the journal of vol. 266, no 32, 15, pp, 21657-21665,1991 ... · the journal of biological chemistry...

THE J O U R N A L OF BIOLOGICAL CHEMISTRY ‘c 1991 hy The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No 32, Issue of November 15, pp, 21657-21665,1991 Printed in U.S.A.

Mechanism-based Inactivation of Alanine Racemase by 3 -Halovinylglycines*

(Received for publication, December 17, 1990)

Nancy A. Thornberry$#, Herbert G. Bull$, David Taubll, Kenneth E. WilsonII, Guillermo Gimenqz-Gallego**, Avery Rosegay$$, Denis D. SodermanOB, and Arthur A. Patchettll From the Departments of $Enzymology, VExploratory Chemistry, IlNatural Product Chemistry, $$Animal and Exploratory Drug Metabolism, and §§Growth Factor Research, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 07065

Alanine racemase, an enzyme important to bacterial cell wall synthesis, is irreversibly inactivated by 3- chloro- and 3-fluorovinylglycine. Using alanine racemase purified to homogeneity from Escherichia coli B, the efficient inactivation produced a lethal event for every 2.2 f 0.2 nonlethal turnovers, compared to 1 in 800 for fluoroalanine. The mechanism of inhibition involves enzyme-catalyzed halide elimination to form an allenic intermediate that partitions between reversible and irreversible covalent adducts, in the ratio 3:7. The reversible adduct (X,,, = 5 16 nm) decays to regenerate free enzyme with a half-life of 23 min. The lethal event involves irreversible alkylation of a tyrosine residue in the sequence -Val-Gly-Tyr-Gly-Gly-Arg. The second-order rate constant for this process with D-chlorovinylglycine (122 & 14 M” s-’), the most reactive analog examined, is faster than the equivalent rate constant for D-fluoroalanine (93 M“ s-’). The high killing efficiency and fast turnover of these mechanism-based inhibitors suggest that their design, employing the haloethylene moiety to generate a reactive allene during catalysis, could be extended to provide useful inhibitors of a variety of enzymes that conduct carbanion chemistry.

Alanine racemase is unique to bacteria, providing D-alanine essential for synthesis of the peptidoglycan layer of the bacterial cell wall (1). This enzyme has long been considered an attractive target for design of antibiotics, and the utility of this approach has been established by fluoro-D-alanine, an inhibitor of the enzyme which is a potent, broad spectrum, orally active antibiotic (2). However, safety issues unrelated to its mechanism of action have hindered clinical application (3).

Continuing the search for novel inhibitors of alanine racemase, we recently described halovinylglycines as a new class of potent mechanism-based inhibitors (4). The mechanistic rationale for this approach, originally proposed by Robert Abeles (Brandeis University), is compared below to the well established mechanism of inhibition by fluoroalanine.

Due to their carbanion chemistry, alanine racemase and other pyridoxal-dependent enzymes have been a productive area for design of “mechanism-based” or “suicide” inhibitors,

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

7120. 1 To whom correspondence should be addressed. Tel.: 908-594-

quez, 144, 28006 Madrid, Spain. ** Present address: Centro de Investigaciones Biologicas, Velaz-

the subject of a recent review by Walsh (5). Such inhibitors are processed by the enzyme as if potential substrates, yet lead, in a diversion from the normal course of the reaction, to inactivation of the enzyme (6). Fluoroalanine and analogous /%substituted alanines are classic examples (7).

The normal catalytic pathway for racemization of alanine proceeds by proton abstraction to give a pyridoxal phosphate- stabilized carbanion intermediate. In the case of haloalanines a, this carbanion expels halide to give the reactive aminoacrylate b, an uncovered nucleophile, which in 1 out of 800 events attacks the pyridoxal-imino linkage rather than diffus- ing harmlessly from the enzyme surface (8,9).

X H I 1

NHZ I

NHZ l

a b

CHz-C-CM3H 5 CHz=C“COOH

X H

NU, I

c d

CHZ=C“C-”OH I I “3. CH,=C=C”COOH N H Z I

In contrast, the expulsion of halide from halovinylglycines c is expected to generate the allene d, a reactive electrophile. This type of enzyme-activated inhibitor design, involving diversion of the substrate analog to an allenic intermediate, resembles the application of acetylenic compounds as irreversible inhibitors of other carbanion-associated enzymatic reactions. The inhibition of /3-hydroxydecanoylthiolester de- hydrase by 3-decanoylthiolester elucidated by Bloch (IO), and the application of propargylglycine and acetylenic thioesters as mechanism-based inhibitors of y-cystathionase (11) and butyryl-CoA dehydrogenase by Fendrich and Abeles (12), are examples. Precedent for the use of the haloethylene moiety for entry to an allenic intermediate comes from inhibition of plasma amine oxidase by 2-chloroallylamine developed by Abeles and co-workers (13). This strategy also has been adopted by a fungus, Aminita pseudoporphyria Hongo, which produces the antibacterial amino acid ~-2-amino-4-chloro-4- pentenoic acid, recently shown to be a mechanism-based inactivator of L-methionine y-lyase (14).

Previous studies in this laboratory established that the halovinylglycines are exceptionally efficient inhibitors of alanine racemase, producing one lethal inactivation event every 2.2 +. 0.2 nonlethal turnovers for the enzyme from Escherichia coli B (4). The purpose of this paper is to describe the detailed kinetic and chemical mechanism of inhibition by these inhibitors, as a basis for understanding their efficiency.

EXPERIMENTAL PROCEDURES

Materials Commercially available compounds were purchased from the fol-

lowing sources: D-alanine and L-alanine from Aldrich; L-alanine

21657

21658 Inactivation of Alanine Racemase

dehydrogenase, D-amino acid oxidase, and lactate dehydrogenase from Sigma; ethyl acetoacetate from Aldrich; and L-1-tosylamido-2- phenylethyl chloromethyl ketone-treated bovine pancreatic trypsin from Worthington, a-Aminoacetone hydrochloride was prepared by a published procedure (17).

