9, 5050 (1970).

[10]

HALOKETONES

135

carbonyl, but it aso provides an ideal method for introducing a stable radioactive isotope into any protein labeled by a haloketone and a means for determining the extent of reagent incorporation. These are important considerations, since in many cases labeled starting materials are either unavailable or prohibitively expensive. Even when labeled starting materials are available, lengthy synthetic routes for preparing a particular affinity label make introduction of label subsequent to the chemical modification more attractive. Meloche 16 used approaches to identify the modified residues after reduction that are fairly representative of those used in characterization of new derivatives of amino acids: The elution positions of the radioactive components in acid hydrolyzates were determined by ion-exchange chromatography on an amino acid analyzer. These elution positions were then compared with those of synthetically prepared standards. Hydrolyzates of inactivated kdGtP aldolase contained two 14C-labeled derivatives, which eluted from the long column with the front and just ahead of aspartic acid, respectively. The compound that was not retarded was ninhydrin negative and thus was assumed to be a decomposition or hydrolytic product. Subsequently, this compound was isolated and identified as glyceric acid. From this finding and studies of the stability of the protein-reagent bond, Meloche 1~ concluded that bromopyruvate esterifies a carboxyl group of either a glutamyl or aspartyl residue. The corresponding tryptic peptide from inactivated enzyme has been recently shown to contain only glutamate as a potential esterification site (personal communication from H. P. Meloche). The compound that emerges just ahead of aspartic acid cochromatographed with 1-carboxy-l-(DL)-hydroxyethylcysteine prepared by alkylation of glutathione with bromopyruvate followed by reduction and hydrolysis (Fig. 2). The alkylated glutathione could also be degraded to give carboxymethylcysteine by oxidation with hydrogen peroxide followed by hydrolysis (Fig. 2), but this approach with the inactivated enzyme was not reported. Peroxide treatment of cystcine thioethers presents the risk of sulfoxide formation. Sulfoxides undergo decomposition during hydrolysis leading to ambiguous results and must be reduced back to the thioether with HI prior to hydrolysis. Whenever labeled protein undergoes extensive loss of radioactivity subsequent to acid hydrolysis and drying thioether sulfoxide existence should be suspected. The conversion of an incorporated a-methylketone to a carboxymethyl group is an attractive method (which was used earlier to characterize histidine modified by bromopyruvate 17) for identifying modified residues, since all carboxy17R. L. Heinrikson, W. H. Stein, A. M. Crestfield, and S. Moore, J. Biol. Chem. 240, 2921 (1965).

136

eE~ERAL METHODOLOGY

COOH I G-SH + C=O I CH2-Br

[10]

0

)

II G-S-CH 2-c-COOH

NoB~ OH I G-S-CH 2-CH- COOH I H NH2 I Glu + Gly + S-CH2-CH-COOH I CH2 I CHOH

~202 0!

G-S-CH2CCOH G-S-CH2COOH l H+

NH2 I

Glu + Gly + S-CH2-CH-COOH I CH2 I COOH

I

COOH Fro. 2. Characterization of glutathione (G-SH) alkylated by bromopyruvate.

methyl amino acids are well characterized on the amino acid analyzer, ls,19 However, if carbon-carbon cleavage between the carbonyl carbon and the methyl carbon occurs, a carboxymethyl residue will not be formed. Although the observed stoichiometry of the reaction of kdGtP aldolase with bromopyruvate was 1:1, both a sulfhydryl and a carboxyl group were modified. This result suggested that, within a given subunit (the enzyme is a trimer), modification of either residue prevents modification of the other and that both residues are in or near the active site. Subsequently, it was shown that at low ionic strength (20 mM citrate) 99% of the incorporated bromopyruvate was present as the ester, and at high ionic strength (250 mM citrate) 73% of the incorporated reagent was present as the thiol ether. The stoichiometry remained constant, even though at intermediate ionic strengths the incorporated label partitioned to varying degrees between two residues. The interpretation of these results offered by Meloche TM is that the salt concentration alters an equilibrium between two conformers of the enzyme. In one conformer the active-site sulfhydryl is brought into position .for alkylation, and in the other conformer the carboxyl is in proper orientation for esterification to occur. Consistent with this interpretation are the observed differential ~8H. J. Goren, D. M. Glick, and E. A. Barnard, Arch. Biochem. Biophys. 126, 607

(1968). ~=F. R. N. Gurd, this series, Vol. 25, p. 424.

