Syntheses of (Z)-and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid and their inactivation of gamma-aminobutyric acid aminotransferase.

(Z)- and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid (4 and 5, respectively) were synthesized and investigated as potential mechanism-based inactivators of gamma-aminobutyric acid aminotransferase (GABA-AT) in a continuing effort to map the active site of this enzyme. The core alpha-trifluoromethyl-alpha,beta-unsaturated ester moiety was prepared via a Reformatsky/reductive elimination coupling of the key intermediates tert-butyl 2,2-dichloro-3,3,3-trifluoropropionate and N,N-bis(tert-butoxy-carbonyl)glycinal. Both 4 and 5 inhibited GABA-AT in a time-dependent manner, but displayed non-pseudo-first-order inactivation kinetics; initially, the inactivation rate increased with time. Further investigation demonstrated that the actual inactivator is generated enzymatically from 4 or 5. This inactivating species is released from the active site prior to inactivation, and as a result, 4 and 5 cannot be defined as mechanism-based inactivators. Furthermore, 4 and 5 are alternate substrates for GABA-AT, transaminated by the enzyme with Km values of 0.74 and 20.5 mM, respectively. Transamination occurs approximately 276 and 305 times per inactivation event for 4 and 5, respectively. The enzyme also catalyzes the elimination of the fluoride ion from 4 and 5. A mechanism to account for these observations is proposed.     

2-({6-[(3R)-3-amino-3-methylpiperidine-1-yl]-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-yl}methyl)-4-fluorobenzonitrile (DSR-12727): a potent, orally active dipeptidyl peptidase IV inhibitor without mechanism-based inactivation of CYP3A

We report on the identification of 2-({6-[(3R)-3-amino-3-methylpiperidine-1-yl]-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-yl}methyl)-4-fluorobenzonitrile (DSR-12727) (7a) as a potent and orally active DPP-4 inhibitor without mechanism-based inactivation of CYP3A. Compound 7a showed good DPP-4 inhibitory activity (IC(50)=1.1 nM), excellent selectivity against related peptidases and other off-targets, good pharmacokinetic and pharmacodynamic profile, great in vivo efficacy in Zucker-fatty rat, and no safety concerns both in vitro and in vivo.     

Mechanism-based irreversible inactivation of horseradish peroxidase at 500 MPa

The effects of high-pressure treatment on the reaction rates of horseradish peroxidase (HRP) with guaethol or guaiacol as a hydrogen donor were evaluated from direct transmission measurements in a high-pressure optical cell at 435 nm. Peroxidases are known to be very barostable and insensitive to heat. With guaethol the reaction velocity was independent of pressure up to 500 MPa, but with guaiacol the cytochrome c oxidase underwent a mechanism-based irreversible inhibition of catalytic activity when subjected to pressure; in the resting states (fully oxidized or reduced), it was insensitive to pressure. The enzyme inactivation took place with an inactivation rate constant of 5.15 x 10(-1) min(-1) at 500 MPa, 25 degrees C and pH 7. The degree of inactivation was correlated to the concentration of guaiacol. This is the first report on a mechanism-based pressure inactivation of HRP triggered at moderate pressure and temperature and mediated by the hydrogen donor.     

Effect of chlorinated naphthalenes and terphenyls on the activities of drug metabolizing enzymes in rat liver

γ-Aminobutyric acid-α-ketoglutarate transaminase from pig brain is irreversibly inactivated by 4-amino-5-halopentanoic acids. Protection from inactivation by the natural substrates, the pH dependence of inactivation and the incorporation of 1.7 moles of radioactive inhibitor per mole of enzyme from (S)-[U-¹⁴C]-4-amino-5-chloropentanoic acid suggest a covalent adduct at the active site of the enzyme. A mechanism-based inactivation is proposed.     