Syntheses Of D-, L-, and DL-3-chloro- and 3-fluorovinylglycine from the corresponding vinylglycine of 2-fluoroacrolein, respectively, have been outlined (4) and described in detail (15). The synthesis of D- [4-3H2]3-chlorovinylglycine was based on that of unlabeled D-3-chlorovinylglycine and proceeded from carbobenzoxy D-[4-3H2]vinylglycine methyl ester, which was in turn obtained via equilibration of D- methionine sulfoxide with Na03H (16). Significant modifications in the general procedure are described below. The specific activity of ~-[4-~H~]3-chlorovinylglycine was 83 Ci/mol. ~-f4-~H~,5-~HR/Methionin~ Sulfoxide-In a stoppered tube, a so-

lution of D-methionine sulfoxide (0.50 g, 3.03 mmol) and NaOH (260 mg, 6.37 mmol) in 3H20 (4.8 ml, 400 Ci) was held in a bath at 97 "C for 36 h. The solution was cooled, concentrated HCI (0.6 ml) was added, and the solvent was evaporated to obtain a solid residue. The residue was dissolved in water and the solution was concentrated several times to remove most of the readily exchangeable tritium. A solution of the tritiated amino acid was applied to a cation exchange resin column (Bio-Rad AG 50 X 8,200-400 mesh, H' form, 25 ml, 42 meq) and the column was washed with water (200 ml). The amino acid was eluted with 4 N NH,OH. The eluate was evaporated under reduced pressure to a pale yellow crystalline solid which was dried at 60 "C under vacuum to afford 0.42 g of tritiated methionine sulfoxide, specific activity 2.1 Ci/mmol. D- f4-3HJ-Carbobenzoxyuinylglycine Methyl Ester-Pyrolysis of

the tritiated carbobenzoxy methionine sulfoxide methyl ester (16) was carried out in an air-cooled apparatus consisting of a bulb connected to a vertical receiving tube containing three annular wells as condensate traps for collecting the distillate. Tritiated sulfoxide (286 mg) was placed in the bottom bulb and the system was evacuated to 2.0-2.5 mm Hg. The bulb was immersed in a bath at 170 "C to a depth at which the lower condensate trap was 1-2 mm above the surface of the bath. The bath temperature was raised to 190 "C over 10 min and held at 190 "C for 10 min, a t which time the bulb was removed from the bath. Using ethyl acetate, the contents of the condensate traps were pooled (Fraction A) and the residue in the bottom bulb was removed (Fraction B). Fraction A was applied to two silica gel plates (Analtech SGF, preabsorbent, 500 p, 20 X 20 cm) and developed twice in ethyl acetakhexane, 1:3. The product bands were eluted with ethyl acetate to afford >90% pure (by radiochromatographic analysis) [4-3H2]carbobenzoxyvinylglycine methyl ester (25 mg, 126 mCi, 1.25 Ci/mmol). An additional 14 mg (70 mCi) was recovered by multiple preparative thin layer chromatography of Frac- tion B. Further purification was carried out by silica gel column chromatography (E. Merck Lobar, run in ethyl acetate:hexane, 1882). At this stage, carrier carbobenzoxyvinylglycine methyl ester was added to adjust the specific activity to approximately 80 mCi/mmol.

~-[4-~HJ3-Chlorovinylglycine-Chlorination of ~-[4-~H~]carbobenzoxyvinylglycine methyl ester was accomplished as previously described for the unlabeled species (15), except that NaIO, in aqueous methanol was the oxidant rather than ozone. Following isolation of the crude ester by silica gel (Lobar) chromatography, final purification was carried out by multiple semipreparative normal phase high pressure liquid chromatography (Zorbax SIL, 2 columns (total 50 cm); ethanokhexane, 0.899.2, flow rate 6.5 ml/min, absorbance de- tection at 210 nm, retention time 13.5 min). The specific activity of the purified product was 83 mCi/mol based on its chromatographic peak area relative to a standard. Following deprotection (6 N HCI, 100 'C, 1 h) on a 0.5-mg scale, ~-[4-~H~]3-chlorovinylglycine was isolated by chromatography on a cation exchange resin (Bio-Rad AG 50 X 8, 1 g, H' form, flow rate 0.4 ml/min) using 40 mM HCl as eluant. The retention volume of the product peak was 80 ml. The specific activity of the purified [~HH]3-chlorovinylglycine is expected to be the same as that of its precursor, since similar deprotection of [4-2H2]vinylglycine is known to occur without loss of deuterium (16). By thin layer radiochromatographic analysis (E. Merck silica gel plate, 1-butanobacetic acid water:ethyl acetate, l:l:l:l, replicative form 0.5), the purity found was 98.6%. Analysis by high pressure liquid chromatography (Zorbax SCX, 1 mM citrate buffer at pH 2.5, flow rate 1 ml/min) showed 98.0% of the radioactivity traveled as a single peak (retention time: 7 min).

Spectrophotometric Assays Alanine racemase activity was measured in both D+L and L ~ D

directions using coupled continuous spectrophotometric assays adapted from the literature (7, 18). A unit is refined as the amount of enzyme that catalyzes formation of 1 &mol of product/min under saturating conditions.

hL-Racemization of D-alanine to L-alanine is coupled with oxidation of L-alanine to pyruvate and ammonia in an NAD-dependent reaction catalyzed by saturating levels of L-alanine dehydrogenase. The NADH produced from the coupled reaction was monitored continuously at 340 nm in a Cary-219 recording spectrophotometer, using an extinction coefficient of 6200 M" cm". A typical reaction (1 ml) contained 2 X units of racemase, 0.01 M NAD, 0.9 units of L-alanine dehydrogenase, and 5 mM D-alanine in 0.1 M CHES' a t pH 9.00 and 25 "C. After a lag period of 5 min, the reactions were monitored for 0.1 A units, corresponding to less than 1% conversion of substrate to product.

-"The conversion of L-alanine to D-alanine is monitored using two coupling enzymes: D-aminO acid oxidase catalyzes the conversion of D-alanine to pyruvate which in turn is reduced to lactate by lactate dehydrogenase. The decrease in absorbance due to consumption of NADH was monitored as above. A typical 1-ml reaction mixture contained 4.35 units of D-amino acid oxidase, 500 units of lactate dehydrogenase, 300 pM NADH, and 10 mM L-alanine in 0.1 M Hepes at pH 8.00 and 25 "C. Under these conditions the initial absorbance of the reaction due to NADH is 1.86. It is crucial to use highly purified L-alanine (>99.9%) to avoid an appreciable background from contam- inating D-alanine. Under these assay conditions, after a brief lag phase (4 min) the reactions were linear for an absorbance range of at least 0.5 units. Lag periods in both directions were in good accord with theoretical expectations based on K, and VmaX values of the coupling enzymes.

Data Analysis All kinetic constants were computed by direct tits of the data to

the appropriate equation using a nonlinear least squares analysis computer program. This program, when furnished with the equation and its partial derivatives with respect to each unknown parameter, uses the Marquardt algorithm to converge on the best estimates of the parameters and provides the standard error of each estimate.

P Us*t + ( u o - v a ) * ( l - exp(-k&,.*t))/k&,, + P o (1)

u = u, + (u, - u,)*exp(-b.*t) (2)

y = a*exp(-kobs*t) + b (3)

The derivation and applications of Equation 1 have been developed fully elsewhere (19). This equation is the integrated form of the simpler Equation 2, that predicts a first-order increase or decrease in the reaction velocity from an initial steady-state, v,, to a final steady- state, v., with a rate constant kobs. The integrated form of the equation permits the raw data ([product], time) to be fit directly to obtain estimates of the parameters.