[10]

HALOKETONES

137

effects of ionic strength on Vmax for the exchange reaction and Vmax for the cleavage reaction. 2° An instructive aspect of this study is the demonstration that rather minor changes in experimental conditions can lead to substantive new findings about the identity of residues at the active site. The close structural resemblance of bromopyruvate to the ketone substrate provides unusual opportunities for ascertaining whether the enzyme really recognizes bromopyruvate as substrate and whether bromopyruvate really alkylates the base that activates an a-hydrogen. Since the reagent contains both a ketone group and a-hydrogen atoms, one can ask if the reagent forms a Schiff base with the essential amino group and if one of the a-hydrogens in the bromopyruvyl-enzyme Schiff-base exchanges with solvent protons. In answering these questions, Meloche 15,~1 has provided an additional criterion of affinity labeling that is applicable to certain other situations and has reinforced a general mechanism of aldolase catalysis. (3R,S)-[3-SH~]Bromopyruvate is a substrate for kdGtP aldolase in the exchange reaction under conditions in which 80% of incorporated reagent is bound as an ester. 15 The enzyme is stereospecific for the Pro-R hydrogen. The kinetics of detritiation and inactivation are consistent with the two processes occurring at the same site according to the equation

E

+

I

~

Ci

/ i Einact ~ E

+

P

where E is free enzyme, I is bromopyruvate, Ci is the enzyme-bromopyruvate complex (a ketimine Schiff base), Einact is inactivated enzyme, and P is detritiated bromopyruvate. Since the rates of both inactivation and exchange are proportional to the concentration of a common intermediate (C~), a constant ratio of moles of bromopyruvate detritiated per mole of enzyme inactivated should be seen irrespective of the concentration of I. Furthermore, the concentration of bromopyruvate giving onehalf the maximal rate of inactivation should also give one-half the maximal rate of exchange. Both predictions were verified~; the ratio of exchange to alkylation was about 50 over a wide range of reagent concentrations, and the half-maximal concentration of bromopyruvate for both processes was 1 raM. It has not been demonstrated directly that bromoH. P. Meloche, unpublished data cited in ref. 16. 21H. P. Meloche and J. P. Glusker, Science 181, 350 (1973).

138

GENERAL METHODOLOGY

J

Eneamine 0 .~H

\~ Corboxylote

~-"H---o~'C ~

[10]

CH2

f77

CH2

./

xylate

CH~ "~"

// 7

Glyceraldehyde 3-phosphate Fro. 3. Single-base mechanism for 2-keto-3-deoxygluconate-6-phosphate aldolase.

pyruvate forms a Schiff base with the enzyme as an obligatory precursor of alkylation. This appears, however, to be an inescapable conclusion, since Schiff-base formation precedes exchange and since both exchange and alkylation require the same complex. Proof that the carboxylate, which is susceptible to esterification by bromopyruvate, is the base that activates a substrate hydrogen comes from results of borohydride reduction. 22 Reduction of the esterified aldolase (in the absence of protein denaturant) produces a Ne-lysyl secondary amine, demonstrating the presence of a Schiff base formed between the covalently fixed carboxyketomethyl group and the essential ~amino group. Thus, when the carbonyl of bromopyruvate is bound at the active site as a Schiff base, C3 of the bound reagent is juxtaposed opposite the carboxylate so that either esterification or carboxylate-catalyzed exchange of protons occurs. In contrast a reagent bridge between the alkylated sulfhydryl and the essential amino group cannot be demonstrated by borohydride reduction. 22 Realizing that a carboxylate is probably the base involved in activation of pyruvate hydrogens, Meloche and Glusker 21 have shown with models that a single base could serve in both proton-transfer steps (see Fig. 1) necessary to effect the condensation of pyruvate and D-glyceraldehyde 3-phosphate to the 6-carbon sugar phosphate. Rotation about the bond between C2 and C3 of a glutamyl side-chain positions the carboxylate so that it can be adjacent to either C3 of pyruvate or C4 of 2-keto-3-deoxygluconate-6-phosphate (Fig. 3). Meloche and Monti 28 have recently extended these studies to the H. P. Meloche, J. Biol. Chem. 248, 6945 (1973). H. P. Meloche and C. T. Monti, Biochemistry 14, 3682 (1975).