Hexanal phenylhydrazone is a mechanism-based inactivator of soybean lipoxygenase 1

Hexanal phenylhydrazone (1; 70:30 E:Z mixture) at micromolar concentration irreversibly inactivates soybean lipoxygenase 1 (L-1) in the presence of dioxygen. L-1 catalyzes the oxidation of 1 into its alpha-azo hydroperoxide 2 [C5H11CH(OOH)N = NC6H5]. 2 is an efficient inactivator of L-1. The aerobic reaction between 1 and L-1 follows a branched pathway leading to the release of 2 into the medium or to L-1 inactivation. The respective parameters corresponding to this inactivation by the (E)-1 and (Z)-1 isomers are Ki = 0.25 and 0.40 microM and kinact = 0.8 and 2.1 min-1. Linoleic acid protection agrees with a mechanism-based inactivation process. The oxidation of a minimum of 13 +/- 3 molar equiv of 1 is required for complete L-1 inactivation, but up to 70 equiv is necessary in the presence of a very large excess of 1. The inactivation is actually the result of two pathways: one is due to a reaction of 2 as soon as it is formed at the active site (20%); the other is due to 2 released into the medium and coming back to the active site (80%). The inactivation is accompanied by the oxidation of 1.8 +/- 0.8 methionine residues of the protein into the corresponding sulfoxide. The inactivated L-1 is electron paramagnetic resonance (EPR) silent with an effective magnetic moment of mu = 5.0 +/- 0.1 Bohr magnetons corresponding to an S = 2 spin state. An inactivation mechanism is proposed on the basis of EPR and magnetic susceptibility data obtained from the anaerobic and aerobic reactions of L-1 with 1 and 2.     

(+/-)-(1S,2R,5S)-5-Amino-2-fluorocyclohex-3-enecarboxylic acid. A potent GABA aminotransferase inactivator that irreversibly inhibits via an elimination-aromatization pathway

Inhibition of gamma-aminobutyric acid aminotransferase (GABA-AT) increases the concentration of GABA, an inhibitory neurotransmitter in human brain, which could have therapeutic applications for a variety of neurological diseases, including epilepsy. On the basis of studies of several previously synthesized conformationally restricted GABA-AT inhibitors, (+/-)-(1S,2R,5S)-5-amino-2-fluorocyclohex-3-enecarboxylic acid (12) was designed as a mechanism-based inactivator. This compound was shown to irreversibly inhibit GABA-AT; substrate protects the enzyme from inactivation. Mechanistic experiments demonstrated the loss of one fluoride ion per active site during inactivation and the formation of N-m-carboxyphenylpyridoxamine 5'-phosphate (26), the same product generated by inactivation of GABA-AT by gabaculine (8). An elimination-aromatization mechanism is proposed to account for these results.     

Metabolite-cytochrome P450 complex formation by methylenedioxyphenyl lignans of Piper cubeba: mechanism-based inhibition

Five methylenedioxyphenyl lignans, (-)-clusin (1), (-)-dihydroclusin (2), (-)-yatein (3), (-)-hinokinin (4), and (-)-dihydrocubebin (5), were isolated from Piper cubeba as potent and selective inhibitors against cytochrome P450 3A4 (CYP3A4). In this study, we investigated the mechanism of inhibition of CYP3A4 by these lignans and the possibility of their mechanism-based inhibition. Using [N-methyl-14C]erythromycin as a substrate, all lignans appear to be showed mixed-type of inhibition with apparent Ki of 1.96-4.07 microM. Furthermore, all lignans (1-5) inhibited CYP3A4 in a time-, concentration-, and NADPH-dependent manners and thus appear to be the mechanism-based inhibitors of CYP3A4. The apparent inactivation parameter, K(I) for these compounds were in the range of 0.054-0.373 microM, whereas the k(inact) values were 0.225-0.320 min-1. Among them, (-)-clusin (1) and (-)-dihydroclusin (2) were found to be the most potent CYP3A4 inactivator with apparent K(I) and k(inact) values of 0.082, 0.054 microM and 0.253, 0.310 min-1, respectively. Spectral scanning of microsomes with these lignans yielded an absorbance at 455 nm, suggesting that all of them appear to inactivate the cytochrome P450 via the formation of a metabolite intermediate complex. This pattern is consistent with the metabolism of the methylenedioxyphenyl compounds. These results indicate that (-)-clusin (1), (-)-dihydroclusin (2), (-)-yatein (3), (-)-hinokinin (4), and (-)-dihydrocubebin (5) are effective mechanism-based inhibitors of CYP3A4.     