Enzyme Purification The method used for purification of alanine racemase was adapted

from the literature (7), and results are summarized in Table I. During purification the D-L assay was employed, as the L-D alternative was subject to appreciable interference in crude preparations. The reported specific activities are based on protein concentrations esti- mated by the absorbance at 280 nm, assuming an extinction coefficient of 1 (mg/ml protein)" cm", with the exception of that for homogeneous enzyme, where the protein concentration is based on an extinction coefficient of 2 (mg/ml of protein)" cm", derived as described below.

Disruption of Cells-A culture of E. coli B (strain no. MB1967) was grown on L-alanine as the sole carbon source. After harvesting, the cells (71.4-g dry cell weight) were pelleted by centrifugation at 10,000 X g for 20 min. To break the cells, the pellet (208 g) was resuspended in 523 ml of 20 mM Tris, 0.05 M NaCI, pH 8.00, buffer and sonicated on ice in 200-ml batches (Branson Sonifier 350, 50% duty cycle, medium probe, micro-tip limit) until maximum enzyme activity was achieved (-45 min). The sonicate (873 ml, 6577 units, 0.073 units/

The abbreviations used are: CHES, 2-(cyclohexylamino)- ethanesulfonic acid; Hepes, 4-(2-hydroxyethyl)-l-piperazineethane- sulfonic acid.

Inactivation of Alanine Racemase 21659 TABLE I

Purification of alanine r a c e m e

Sample Volume Protein Units Purification Recovery Specific activity

ml mg unitslmg -fold 75 Sonicated cells 873 90,000" 6,577 0.073 1 100 25,000 X g supernatant 763 40,000" 5,540 0.138 2 84 1st DEAE step 413 4,130 2,858 0.67 9 42 2nd DEAE step 91 1,050 2,058 2.0 27 31 Low-salt precipitate 3 11 1,423 133.0 1,822 22

Values are approximate.

mg of protein) was dialyzed overnight against 12 liters of 20 mM Tris, 0.05 M NaCl, pH 8.00, buffer. Cell debris was removed by centrifugation at 25,000 X g for 1 h.

DEAE Chromatography-The supernatant (763 ml, 5540 units, 0.138 units/mg of protein) was applied to a DEAE-52 (Whatman) anion exchange column (Pharmacia K26, 2.6 X 40 cm, 200 ml total volume) equilibrated with 20 mM Tris, 0.05 M NaC1, pH 8.00, at a flow rate of 1 ml/min at 4 'C. After washing the column with 5 volumes of equilibration buffer, the enzyme was eluted by application of an 1100-ml gradient of 0.05-0.5 M NaCl. A single peak of enzymatic activity was obtained, containing 2858 units and corresponding to 50% recovery. The fractions containing active enzyme were pooled and dialyzed overnight against 4 liters of 0.01 M Hepes, pH 7.00, to remove salt. The anion exchange chromatography was repeated with the dialysate (413 ml, 2858 units, 0.67 units/mg of protein) under different buffer conditions (0.01 M Hepes, pH 7.00). In this case a single peak of activity was again eluted containing 75% of the applied enzymatic activity. The active fractions were pooled (91 ml, 2058 units, 2.0 units/mg of protein) and stored at -70 "C.

Precipitation of Enzyme-The chromatography described above results in purification of the enzyme to 2% homogeneity. The remaining purification was accomplished through the fortuitously dis- covered precipitation of virtually homogeneous enzyme that occurred upon dialysis against low ionic strength buffer. To precipitate the enzyme, the pooled fractions were thawed and dialyzed overnight against 4 liters of 20 mM Tris, 0.01 M NaC1, pH 7.50, buffer at 4 "C. The sample was then concentrated 12-fold to 7.75 ml in an Amicon ultrafiltration apparatus using a PM-10 membrane. The resulting cloudy solution was centrifuged at 100,000 X g for 1 h. Under these conditions 72% of the total enzymatic activity was precipitated. Resuspension of the pellet in 0.1 M Hepes, 0.5 M NaC1, pH 8.00, buffer gave quantitative recovery and complete solubilization of the enzyme activity, as determined by centrifugation at 100,000 X g, for 1 h. The specific activity of this sample, containing 1423 units, was 133 units/mg of protein, corresponding to a recovery of 70% in a 66- fold purification step. The overall recovery of enzymatic activity is 22% and the purification is 1800-fold. The enzyme is stable in 0.1 M Hepes, 0.5 M NaCl, pH 8.00, buffer for at least 2 years a t -70 "C, and for several weeks at 4 "C.

Enzyme Characterization The purification scheme described above yields a solution that

gives a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and corresponds to a molecular weight of approximately 50,000 g/mol, in good agreement with the subunit molecular weight of 49,000 reported by Wang and Walsh for E. coli racemase (7). The spectrum of the purified enzyme is characteristic of pyridoxal phosphate-dependent enzymes, having a chromophore at 418 nm attributed to the enzyme-cofactor Schiff s base linkage. An accurate measure of enzyme concentration was obtained by determining pyridoxal phosphate content, as described below. Based on this method, extinction coefficients for homogeneous enzyme are 2 (mg/ml of protein)" cm" at 280 nm and 9500 M" cm" at 418 nm. The latter value agrees closely with those reported for Salmonella typhimurium encoded alr and dadB alanine racemases: 9300 M" cm" and 10,000 M" cm", respectively (20, 21).

Measuring conversion of D-alanine to L-alanine at pH 9.00, the specific activity of homogeneous enzyme is 133 units/mg. The kinetic constants K, and kcat are 1.18 f 0.07 mM and 111 f 2 s-l, respectively. In the L-alanine to D-alanine direction at pH 8.00, the corresponding kinetic constants are 6.0 f 0.3 mM and 390 +. 8 s" and the specific activity of homogeneous enzyme is 460 units/mg.

Determination of Enzyme Concentration Concentration of homogeneous enzyme was determined by meas-

urement of pyridoxal phosphate content. This was accomplished by heat denaturation of the enzyme at 100 "C for 2 min, followed by centrifugation of precipitated protein at 500,000 X g for 15 min. Spectra of the supernatants were identical to that of native pyridoxal phosphate, having a maximum absorbance at 389 nm under identical conditions (0.1 M Hepes, pH 8.00). Enzyme concentration was calculated based on an extinction coefficient for native pyridoxal phosphate at 389 nm of 5,476 M" cm" and assuming 1 mol of pyridoxal phosphate/49,000 M, subunit active enzyme.