[10]

HALOKETONES

139

TABLE I EXAMPLES OF ACTIVE-SITE LABELING WITH BROMOPYRUVATE

Enzyme

Residue modified

References

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase Isocitrate lyase Carbonic anhydrase N-Acetylneuraminate lyase

--

a

-His Cys (chloropyruvate used) Cys Cys Cys

b c d

Pyruvate carboxylase Glutamate decarboxylase Malic enzyme

e f g, h

a M. Staub and G. D6nes, Biochim. Biophys. Acta 189, 519 (1967). b T. E. Roche, B. A. McFadden, and J. O. Williams, Arch. Biochem. Biophys. 147, 192 (1971). P. O. GSthe and P. O. Nyman, FEBS Left. 21, 159 (1972). J. E. G. Barnett and F. Koliss, Biochem. J. 143, 487 (1974). P. J. Hudson, D. B. Keech, and J. C. Wallace, Biochem. Biophys. Res. Commun. 66, 213 (1975). s M. L. Fonda, J. Biol. Chem. 251, 229 (1976). g G. Chang and R. Y. Hsu, Biochem. Biophys. Res. Commun. 55, 580 (1973). ^ G. Chang and R. Y. Hsu, Biochemistry, in press. reaction of b r o m o p y r u v a t e with 2-keto-3-deoxygalactonate-6-phosphate aldolase to compare the geometry and chirality of the p y r u v a t e portions of the active sites of two different aldolases. B r o m o p y r u v a t e has proved to be an exceedingly versatile affinity label; some of the more successful examples of its use as an active-site probe are listed in T a b l e I. Especially interesting are recent studies by Chang and Hsu :4,26 in which the reagent is shown to be both an inactivator and substrate for pigeon liver malic enzyme, and half-of-sites stoichiomet r y is observed. 3-Haloacetol Phosphates

These compounds are close structural analogs of dihydroxyacetone phosphate, differing only in replacement of the hydroxyl with a halogen. The rationale for designing haloacetol phosphates was much the same as t h a t which led Meloche 13,14 to believe t h a t b r o m o p y r u v a t e would be a likely affinity label for k d G t P aldolase. Reactions catalyzed by both 5, G. Chang and R. Y. ttsu, Biochem. Biophys. Res. Commun. 55, 580 (1973). G. Chang and R. Y. Hsu, Biochemistry, in press.

140

GENERAL METHODOLOGY

(A)

0 fl

?H2-OC(~

CH20H I

C=O

CH--OH l

CH2-Br CHzOH

[10]

I

0

CH2 - Br

(i) @c-cl •

® [o]

o

0 II/OCH2-OP\o_

c~-o~¢

?

CH-OH I

CHz- CI

C=O I

(D HC(OCH3)3, H+

CH2-CI

OHIO) POCI3

I

)

CH30- C-OCH 3 I

CH2 - X

Nol 0~l

0rl/0-

CHz--OC¢

ICH2-OP \ 0 C=O

C=O I

I

CH~,-I

CH2 - X

X: CI, Br o r I

(B) BrCHaCOOH

O0 II II CICCCI

H3PO4

0 0 II CH2N2) II • BrCH2CCI BrCH2CCHN 2

+ SF3

0 II ~, BrCH2CCH2OPO3H 2

(c) CH2~" /H~O

KHF2 •

I

CH2CI

rCH2F

CH2F I CHOH

Or20 ~~

I

t l '

CH2CI

C=O I

CH2CI 0

I ((~CH20)2pIo-Ag+ CH2F I C=O

NoHC03 (:

CHzF

Hz

C=O ~

I

I

CH2OPO2H-NO+ 0

CH2OIP(OH) 2 0

~ z ~

CH2F C=O I

CH2OIP(OCH2*) z 0

FIG. 4. Syntheses of haloacetol phosphates.

fruetose-bisphosphate aldolase and triosephosphate isomerase involve stereospecific removal of one of the prochiral protons from C3 of dihydroxyacetone phosphate. Thus, the possibility existed that a single reagent could be used to identify active-site residues with common catalytic functions in different enzymes. Iodoacetol phosphate was the first haloacetol phosphate synthesized, 26,2~ and the sequence of reactions used (Fig. 4A) also made availF. C. Hartman, Fed. Proc., Fed. Am. Soc. Exp. Biol. 27, 454 (1968). F. C. Hartman, Biochem. Biophys. Res. Commun. 33, 888 (1968).