Inactivation of dihydropyrimidine dehydrogenase by 5-iodouracil

5-Iodouracil was a substrate for bovine liver dihydropyrimidine dehydrogenase (DHPDHase) and was a potent inactivator of the enzyme. NADPH increased the rate of inactivation and thymine protected against inactivation. These findings suggest that 5-iodouracil was a mechanism-based inactivator. However, dithiothreitol and excess 5-iodouracil protected the enzyme against inactivation. Thus, a reactive product, presumably 5-iodo-5,6-dihydrouracil generated through the enzymatic reduction of 5-iodouracil, was released from DHPDHase during processing of 5-iodouracil. Since only 18% of [6-3H]5-iodouracil reduced by DHPDHase was covalently bound to the enzyme and radiolabel was not lost to the solvent as tritium, the partition coefficient for inactivation was 4.5. However, the enzymatic activity was completely titrated with 1.7 mol of 5-iodouracil per mol of enzyme-bound flavin. These results indicate that there was 0.31 mol of enzyme-bound inactivator per mol of enzyme flavin. This suggests there were 3.2 flavins per active site, which is consistent with the report of multiple flavins per enzymic subunit (Podschun, B., Wahler, G., and Schnackerz, K. D. (1989) Eur. J. Biochem. 185, 219-224). DHPDHase was inactivated by 2.1 mol of racemic 5-iodo-5,6-dihydrouracil per mol of active sites. The stoichiometry for inactivation of the enzyme by the nonenzymatically generated enantiomer of 5-iodo-5,6-dihydrouracil was calculated to be 1. Two radiolabeled fragments were isolated from a tryptic digest of DHPDHase inactivated with radiolabeled 5-iodouracil. The amino acid sequences of these peptides were Asn-Leu-Ser-X-Pro-His and Asn-Leu-Ser-X-Pro-His-Gly-Met-Gly-Glu-Arg where X was the modified amino acid containing radiolabel from [6-3H]5-iodouracil. Fast atom bombardment mass spectral analysis of the smaller peptide yielded a protonated parent ion mass of 782 daltons that was consistent with X being a S-(hexahydro-2,4-dioxo-5-pyrimidinyl)cysteinyl residue.     

Haloenol lactones as inactivators and substrates of aldehyde dehydrogenase

Human aldehyde dehydrogenase (EC 1.2.1.3) isozymes E1 and E2 were irreversibly inactivated by stoichiometric concentrations of the haloenol lactones 3-isopropyl-6(E)-bromomethylene tetrahydro-pyran-2-one and 3-phenyl-6(E)-bromomethylene tetrahydro-pyran-2-one. No inactivation occurred with the corresponding nonhalogenated enol lactones. Both the dehydrogenase and esterase activities were abolished. Activity was not regained on dialysis or treatment with 2-mercaptoethanol. The inactivation was subject to substrate protection: NAD afforded protection which increased in the presence of the aldehyde-substrate competitive inhibitor chloral. Saturation kinetics gave positivey-axis intercepts, allowing the determination of binding constants. Inactivation stiochiometry determined with¹⁴C-labeled 3-(1-naphthyl)-6(E)-iodomethylene tetrahydropyran-2-one was found to correspond to the active-site number. The nonhalogenated lactone, 3-(1-naphthyl)-6(E)-methylene tetrahydropyran-1-one was shown to be a substrate for aldehyde dehydrogenase via its esterase function. Inactivation and enzymatic hydrolysis occurred within a similar time frame. Opening of the lactone ring to form enzyme-acyl intermediate with active site cysteine appears to be a necessary prerequisite to inactivation, since halogen in the lactone ring is nonreactive. Thus, the inactivation of aldehyde dehydrogenase by haloenol lactones is mechanism-based. Inactivation by haloenol lactones occurs in a manner analogous to that of chymotrypsin with which aldehyde dehydrogenase shares esterase activity and binding of haloenol lactones at the active site.     

Studies of neuronal nitric oxide synthase inactivation by diverse suicide inhibitors.