Kinetic Studies Unless otherwise indicated, all determinations of rates of inacti-

vation were performed in 0.1 M Hepes, 0.1 M NaCI, 1 mg/ml of bovine serum albumin at pH 8.00 and 25 "C using homogeneous enzyme. Typically, the kinetic measurements were performed discontinuously by incubating small solutions of enzyme and inhibitor for various lengths of time prior to 200-fold dilution into the continuous D+L assay. This discontinuous method was used because the coupling enzyme, L-alanine dehydrogenase, is susceptible to inhibition by halovinylglycines,

Equilibrium Dialysis The partition ratio of ~-[~H]chlor~vinylglycine was measured by

equilibrium dialysis in paired 250-p1 chambers separated by dialysis tubing (Spectrapor 12,000-14,000 molecular weight cutoff). Concen- trations of enzyme and inhibitor were selected to insure complete processing of inhibitor, as verified by the presence of active enzyme at infinite time. Under these conditions the ratio of bound to free radioactivity represents the ratio of moles of enzyme inactivated to moles of product formed. Inhibitor (0.3 nmol, 5.7 X 104 dpm) and enzyme (0.5 nmol) were placed in the equilibrium dialysis chambers. Standard conditions were 0.1 M Hepes, 0.1 M NaCl, 1 mg/ml of bovine serum albumin at pH 8.00 and 25 "C. Aliquots from each chamber were assayed periodically for enzymatic activity and radioactive content. The reaction was complete after 24 h and the distribution of radioactivity corresponded to 0.10 nmol of bound and 0.21 nmol of free inhibitor. Analysis of residual enzyme activity indicated that 22% of the total activity, corresponding to 0.11 nmol, was inhibited, in good agreement with the measure of bound radioactivity. The partition ratio, given by the ratio of free to bound radioactivity, was calculated to be 2.20 f 0.09 from duplicate determinations.

Actiue Site Labeling Alanine racemase (13 nmol, 0.65 mg) was treated with a 5-fold

excess of ~-[~H]chlorovinylglycine (65 nmol, 83 pCi/pmol) in a total reaction volume of 625 wl in 0.1 M Hepes, 0.1 M NaCl, pH 8.00, buffer at 25 "C. Incubation for 14 h resulted in irreversible inhibition of >99% of the total activity. The labeled enzyme was desalted on a gel filtration column (Sephadex G-25, fine, 0.9 X 60 cm) equilibrated with 0.1 M NHdHC03 at a flow rate of 0.5 ml/min. Analysis of the fractions (1 ml) for radioactive content revealed two well resolved peaks. The protein fraction, eluting in the void volume, contained 20% of the applied radioactivity, and the small molecule fraction contained the remaining 80%.

Reductive Alkylation A sample (13 nmol, 0.65 mg) of the lyophilized salt-free D-[%]

chlorovinylglycine-inactivated alanine racemase was denatured with 6 M guanidine-HCl, 0.5 M Tris, 2 mM EDTA, and 10 mM dithiothreitol in a 1-ml total volume at pH 8.5. The sample was purged with N, and

2 1660 Inactivation of Alanine Racemase incubated in a sealed tube at 50 "C. After 2 h, 500 pl of 0.1 M iodoacetic acid in 0.5 M Tris at pH 8.5 was added and the incubation continued in the dark for 30 min at 25 "C. The carboxymethylation reaction was quenched by the addition of 500 pl of 0.1 M dithiothreitol.

Trypsin Digestion To prepare the sample for trypsin digestion, the reduced carboxy-

methylated protein (13 nmol, 0.65 mg) was diluted 6.5-fold to 12.45 ml with 0.1 M Tris at pH 8.00. Under these conditions a majority of the protein precipitates and can be quantitatively recovered in the pellet of a 100,000 X g centrifugation. The final concentrations of reagents in the pellet are acceptable conditions for successful digestion with trypsin. It was necessary to use this method instead of the conventional methods for removal of reagents (dialysis or chromatography) since denatured carboxymethylated protein precipitated when the guanidine hydrochloride was removed, leaving the protein stuck to dialysis tubing or column packing. The pellet from the 100,000 X g centrifugation was treated with 200 pl of 0.125 mg/ml of trypsin in 0.1 M Tris at pH 8.00 overnight at 37 "C with slow mixing. The resulting clear solution contained 8 nmol of peptide based on radioactive content, corresponding to recovery of 62% of the label.

Peptide Purification The tryptic digest (8 nmol peptide) was applied to a Vydac C,

reverse-phase high pressure liquid chromatography column (300 A pore size, 5-pm particle size, 4.6 mm X 25 cm) equilibrated with 10 mM trifluoroacetic acid at a flow rate of 0.5 ml/min. The effluent was monitored continuously for absorbance at 230 nm and peaks were collected manually. Analysis of the peaks (and all other fractions) for radioactive content revealed that one, eluting at 8 min, contained 34% of the total radioactivity applied. Elution of the remaining peptides was accomplished with a 2-h linear gradient to 66% CH&N containing 4 mM trifluoroacetic acid, but none of these fractions was found to contain appreciable further radioactivity.

Peptide Sequencing Peptide samples (75 pmol) were subjected to amino-terminal Ed-

man degradation on Polybrene-coated filters using an Applied Bio- systems 470A/120A microsequenator. A portion of the sample at each cycle was analyzed for radioactive content. Duplicate determinations on the radioactivity containing fraction revealed a single peptide of 6 amino acid residues: H2N-Val-Gly-X-Gly-Gly-Arg-COOH. The sequence was determined with a high yield of the first residue (96%) and a very good repetitive yield (95%). Radioactivity was released with the amino acid detected in cycle 3, which has the same elution time as Tyr. However, only a portion (15%) of the total radioactivity expected was recovered, suggesting instability of the inactivator- derived adduct to sequencing conditions. Consistent with this dis- crepancy, the amount of phenylthiohydantoin-derivatives recovered at this cycle was only 70% of that expected based on initial and repetitive yield calculations. Thus, this residue was tentatively as- signed to tyrosine. A second sample of this peptide was prepared and subjected to amino acid analysis for confirmation.

Amino Acid Analysis Peptide samples (400 pmol) were vacuum-centrifuged to dryness

in glass vials (22), hydrolyzed, derivatized with phenylisothiocyanate, and chromatographed and quantitated as described (23). In this analysis, no detectable radioactivity was associated with any of the fractions, indicating the hydrolysis conditions (constant boiling HCI vapors at 110 'C for 20 h) were harsh enough to completely dissociate the inactivator.