[10]

HALOKETONES

141

able the bromo and chloro compounds. ~ Two other syntheses were independently devised, one for bromoacetol phosphate (Fig. 4B)2s and one for the corresponding fluoro and chloro analogs (Fig. 4C).29 The reagents can be labeled with a2p (Hartman3O) or 14C (Coulson et a/.2s). Since both the halogen and phosphate groups of haloacetol phosphates are quite labile, their concentrations in solution should be carefully determined just prior to the chemical modification study. This is conveniently accomplished by quantitating base-labile phosphate content and reactive halogen content2 In the latter method a molar excess of glutathione, whose sulfhydryl group is readily alkylated by chloro- or bromoacetol phosphate, is added to a solution of the reagent, and the amount of glutathione remaining is determined by titration with p-chloromercuribenzoate 81 or 5,5'-dithiobis (2-nitrobenzoic acid).32 The most successful application of haloacetol phosphates as affinity labels has been in the partial characterization of the active site of triosephosphate isomerase. Very similar studies, carried out independently in the laboratories of F. C. H a r t m a n and J. R. Knowles, demonstrated an essential glutamyl ~,-carboxylate (esterified by the reagent) in the enzyme. ~3-3s The recently determined primary structure of the enzyme from rabbit muscle places the glutamyl at position 165. 39 All the usual criteria of affinity labeling were satisfied and have been well documented. Certain aspects of these studies that either relate to the use of haloketones in general or have provided evidence of the carboxylate's intimate role in the catalytic process will be considered. Characterization of the product of the reaction of chloroacetol phosphate with triosephosphate isomerase was facilitated by reduction of the derivatized protein with sodium borotritride. Initially the only isotopically labeled chloroacetol phosphate available was the 32P-labeled material, 28A. F. W. Coulson, J. R. Knowles, and R. E. Offord, Chem. Commun. 1, 7 (1970). ~J. B. Silverman, P. S. Babiarz, K. P. Mahajan, J. Buschek, and T. P. Fondy, Biochemistry 14, 2252 (1975). F. C. Hartman, Biochemistry 9, 1783 (1970). 3~p. D. Boyer, J. Am. Chem. Soc. 76, 4331 (1954). 32G. L. Ellman, Arch. Biochem. Biophys. 82, 70 (1959). F. C. Hartman, J. Am. Chem. Soc. 92, 2170 (1970). ~4F. C. Hartman, Biochem. Biophys. Res. Commun. 39, 384 (1970). 35F. C. Hartman, Biochemistry 10, 146 (1971). F. C. Hartman, this series, Vol. 25, p. 661. 3~A. F. W. Coulson, J. R. Knowles, J. D. Priddle, and R. E. Offord, Nature (London) 227, 180 (1970). 3~S. De La Mare, A. F. W. Coulson, J. R. Knowles, J. D. Priddle, and R. E. Offord, Biochem. J. 129, 321 (1972). ~ P. H. Corran and S. G. Waley, FEBS Lett. 30, 97 (1973).

142

GENERAL METHODOLOGY

[10]