N(G)-Amino-l-arginine, N(5)-(1-iminoethyl)-l-ornithine, N(6)-(1-iminoethyl)-l-lysine, and aminoguanidine were studied for the mechanisms by which they produce suicidal inactivation of the neuronal nitric oxide synthase isoform (nNOS). All of the inactivators that were amino acid structural analogs targeted the heme residue at the nNOS active site and led to its destruction as evidenced by the time- and concentration-dependent loss of the nNOS heme fluorescence, which reflects the disruption of the protoporphyrin-conjugated structure. The loss of heme was exclusively associated with the dimeric population of the nNOS. This inactivator-mediated loss of the nNOS heme never reached more than 60%, suggesting that only half of the dimeric heme is involved in catalytic activation of mechanism-based inactivators studied. Aminoguanidine-induced nNOS inactivation produced covalent modification of the nNOS protein chain with a stoichiometry of 0.8 mol of aminoguanidine per mole of the nNOS monomer. Specific covalent modification by aminoguanidine was exclusively associated with the oxygenase domain of the nNOS. The mechanisms by which N(6)-(1-iminoethyl)-l-lysine and aminoguanidine inactivate the nNOS and iNOS do not differ between the isoforms. The selectivity of these inactivators toward the iNOS isoform is a reflection of their much lower partition ratios, which were determined to be 0.16 +/- 0. 1 for N(6)-(1-iminoethyl)-l-lysine and 12 +/- 1.5 for aminoguanidine in case of the iNOS isoform while the same inactivators produced the partition ratios of 17 +/- 2 and 206 +/- 4, respectively, for the nNOS isoform.     

Mechanism of inactivation of S-adenosyl-L-homocysteine hydrolase by 5'-deoxy-5'-S-allenylthioadenosine and 5'-deoxy-5'-S-propynylthioadenosine

5'-Deoxy-5'-S-allenylthioadenosine 1 and 5'-deoxy-5'-S-propnylthioadenosine 2, derived from adenosine, were prepared. 1 and 2 caused irreversible inactivation of AdoHcy hydrolase. ESI mass spectra analysis of the inactivated enzyme demonstrated that 1 and 2 were type II "mechanism-based" inhibitors.     

Isolation and characterization of Pseudomonas aeruginosa mutants blocked in the synthesis of pyoverdin.

We have isolated and characterized by chemical and enzymatic analyses three distinct types of pyoverdin-defective (pvd) mutants of Pseudomonas aeruginosa PAO1. The pvd-1 mutant is an L-N5-hydroxyornithine (L-N5-OH-Orn) auxotroph unable to hydroxylate L-ornithine (L-Orn) in a cell-free system and requiring L-N5-OH-Orn for pyoverdin production. The other two types of mutants appear to be blocked in further steps of the biosynthetic pathway leading to pyoverdin, namely, the acylation of L-N5-OH-Orn (pvd-2) and chromophore synthesis (pvd-3). The different pvd mutations were all found to be located in the catA1 region at 47 min of the genetic map of P. aeruginosa PAO1.     

Inactivation of gamma-aminobutyric acid aminotransferase by (Z)-4-amino-2-fluorobut-2-enoic acid

(Z)-4-Amino-2-fluorobut-2-enoic acid (1) is shown to be a mechanism-based inactivator of pig brain gamma-aminobutyric acid aminotransferase. Approximately 750 inactivator molecules are consumed prior to complete enzyme inactivation. Concurrent with enzyme inactivation is the release of 708 +/- 79 fluoride ions; transamination occurs 737 +/- 15 times per inactivation event. Inactivation of [3H]pyridoxal 5'-phosphate ([3H]PLP) reconstituted GABA aminotransferase by 1 followed by denaturation releases [3H]PMP with no radioactivity remaining attached to the protein. A similar experiment carried out with 4-amino-5-fluoropent-2-enoic acid [Silverman, R. B., Invergo, B. J., & Mathew, J. (1986) J. Med. Chem. 29, 1840-1846] as the inactivator produces no [3H]PMP; rather, another radioactive species is released. These results support an inactivation mechanism for 1 that involves normal catalytic isomerization followed by active site nucleophilic attack on the activated Michael acceptor. A general hypothesis for predicting the inactivation mechanism (Michael addition vs enamine addition) of GABA aminotransferase inactivators is proposed.     

Probing the radical mechanism of galactose oxidase using an ultrafast radical probe

Processing of trans-2-phenylcyclopropylmethanols 5 and 6 by the monocopper/tyrosine radical enzyme galactose oxidase led to mechanism-based inactivation with a partition ratio, (k(cat) + k(inact))/k(inact), of approximately 1 and a primary deuterium isotope effect, k(inact(H))/k(inact(D)), of 3.2. The data are consistent with a radical mechanism for galactose oxidase with a short lived ketyl radical anion intermediate.     