Identification of a-Aminoacetone A high pressure liquid chromatography method was developed to

assay for a-aminoacetone in a sampie containing the low molecular weight components from the ~-[~H]chlorovinylglycine inactivation mixture (15.3 ml, 1.8 X lo' dpm, 98 nmol of tritium) that was isolated from labeled enzyme by gel filtration. This procedure involved pre- column derivatization of a-aminoacetone with ethylacetoacetate to form 2,4-dimethyl-3-ethoxycarbonylpyrrole, which could be monitored colorimetrically (24, 25). A 300-pl aliquot of the sample, containing approximately 3 x lo5 dpm, was treated successively with: 1) 30 pl of 1 N H2S0,; 2) 30 p1 of an 870 pg/ml of aqueous solution of cold aminoacetone hydrochloride as an internal standard; and 3) 150 pl of a reagent solution containing 3 M sodium acetate, pH 4.6,

ethanol, acetonitrile, and ethyl acetoacetate (5:l:l:l). The reaction mixture was incubated at 100 "C for 1 h in a sealed tube. Under these conditions, the conversion of aminoacetone to the pyrrole is quantitative within experimental error. The sample was applied to a Dy- namax C,a reverse phase high pressure liquid chromatography column (3-pm particle size, 3 mm X 4 cm) and run isocratically at ambient temperature with 33:67 acetonitrile, 10 mM potassium phosphate, pH 7, conditions under which the pyrrole is separated with base-line resolution from unreacted D-chlorovinylglycine. The effluent was monitored continuously for absorbance at 270 nm and 0.1-ml fractions were evaluated for radioactive content. Analyses were performed on mixtures predicted to contain 43.8% total low molecular weight product and 56.2% unreacted D-chlorovinylglycine, based on the partition ratio and moles of labeled enzyme recovered by gel filtration. In a typical run, the total recovery of tritium injected onto the column was 92%, of which 9.1% of the predicted tritium due to product coeluted with the pyrrole internal standard. Replicate analyses on this and another sample from a second active site labeling experiment gave comparable levels of a-aminoacetone (+1.6% error), and appropriate controls indicated there was no nonenzymatic formation of a- aminoacetone from D-chlorovinylglycine under the labeling conditions.

RESULTS

Irreversible Inhibition-Alanine racemase was purified to homogeneity from E. coli B, as described in detail under "Experimental Procedures." Four members of the 3-halovinylglycine series were synthesized and tested for inhibition of this enzyme: D-chloro, L-chloro, D-fluoro, and L-fluorovinylglycine. All show time-dependent inhibition of the enzyme that is irreversible, as demonstrated by exhaustive dialysis of preformed enzyme-inhibitor complex.

For irreversible mechanism-based inhibitors an important indicator of potency is the partition ratio, the number of turnovers per inactivation event. The values for D- and L- chlorovinylglycine were determined using two different methods: kinetically by titration of enzymatic activity, and physi- cally by equilibrium dialysis with radiolabeled inhibitor. The results from both methods are collected in Table 11. Parallel titration experiments with D-chlorovinylglycine and, for comparison, D-fluoroalanine are shown in Fig. 1 and indicate that the partition ratio for the halovinylglycine, 2.2 & 0.5, is 450- fold lower than that for D-fluoroalanine. The value obtained for D-fluoroalanine, 980 f 34, is in good agreement with the value of 800 reported by Wang and Walsh (7). The partition ratio for L-chlorovinylglycine was 3, which is identical to that obtained for the D-isomer, within experimental error. The partition ratio for D-chlorovinylglycine was confirmed by equilibrium dialysis using radiolabeled compound. This method is more accurate since the partition ratio is calculated directly from the ratio of free to bound radioactivity and does not rely on an accurate knowledge of enzyme or inhibitor concentration. Results of these studies gave a partition ratio of 2.20 & 0.09, securing the results obtained kinetically by titration.

The fluorovinylglycines are much less reactive than the chlorovinylglycines, as discussed below. This low reactivity precluded accurate determination of their partition ratios, since inordinately high concentrations of enzyme and long incubation times would have been required. However, an upper limit of 40 was established for the racemic mixture.

TABLE I1 Partition ratios

Compound Method Partition ratio

D-Chlorovinylglycine Titration 2.2 k 0.5 ~-[~H]Chlorovinylglycine Equilibrium dialysis 2.2 k 0.1 L-Chlorovinylglycine Titration 3.0 D-Fhoroalanine Titration 937.0 & 32.0

Inactivation of Alanine Racemase 21661

Mluoro-D-alanine, pM

1 2 3 4 5 6 D-chlomvinylglycine, pM

FIG. 1. Titration of alanine racemase by D-chlorovinylglycine and D-fluoroalanine. The two plots compare enzyme activity remaining at infinite time (10 half-lives) after treatment of identical solutions of alanine racemase (1.6 pM) with increasing concentrations of D-chlorovinylglycine or D-fluoroalanine under standard conditions. The residual activity was determined by diluting small portions of the reaction mixtures 2000-fold into the assay solution (D-L). The x intercepts represent the concentration of inhibitor required to inac- tivate all of the enzyme. Based on the results from two similar experiments the partition ratio for D-chlorovinylglycine (2.2 * 0.5) is 450-fold lower than that for D-fluoroalanine (980 f 30). These data are reproduced from Ref. 4.

+ E + P

SCHEME 1

Kinetic Mechanism-The kinetics of inactivation indicate that the 3-halovinylglycines interact in a more complex way with alanine racemase than do classical 8-substituted alanines in that they form both irreversible and reversible adducts, as shown in Scheme 1. This model proposes that halide elimination takes place to give a common reactive intermediate (EX) that partitions three ways: innocuous turnover to product, lethal inactivation (EXinac,ive), and reversible inactivation

Reversible inhibition is evident in the progress curve shown in Fig. 2, which is observed when enzyme is treated with excess inhibitor and diluted into the assay solution. The velocity initially is less than 5% of control, indicating that nearly all of the enzyme is tied up in the form of covalent intermediates or end products, and increases by a first-order process as free enzyme is regenerated. The final steady-state velocity attained in these progress curves is a measure of the quantity of enzyme that becomes irreversibly inhibited during preincubation with inhibitor. Increasing periods of preincubation leave the rate constant unchanged but suppress the final steady-state velocity achieved, and prolonged preincubation (10 h) finally results in no return of activity, indicating

( E X I r a n a i e n t ) .

Time, 8

FIG. 2. Partial recovery from inhibition. This typical absorbance tracing shows the partial recovery of activity (30%) that occurs with time when preformed enzyme-inhibitor complex is diluted soon after its formation into the continuous assay ( U D ) . Data are for 0.1 p~ enzyme and 100 p~ D-chlorovinylglycine, incubated 10 min, and then diluted 1000-fold into the assay. The solid l i n e is predicted by an integrated first-order rate expression (Equation 1) with a rate constant for recovery of activity of 5 X lo-* s” ( t /2 = 23 min). Comparable tracings were seen for all the halovinylglycines, indicating this process is independent of halogen substituent and enantiomeric configuration.