which is not well suited for characterization of modified residues because of the lability of the phosphate group. Reduction with borotritride provided a stable isotopic label on the carbon chain of the reagent that survives conditions used to hydrolyze proteins and also stabilized the phosphate group so that it was not liberated as Pl during proteolytie digestion of the modified protein, s~,35 In addition to providing a marker, the incorporated tritium can be quantitated, thereby revealing the amount of reagent covalently attached to the protein. The specific radioactivity of NaBT4 must be measured indirectly, since lack of purity and stability preclude preparing stock solutions of known concentration based on weight. One approach is to reduce glutathione that has been alkylated with chloroacetol phosphate with a portion of the same borotritride solution used to reduce the modified protein. The glutathione derivative is easily purified on Dowex 50 (H ÷) and quantitated on the amino acid analyzer, thus providing an accurate measurement of the specific radioactivity of the borotritride. 4°,41 The extent of incorporation of chloroacetol phosphate into triosephosphate isomerase determined indirectly by reduction is identical to that determined with 32P-labeled reagent. 42 This indirect method has also been used in the case of ribulose-bisphosphate carboxylase labeled with 3-bromo1,4-dihydroxy-2-butanone 1,4-bisphosphate2 °,41 Knowles and colleagues3s discovered that if the inactivated isomerase was not reduced with borohydride, the phosphate group of the incorporated reagent was displaced by the phenolic hydroxyl group of an adjacent tyrosyl residue, thereby forming a cross-link. After proteolytie digestion, the reagent moiety was found attached to the tyrosyl residue through an ether linkage rather than to the glutamyl carboxylate, the initial site of reaction. Thus, with any haloketone containing an additional group that can be activated by the adjacent carbonyl, possibilities exist for cross-linking two active-site residues or for migration to a second site. As indicated by Knowles, this latter possibility raises the danger of an incorrect identification of the initial site of reaction and therefore an incorrect conclusion as to the identity of an active-site residue. A difficult question to answer from chemical modification studies alone, even when a high degree of selectivity is achieved, is whether the residue modified plays an intimate role in the catalytic process. Two approaches have been used in the case of inactivation of triosephosphate isomerase by chloroacetol phosphate that provide rather convincing, albeit indirect, 4°I. L. Norton, M. H. Welch, and F. C. I-Iartman, J. Biol. Chem. 250, 8062 (1975). ~1I. L. Norton and F. C. I-Iartman, this volume [42]. 42F, C. Hartman, unpublished data.

[10]

HALOKETONES

143

evidence that the implicated carboxyl group is essential to catalysis. The rate of esterification of Glu-165 by chloroacetol phosphate is extremely rapid [at 2 ° and pH 6.5, the apparent k2nd is 2300 M -1 sec-1 (Hartman zS) ; at 25 ° and pH 7.0, k2nd is 14,000 M -1 sec-~ (Davis et al. 4~) ]. In contrast, esterification of glutamic acid by chloroacetol phosphate has not been demonstrated. At 25 ° and pH 8.1, incubation of chloroacetol phosphate (20 mM) with glutamic acid (1 mM) for 12 hr resulted in no detectable loss of glutamic acid as measured with the amino acid analyzer. 5 If, for purposes of calculation, one assumes a 10% loss of glutamic acid (a 5% loss would have been readily detected) in the 12-hr period, k2nd would be 9 X 10-5 M -1 sec -1. Thus, the rate enhancement for the reaction of Glu165 of triosephosphate isomerase with chloroacetol phosphate as compared with free glutamic acid is ~ 1.5 X 108. Such an enhancement is as large as the difference in rate of some enzyme-catalyzed reactions vs. their nonenzymic counterparts, so that the esterification of Glu-165 might be said to be "enzyme-catalyzed." The other type of evidence that strongly suggests a catalytic role of Glu-165 comes from comparative studies. The isomerases from yeast, 44 chicken muscle,as rabbit muscle, 35 and human red blood cells 45 have all been subjected to modification with haloacetol phosphates; in each case inactivation results from esterification of a glutamyl ~-carboxylate. Furthermore, in each case the amino acid sequences of hexapeptides containing the essential residue are identical: -Ala-Tyr-Glu-Pro-Val-Trp-. This high degree of species invariance of the active-site glutamyl residue and the adjacent primary structure strongly implies that the carboxyl group is functional in catalysis. Banner et al. 46 have recently published the three-dimensional structure of chicken muscle triosephosphate isomerase. If it is assumed that the binding site for dihydroxyacetone phosphate is in the same region as the binding site for sulfate, which was visualized, Glu-165 is at the active site. The interconversion of D-glyceratdehyde-3-phosphate and dihydroxyacetone phosphate as catalyzed by triosephosphate isomerase involves proton abstraction from C3 of the ketonic substrate by an acid-base group of the enzyme to generate an enediol intermediate, followed by proton transfer from the acid-base group to C2 of the enediol to form the alde4aR. H. Davis, Jr., P. Delaney, and C. S. Furfine, Arch. Biochem. Biophys. 159, 11 (1973). 4~I. L, Norton and F. C. Hartman, Biochemistry 11, 4435 (1972). ~5F. C. Hartman and R. W. Gracy, Biochem. Biophys. Res. Commun. 52, 388 (1973). ,8D. W. Banner, A. C. Bloomer, G. A. Petsko, D. C. Phillips, C. I. Pogson, I. A. Wilson, P. H. Corran, A. J. Furth, J. D. Milman, R. E. Offord,J. D. Priddle, and S. G. Waley, Nature (London) 255, 609 (1975).

144

GENERXL METHODOLOGY

[10] J J J J J

J

SJ

Jj~

I

/o- P / /

Fro. 5. Mechanism for triosephosphate isomerase.

hyde (Fig. 5).'7,48 Based on the observation of esterification of Glu-165, the carboxylate is considered a likely candidate for the essential base. Although this postulate has been generally accepted and also supported by results with glycidol phosphate49,5° (an affinity label that also esterifies Glu-165), definitive proof will probably have to await description of the crystallographic structure of the enzyme-substrate complex. However, data are available that permit calculations of the maximal value for the pK~ of the acid-base group. During the enzyme-catalyzed conversion of dihydroxyacetone phosphate (stereospeeifically tritiated at C3) to D-glyceraldehyde 3-phosphate, > 9 8 ~ of the tritium is exchanged for solvent protons and < 2 % is transferred to C221 An explanation for this result is that the conjugate acid of the essential base ionizes (i.e., exchanges its proton for a water proton) at least 50 times more rapidly ~E. A. Noltmann, in "The Enzymes" (P. D. Boyer, ed.), 3rd ed., Vol. 6, p. 271. Academic Press, New York, 1972. I. A. Rose, Adv. Enzymol. 43, 491 (1975). 4oj. C. Miller and S. G. Waley, Biochem. J. 123, 163 (1971). ~°K. J. Schray, E. L. O'Connell, and I. A. Rose, J. Biol. Chem. 248, 2214 (1973). See also this volume [41]. ulB. Plaut and J. R. Knowles, Biochem. J. 129, 311 (1972).

[10]

HALOKETONES

145

than it transfers the proton to C2 of the enediol intermediate. With this value and the value for kcat , Rose 4s has calculated that the group involved in proton transfer must have a pK~ less than 5.0. Based on the pH dependency of the inactivation rate of rabbit muscle triosephosphate isomerase with glycidol phosphate, the pKa for Glu-165 was calculated to be less than 5.5 in one study 5° and 6.0 in another22 Since this difference could be due to the complication of a variable affinity of the reagent for the enzyme as a result of the change in ionization state of the reagent over the pH range examined, the pH dependency of inactivation rate was also studied with the strong, monoprotic acid chloroacetol sulfate, another compound which selectively esterifies Glu-165. ~a With this compound, the pKa of Glu-165 in the rabbit muscle enzyme was found to be less than 5.0. An exact value could not be determined because of the instability of the enzyme to acid; however, with the more stable yeast enzyme, a pKa of 3.9 was calculated23 Thus, the acidity of the essential carboxyl group is consistent with its postulated role in catalysis.

Haloketone Analogs o] N A D

Reactive analogs of coenzymes are of special interest because they provide the opportunity to probe the active sites of a multitude of dehydrogenases and thus possibly answer questions concerning the degree of homology among nucleotide binding sites and the identity of basic residues involved in hydrogen transfer from the alcohol substrate to the coenzyme. Several a-haloketones that resemble pyridine nucleotides have been designed (primarily by C. Woenckhaus and colleagues) and used successfully to label active-site residues in a number of enzymes, including alcohol dehydrogenase, lactate dehydrogenase, and estradiol 17fl-dehydrogenase (Table II). A very simple derivative of nicotinamide, 3-bromoacetylpyridine, preferentially alkylates both an essential cysteinyl and an essential histidyl residue in pig heart lactate dehydrogenase. ~4 The reagent is selective for the histidyl residue if the enzyme is pretreated with mercuric ions to protect the sulfhydryl group. Subsequent to these investigations the three-dimensional structures of dogfish lactate dehydrogenase and 5~S. G. Waley, Biochem. J. 126, 255 (1972). ~sF. C. Hartman, G. M. LaMuraglia, Y. Tomozawa, and R. Wolfenden, Biochemistry 14, 5274 (1975). 54C. Woenckhaus, J. Berghiiuser, and G. Pfleiderer, Hoppe-Seyler's Z. Physiol. Chem. 350, 473 (1969).