Time. min.

FIG. 3. Time course for irreversible inactivation. The steady-state velocities attained after dilution of preformed enzyme- inhibitor complex into the assay (as in Fig. 2) are shown divided by the uninhibited control velocity and plotted as a function of incubation time. Data are for 52 nM alanine racemase treated with 50 p~ D-chlorovinylglycine. The solid l ine is theoretical for two consecutive first-order processes with rate constants of 6.07 +- 0.36 X s” and 1.54 f 0.18 X lo-* s-’. The rate constant for the fast primary event is a function of inhibitor concentration, leaving group, and enantiomeric configuration; but that for the slow secondary event is independent of all these parameters.

that all of the enzyme is irreversibly inhibited. The first-order rate constant for return of enzyme activity, 5 X s”, corresponds to a half-life of 23 min, and is independent of inhibitor concentration, stereochemistry, and leaving group, supporting the proposed kinetic model.

The transient complex may be regarded as a “waiting room” in which a portion of the enzyme is temporarily protected from irreversible inhibition. This protection is evident in the biphasic irreversible inactivation that is observed when steady-state velocities from the progress curves are plotted as a function of time of incubation. Data for D-chlorovinylglycine, shown in Fig. 3, indicate that 70% of the total activity is lost rapidly (primary event) while the remaining 30% is destroyed slowly (secondary event). This ratio of primary to secondary inactivation events is preserved for all 4 halovinylglycines, which is evidence that the biphasic kinetics are not due to the presence of a rapidly reacting impurity in the inhibitor solutions. The rates of the rapid and slow phases of inactivation each conform separately to first-order processes, as indicated by the solid, theoretical curves in the Figure, and have been measured for each halovinylglycine at several inhibitor concentrations. Additionally, the nucleophilic scav-


enger P-mercaptoethanol (0.1 M) was shown to have no influence on these time courses and biphasic kinetics.

As predicted by the kinetic model, the rate of the primary event is a function of inhibitor concentration. Data for D- chlorovinylglycine, shown in Fig. 4, indicate that the highest accessible concentration M) is still far below saturation for this inhibitor. Similarly, the highest practical concentrations of L-chlorovinylglycine M) also failed to give evidence of saturation. Consequently, only second-order rate constants were determined for these compounds. In contrast, the fluorovinylglycines are much less reactive, and at the highest concentrations employed the rate of the fast event is comparable to that for the slow phase of inactivation, placing a lower limit of 1 X s-' on the maximum velocity of inactivation. Second-order rate constants for the F-analogs were determined at low concentrations M), where the first phase of irreversible inactivation becomes rate-limiting and the second phase is not observed.

A comparison of second-order rate constants for inactivation (k l ) for all 4 halovinylglycines, collected in Table 111, indicates that this parameter is extremely sensitive to halogen substituent. Specifically, the fluoro compounds are more than 3000-fold less reactive than D-chlorovinylglycine. This influence on reactivity correlates with the relative leaving group potential of F- versus C1-. In the case of the chloro-analogs, the rate of inactivation is also sensitive to enantiomeric configuration. The second-order rate constant for L-chlorovinylglycine (3.2 M-' s-I) is 30-fold less than for its D-anomer (122 M" S - ' ) .

The rate of the slow secondary inactivation event is gov- erned by the rate of regeneration of free enzyme from transient complex. In concert with the Scheme, this rate constant (kz = 1 x s-') is independent of inhibitor concentration, halogen leaving group, and enantiomeric configuration. Also as predicted by the Scheme, this rate constant is comparable to the rate constant for partial recovery of activity that is observed when preformed enzyme-inhibitor complex is diluted into the assay solution (5 x s"), which is likewise

,012 1 1

,009 c / I

.02 . 0 4 .06 .08 .10 D-chlorovinylglycine, mM

FIG. 4. Concentration dependence of kprimary for chlorovinylglycines. The observed first-order rate constant for the primary inactivation event is plotted as a function of D-chlorovinylglycine concentration. There is no evidence of saturation at the highest practical concentrations of inhibitor, placing a lower limit on Ki of 100 pM.

TABLE 111 Kinetic constants for inhibition of E. coli B. alanine racemase

Compound kl 10' X k2 "I s-l S"

D-Chlorovinylglycine 122.0 1.2 L-Chlorovinylglycine 3.2 1.2 D-Fluorovinylglycine 0.019 1.0 L-Fluorovinylglycine 0.041 1.0 D-Fluoroalanine 93.0

independent of halogen substituent and enantiomeric configuration.

Proof of Single Enzyme-It was important to be certain that biphasic inhibition was neither the result of having two species of enzyme in the preparation with different suscepti- bilities to inactivator, nor a consequence of half-site reactivity of dimeric enzyme. To rule out these proposals, the enzyme was treated with inhibitor until the primary phase had been completed, and then rescued by gel filtration and treated again with fresh inhibitor. As shown in Fig. 5, the inactivation time course was again biphasic and indistinguishable from that of native enzyme, providing evidence for a single catalytic species.

Characterization of the Transient Complex-Further evidence for the proposed kinetic model, and for the structure of the transient intermediate, comes from spectra taken during inactivation with D-chlorovinylglycine, as shown in Fig. 6. The most striking feature is the rapid formation of an intermediate with an intense long wavelength absorption (X,,, = 516 nm), indicative of extended conjugation with the pyridoxal group, which slowly disappears with a rate constant (0.94 -+ 0.03 X s-'; shown in inset) virtually identical to that of the secondary inactivation event (1.25 +. 0.04 X SF).

Consideration of the kinetic behavior and spectral charac- teristics of this transient complex led to the proposal of chemical structures that would be expected to hydrolyze to release a-aminoacetone. Analysis of the reaction products from inactivation by ~-[~HH]chlorovinylglycine confirmed that [3H]a-aminoacetone is a biproduct and accounts for at least 8.0 +. 1.6% of the total turnovers. Assuming the transient intermediate only decays by hydrolysis and escape, the model predicts this route should account for 16.8% of all turnovers, in reasonable agreement with the amount of a-aminoacetone detected.

Characterization of the Irreversible Complex-Heat denaturation of the irreversible enzyme-inhibitor complex formed from ~-[~H]chlorovinylglycine causes quantitative release of free pyridoxal phosphate, as shown by the spectra in Fig. 7, with the "H-labeled inhibitor remaining firmly attached to the protein. In the native enzyme, this protein-inhibitor linkage is also stable to exposure to mild acidic (pH 2) and basic

*o 4 4 0 , 0

100 200 300 400 500 Time, min.

FIG. 5. Proof of single enzyme species. An overlay of time courses for irreversible inactivation of native enzyme (circles) and enzyme rescued after partial inactivation with inhibitor (triangles) is shown as the mole fractions of active enzyme versus time. In this experiment enzyme (1.6 pM, 790 pmol) and D-chlorovinylglycine (100 PM) were incubated together until the rapid phase of irreversible inactivation was complete (10 min). Then a portion of the enzyme (630 pmol) was quickly separated from excess inhibitor on a Sephadex G-25 column (1 X 40 cm, run in the standard buffer a t a flow of 1.23 ml/min), during which it underwent 30% reactivation (as in Fig. 2). Recovered enzyme (290 pmol active) was then treated with fresh inhibitor (100 p ~ ) and the time course re-determined. The superim- posable curves rule out the presence of a second catalytic species as an explanation for the biphasic kinetic behavior.


.3

Native Enzyme P .2

a

.1

I

300 400 500 600

Wavelength, nm

FIG. 6. Inactivation spectra. Spectra are shown at various times for inactivation of 19.5 pM alanine racemase with 111 pM D-chlorovinylglycine in 0.1 M Hepes, 0.1 M NaC1, pH 8.00, buffer. Times indicated are: a, 10 min; b, 60 min; c, 140 min; and d, 341 min. The fast primary phase of irreversible inactivation is accompanied by three spectral changes: 1) loss of the characteristic lysine-pyridoxal Schiffs base absorbance at 418 nm; 2) appearance of a long wavelength chromophore at 516 nm; and 3) formation of a shoulder at 325 nm. The secondary phase of irreversible inactivation is characterized by disappearance of the long wavelength chromophore and a concur- rent decrease in the intensity of the shoulder at 325 nm. Shown in the inset is the absorbance at 516 nm plotted as a function of time of reaction. The solid line is theoretical for a fit of the data to a first- order rate equation. The rate constant, 0.94 f 0.03 X s”, is essentially identical to that of the slow secondary inactivation event measured in activity profiles of the reaction mixture, 1.2 X s-I.

At infinite time, the spectrum is similar to that of native enzyme, although the chromophore in the region of the pyridoxal aldimine is less intense and shifted upfield to 424 nm, and the shoulder at 325 nm persists.

.os 1 I

Wavelength, nm

FIG. 7. Quantitative liberation of pyridoxal phosphate from enzyme-inhibitor complex. Comparison of spectra for heat-denatured native enzyme (a) and heat-denatured D-[3H]chlorovinylglycine-inactivated enzyme (b) with that of control pyridoxal phosphate (c) indicates that pyridoxal phosphate is quantitatively liberated from inactivated enzyme. The samples, containing 13 p~ enzyme or enzyme-inhibitor complex, or 15 PM pyridoxal phosphate (each in 0.1 M Hepes, 0.1 M NaCl, pH 8.00) were heated at 100 “C for 2 min, then clarified by centrifugation at 500,000 X g for 15 min to obtain these spectra. In contrast to pyridoxal phosphate, the 3H-labeled inhibitor remains firmly attached to the denatured enzyme.

(pH 12) solutions overnight, and to treatment with dithiothreitol in 6 M guanidine hydrochloride.

Trypsin digestion of the enzyme-”-inhibitor complex, as described under “Experimental Procedures,” gave a single radioactive peptide, which was isolated in 35% overall yield. The sequence of this peptide fragment was established to be

H2N-Val-Gly-X*-Gly-Gly-Arg-COOH X = tyrosine

where X indicates the cycle from which radioactivity was released during sequencing. This residue was tentatively as- signed to tyrosine. Amino acid analysis of an independent preparation of the peptide, under conditions that completely

hydrolyzed off the label, confirmed the composition and established unequivocally that the labeled amino acid is a tyrosine: Arg (340 pmol), Gly (1010 pmol), Tyr (370 pmol), and Val (400 pmol).

DISCUSSION

Inhibition of alanine racemase by halovinylglycines meets all the criteria for mechanism-based inactivation developed by Walsh (5). These include time dependent loss of activity that shows saturation kinetics, direct spectrophotometric evidence that the inhibitors interact with the pyridoxal phosphate as a measure of a process occurring in the active site, chemical stoichiometry of modification corresponding to 1 mol of inhibitor bound per mol of enzyme, and demonstration that nucleophilic scavengers have no influence on either the kinetics or partition ratio.

A mechanism-based inhibitor’s partition ratio, the number of turnovers per lethal event, is indicative of its potency and potential in vivo selectivity (5). With a lower partition ratio, less drug is required to kill a given amount of enzyme, and fewer activated metabolites are released that could affect other cellular constituents. The partition ratio for the halovinylglycines, 2.2 & 0.2, is exceptionally favorable, and only a few other mechanism-based inhibitors display better efficiency (26).

The kinetics of inactivation indicate that the 3-halovinylglycines interact in a more complex way than do other mechanism-based inhibitors of alanine racemase, in that they form both irreversible and reversible adducts. Formation of a covalent reversible adduct is evident in slow partial return of activity upon dilution of enzyme-inhibitor complex into the assay and in biphasic inhibition time courses. In these time courses, 70% of the enzyme is quickly inactivated at a rate that is dependent on inhibitor concentration, leaving group, and stereochemistry, but the residual 30% becomes irreversibly inactivated at a rate (t8,z = 96 min) that is independent of these parameters. As shown in Scheme 1, these considerations suggest that the inhibition proceeds through a common achiral intermediate that partitions between three fates: harmless dissociation to free solution, lethal inactivation of the enzyme, and diversion to a transient, slowly reversible adduct with the enzyme. Serving as a waiting room to protect the enzyme from irreversible inactivation, the transient intermediate provides a rationale for the second phase. Slow turnover of this species regenerates free enzyme, which then can react with inhibitor to repeat the process, eventually leading to complete irreversible inactivation. The partial diversion of the reaction to a waiting room has precedence in the work of Knowles (6) on P-lactamase inhibitors.

The second-order rate constant (122 k 14 M” s - I ) for the initial phase of inactivation by D-chlorovinylglycine, the most reactive analog examined, is comparable to the corresponding rate constant for D-fluoroalanine (93 M” s”). The favorable reactivity of D-chlorovinylglycine compared to D-fluoroalanine is the product of a 345-fold slower second-order rate constant for turnover and a 450-fold higher killing efficiency. Based on second-order rate constants, L-chlorovinylglycine reacts 38 times more slowly than the D-isomer; and L- and D- fluorovinylglycine react some 3000 and 6400 times more slowly, respectively.