146

GENERAL METHODOLOGr

0

Z O

0

0

r. 0 r~

I

[10]

[10]

HA.LOKETONES

147

3"

r..)

8~

=Z

I

0--'0

o

o~

o=~-o

[~

o:~

~

~o O--m--O

I 0

o=~-~ 0

O~q

o

~'

ro

[0--0 o

~

-=o H ~---

o" .~ ~0

148

[10]

GENERAL METHODOLOGY

cO D,-

D,.

v

e~

~.~

. ~=~ D,. ~

O--co NM

~Q

E~

I

0

0----~--0 0

I

0"-~--0 I m 0 0

r~

0

.

~

0

0

~

0

0

,,0 ~

dN~ddNddN-~4

~

[10]

ttALOKETONES

149

its ternary complex with NAD-pyruvate were determined. 5~,~8 The histidyl residue (His-195), first implicated in catalysis by its selective modification with 3-bromoacetylpyridine,~4 appears to be involved in proton transfer to or from the substrate, since in the three-dimensional structure the imidazole ring of His-195 is oriented toward the carbonyl of pyruvate27 Although numerous chemical modification studies, 58-61 in addition to the one with 3-bromoacetylpyridine, have implicated Cys-165 in catalysis, the crystallographic work suggests that this sulfhydryl does not play a direct role in catalysis but occupies a position near the active site. 57 Bromoacetylpyridine is prepared by bromination of commercially available [14C]acetylpyridine (labeled in the carbonyl carbon). With the other compounds shown in Table II, the halogen is also inserted (usually by direct halogenation) in the last step of the synthesis. In the recently described 62 synthesis of 3-chloroacetylpyridine adenine dinucleotide, the immediate precursor is the corresponding 3-diazomethylketone. The diazo group is then exchanged for chlorine by treatment with lithium chloride in hydrochloric acid, a procedure similar to one used for the preparation of halomethylketone derivatives of amino acidsY -4 Amino acid residues of dehydrogenases modified by coenzyme analogs have been identified in several cases by the laborious but unequivocal sequential procedures of reduction with borohydride, proteolytic digestion, and characterization of purified peptides carrying the reagent moiety. If the primary sequence of the enzyme is known, the amino acid compositions of the pure peptides normally reveal the kind of residue modified (by the absence of one residue equivalent of a free amino acid and its replacement with an amino acid derivative) and its location in 5~M. J. Adams, M. Buehner, K. Chandrasekhar, G. C. Ford, M. L. Hackert, A. Liljas, P. Lentz, Jr., S. T. Rao, M. G. Rossmann, I. L. Smiley, and J. L. White, in "Protein-Protein Interactions" (J. Jaenicke and E. Helmreich, eds.), p. 139. Springer-Verlag, Berlin and New York, 1972. M. G. Rossmann, M. J. Adams, M. Buehner, G. C. Ford, M. L. ttackert, P. J. Lentz, Jr., A. McPherson, Jr., R. W. Schevitz, and I. L. Smiley, Cold Spring Harbor Symp. Quant. Biol. 36, 179 (1971). ~7M. J. Adams, M. Buehner, K. Chandrasekhar, G. C. Ford, M. L. Hackert, A. Liljas, M. G. Rossmann, I. E. Smiley, W. S. Allison, J. Everse, N. O. Kaplan, and S. S. Taylor, Proc. Natl. Acad. Sci. U.S.A. 79, 1968 (1973). A. H. Gold and H. L. Segal, Biochemistry 4, 1506 (1965). ~gj. j . Holbrook, Biochem. Z. 344, 141 (1966). T. P. Fondy, J. Everse, G. A. Driscoll, F. Castillo, F. E. Stolzenbach, and N. O. Kaplan, J. Biol. Chem. 240, 4219 (1965). 61j . j . Holbrook and R. A. Stinson, Biochem. J. 129, 289 (1970). ~ J. Biellmann, G. Branlant, B. Y. Foucaud, and M. J. Jung, FEBS Lett. 40, 29 (1974).