The large decrease in rate of processing caused by fluoride substitution was unexpected by comparison with P-substituted alanines (7), and suggests halide elimination may be rate-limiting for inactivation by the halovinylglycines. A detailed picture of free energy barriers for racemization of alanine by several enzymes (alr and dadB alanine racemases


from S. typhimurium, and an alanine racemase from Bacillus stearothermophilus) has been provided by a kinetic isotope effect study of Faraci and Walsh (27), who conclude that trans-imination with the pyridoxal phosphate is partially or entirely rate-limiting in both directions. This picture probably holds for the E. coli alanine racemase as well and extends to mechanism-based inhibition, since this enzyme processes C1- alanine, F-alanine, and 0-acetyl serine with kc,, values comparable to those for alanine, and since there is no kinetic isotope effect for inactivation by [a-”HJfluoroalanine (7). The kc,, values for the fluorovinylglycines, being only 0.0001% those of alanine, are a remarkable departure from this series.

A chemical mechanism that accounts for all our observa- tions is shown in Scheme 2. We propose that proton abstraction occurs along the normal pathway for racemization, at which point the would be substrate is diverted by halide elimination to the reactive allene 2. The kinetics of inactivation provide secure evidence for the intermediacy of the allene, since they indicate that all reactions proceed through a common intermediate that is planar at the a-carbon and has undergone halide elimination. This is consistent with identical partition ratios for D- and L-chloroalanine, and identical partitioning between harmless turnover, lethal inactivation, and the same waiting room complex for both chloro- and fluoro-analogs despite initial inactivation rate constants differing by up to 6400-fold.

The allene 2 partitions between three fates as explained below.

1) In 57% of the turnovers, the allene escapes to free solution and undergoes hydrolysis to the reactive Michael acceptor vinylglyoxylate 3. No attempt was made to identify this product.

+; “j Turnover

1

/ Inactivation /

H

6 4 + C q

t

SCHEME 2

2) Another 12% of the time, the allene is diverted to a transient waiting room adduct (X,,, = 516 nm) that decays with a half-life of 23 min to regenerate active enzyme. This intermediate is proposed to be the paraquinoid 4, formed by attack of the active site lysine on the allenic center, or its prototropic tautomer 5. Either of these highly conjugated structures could account for the intense long wavelength absorbance that characterizes this species.2

The lysine-enamine linkage to the inhibitor would be expected to decay to produce a-aminoacetone, thus allowing for regeneration of active enzyme. In confirmation, [3H]a-aminoacetone was demonstrated to be present in solution in at least 47% of predicted yield following reaction of the racemase with D-[3H]chlorovinylglycine. It is not known if the less than theoretical recovery of [3H]a-aminoacetone reflects partial return of the transient adduct to the allene, or possible exchange of ‘H label with solvent during the analysis; our calculations of partition frequencies assume there is no return to the allene.

3) The remaining 31% of the time, the allene takes a lethal course leading to alkylation of a tyrosine residue located in the sequence -Val-Gly-Tyr-Gly-Gly-Arg-. The failure of ex- ogenous nucleophiles to compete for capture of the allene argues this tyrosine is intimately associated with the active site.

The irreversible enzyme-inhibitor complex, from which pyridoxal phosphate is quantitatively liberated upon denaturation, is proposed to be 8. To account for the less intense pyridoximine absorbance at 425 nm than is found in native enzyme and the appearance of a shoulder at 325 nm, it is proposed that this adduct is also partially bonded as the lysine aldimine.

The point of attachment to tyrosine is presumably the terminal carbon, which would be favored if the active site lysine participates on the normal trans-imination pathway to give the Michael acceptor 7. This Michael addition also is consistent with the lability of the peptide-bound hydrolysis product 9 to acid and base extremes observed during amino acid analysis and gas phase sequencing. Unfortunately, posi- tive identification of 9 by fast atom bombardment and elec- tron impact mass spectroscopy gave inconclusive results.

In conclusion, the mechanism of inactivation of alanine

h, =a5 &.‘am &-550

a b C

As good models for 5, an intermediate with structure a and Amax = 460-485 has been convincingly argued by Johnston et al. (28) to occur during inactivation of cystathionine y-synthase by erythro-L- fluoroaminobutyrate; and recently Faraci and Walsh (29) have proposed structure b with Amax = 460-490 to account for the long wavelength intermediate seen during inactivation of alanine racemase by D-triflUOrOalanine. On the other hand, quinoid structures analogous to 4 have been postulated as intermediates in a number of pyridoxal-dependent reactions and, as reviewed by Davis and Metzler (30), are predicted to have Amax “greater than 500 nm.” A reasonable model may be the aluminum chelate of 2-amino-3-butenoic acid, structure c, which is reported by Karube and Matsushima (31) to have Amax = 550 in alkaline methanol solutions. Thus, available models do not allow a compelling choice between 4 and 5.


racemase by halovinylglycines differs fundamentally from that of analogous nonvinylic @-substituted alanines, proceed- ing through an electrophilic allene rather than a nucleophilic aminoacrylate. The high killing efficiency of the halovinylglycines is in concert with the favorable partition ratios usually found for acetylenic mechanism-based inhibitors, which likewise produce electrophilic allenic intermediates. Along the same lines, Silverman and Abeles (32) have argued, and Faraci and Walsh (29) have recently shown, that the increased electrophilic nature of the @-carbon in trifluoroalanine serves to re-route the fate of the incipient aminoacrylate species and produce a close to theoretically ideal partition ratio of 3 turnovers per inactivation event for alanine racemase.

The design of halovinylglycines as mechanism-based inhibitors, employing the haloethylene moiety to generate a reactive allene during catalysis, may have broad application to other medicinally important enzymes carrying out carbanion chemistry. For instance, in the inhibition of ornithine decarboxylase, 3,4-dihydroxyphenylalanine decarboxylase, and a- aminobutyric acid transaminase, which are pyridoxal-dependent enzymes that are proven targets in treatment of cancer, hypertension, and epilepsy (33), the alternative method for entry to an allenic intermediate is via an acetylenic function- ality, which in the case of ethynylglycine is a short-lived natural antibiotic (34).

The antibacterial properties of the halovinylglycines have been reported elsewhere (35). Unfortunately, their potency as antibiotics in vivo is not commensurate with their efficiency as inactivators of alanine racemase in vitro. Although accepted as good substrates by alanine racemase, these inhibitors ap- parently are not recognized well by bacterial amino acid transport systems. Their application as antibiotics awaits a better understanding of bacterial cell permeability.

Acknowledgments-We thank Lauretta Zitano and Joe King of these laboratories for growing the E. coli cells, Robert Abeles for proposing the design of these inhibitors, and Don Hupe and Jack Kirsch for creative suggestions concerning chemical mechanisms. We are especially indebted to Richard Schowen for proposing the concept of a waiting room intermediate to explain the biphasic kinetics of inhibition.

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the journal of vol. 266, no 32, 15, pp, 21657-21665,1991 ... · the journal of biological chemistry...

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