150

GENERAL METHODOLOGY

[10]

NH2 N ~ I I C-CHzBr+ HS- CH2-CH-CO2H I 0

I

-HBr

No oC_CH2~S_CH2_CH_CO2H -H20~ II

f

Fie. 6. Reaction of cysteJnewith bromoacety]pyridine. the primary sequence. For example, in a study of the interaction of horse liver and yeast alcohol dehydrogenases by nicotinamide [5-(bromoacetyl)-4-methylimidazol-l-yl] dinucleotide (which is active as a coenzyme), peptides were isolated whose amino acid compositions were consistent with Cys-43 (which corresponds to Cys-46 in the liver enzyme) as the site of alkylation in the yeast enzyme and Cys-174 as the site of alkylation in the horse liver enzyme23 Recent crystallographic data show that Cys-46 and Cys-174 in the horse liver enzyme function in binding of the catalytic zinc atom. e4 Two other approaches have been used to identify the residues modified by these nucleotide derivatives: (a) oxidation with hydrogen peroxide and subsequent identification of carboxymethyl amino acids in acid hydrolyzates, and (b) reduction with borohydride and comparison of derivatives in the hydrolyzates to synthetically prepared standards. The second approach was used in identifying a sulfhydryl and an imidazole group as the sites of modifiication of lactate dehydrogenase by bromoacetylpyridine2~ An interesting observation was the spontaneous formation of a thiazine (by intramolecular condensation between the ketone group of the reagent moiety and the amino group of cysteine) from the initial S-alkylated product (Fig. 6). This serves to emphasize that small peptides are generally preferable to free amino acids in the synthesis of derivatives in which selective side-chain substitution is desired. In a very nice example of affinity labeling in which 3-chloroacetylpyridine adenine dinucleotide (a compound that is active as a hydrogen acceptor) is shown to be active-site-specific for estradiol 17fl-dehydrogenase from human placenta, Biellmann et al. 6~ have identified the u It. JSrnvall, C. Woencklaus, and G. Johnscher, Eur. J. Biochem. 53, 71 (1975). H. Eklund, B. Nordstr6m, E. Zeppezauer, G. S6derlund, I. Ohlsson, T. Boiwe, and C.-I. Br~inddn,FEBS Lett. 44, 200 (1974). J. Biellmann, G. Branlant, J. Nicolas, M. Ports, B. Descomps, and A. Crastes de Paulet, Eur. J. Biochem. 63, 477 (1976).

[10]

I-IALOKETONE8

151

TABLE I I I SELECTED EXAMPLES OF SITE-SPECIFIC HALOKETONE$

Reagent

Protein

Residue modified References

r CHuB C---O

Transglutaminase

Cys

a

Elongation factor Tu

--

b, c

Cys

d

OH 2-Bromo-4'-hydroxy-3'nitroacetophenone O

/~\

HsC-"~

II t

C--CHiC1

0

If

~>----S - - N H - - C - - H

° d

Luciferase

(S) -N- [a - (Chloroacetyl)phenethyl]p - to luene sullonamide O tl

C--CHzC1

] NH2~C--H I

T y r / P h e transport protein in Bacillus subtilis

(S)-3-Amino- 1-ehloro4-phenyl- 2-butanone

C~CH2C1

I

CI'I~NH2 1-Amino- 3- chloro2-propanone

Neutral amino acid transport protein in Trypanosoma brucei

152

GENERAL METHODOLOGY

[10]

TABLE I I I (Continued)

Reagent

Protein

Residue modified References

C--CH~Br I NI~-- --H CHs--~H ]

L-Isoleucine: tRNA ligase

Cys

CHs (3S, 4S)-3-Amino-l-bromo4- methy 1- 2- hexanone O II C--OH

NH2--~--H Carbamylphosphate synthetase c~ct

(S)- 2-Amino- 5chlorolevulinic acid

011 c - - c H , ct I NH~--C--H )

i,i

L-Valine: tRNA ligase

HsC/HC"CHs ($)-3-Amino-l-chloro4-methyl-2-pentanone O II/O-

c,H,--OP