Novel inactivators of serine proteases based on 6-chloro-2-pyrone

The interaction of serine protease (esterases) with 6-chloro-2-pyrones was investigated. Time-dependent inactivation of chymotrypsin, alpha-lytic protease, pig liver elastase, and cholinesterase was found with 3- and 5-benzyl-6-chloro-2-pyrone, as well as 3- and 5-methyl-6-chloro-2-pyrone. No inactivation was observed with the unsubstituted 6-chloro-2-pyrone. The substituted pyrones did not inactivate papain or carboxypeptidase A, as well as a number of other nonproteolytic enzymes. The substituted chloropyrones, therefore, show considerable selectivity toward serine proteases. Analogues in which the 6-chloro substituent is replaced by H or OH do not inactivate. The presence of the halogen is, therefore, essential for inactivation. Chymotrypsin catalyzes the hydrolysis of 3-benzyl-6-chloro-2-pyrone. At pH 7.5, (E)-4-benzyl-2-pentenedioic acid is the major product, and 2-benzyl-2-pentenedioic anhydride is a minor product. The ration of hydrolysis product found to the number of enzyme molecules inactivated varies from 14 to 40. The enzyme inactivated with the 3-benzyl compound does not show a spectrum characteristic of the pyrone ring. This suggests that inactivation by 3-benzyl-6-chloro-2-pyrone occurs in a mechanism-based fashion after enzymatic lactone hydrolysis. When the enzyme is inactivated with the 5-benzyl compound, absorbance due to the pyrone ring is observed. We suggest that inactivation occurs through an active site directed mechanism involving a 1,6-conjugate addition of an active site nucleophile to the pyrone ring.     

Structure of ACC synthase inactivated by the mechanism-based inhibitor L-vinylglycine

L-Vinylglycine (L-VG) is both a substrate for and a mechanism-based inhibitor of 1-aminocyclopropane-1-carboxylate (ACC) synthase. The ratio of the rate constants for catalytic conversion to alpha-ketobutyrate and ammonia to inactivation is 500/1. The crystal structure of the covalent adduct of the inactivated enzyme was determined at 2.25 Angstroms resolution. The active site contains an external aldimine of the adduct of L-VG with the pyridoxal 5'-phosphate cofactor. The side chain gamma-carbon of L-VG is covalently bound to the epsilon-amino group of Lys273. This species corresponds to one of the two alternatives proposed by Feng and Kirsch [Feng, L. and Kirsch, J.F. (2000) L-Vinylglycine is an alternative substrate as well as a mechanism-based inhibitor of 1-aminocyclopropane-1-carboxylate synthase. Biochemistry 39, 2436-2444] and presumably results from Michael addition to a vinylglycine ketimine intermediate.     

Acyloxybenzyl halides, inhibitors of elastases

3-Benzyl-6-chloromethyl-3,4-dihydrocoumarin inhibits human leucocyte elastase (HLE) and porcine pancreatic elastase (PPE) through a mechanism-based process characterized by the following apparent enzyme-inhibitor dissociation constants, Ki, and limiting inactivation rate constants k2: 200 microM (HLE), 69 microM (PPE) and 5.10(-2) s-1 (HLE), 17.7.10(-2) s-1 (PPE) at pH 8.0, 37 degrees C. Bis(4-acyloxyphenyl)methane derivatives with a benzylic halogen as potential leaving group have also been synthesized and studied. They transiently inactivate PPE and HLE through the formation of an acyl-enzyme.     

Ticlopidine as a selective mechanism-based inhibitor of human cytochrome P450 2C19

Experiments using recombinant yeast-expressed human liver cytochromes P450 confirmed previous literature data indicating that ticlopidine is an inhibitor of CYP 2C19. The present studies demonstrated that ticlopidine is selective for CYP 2C19 within the CYP 2C subfamily. UV-visible studies on the interaction of a series of ticlopidine derivatives with CYP 2C19 showed that ticlopidine binds to the CYP 2C19 active site with a K(s) value of 2.8 +/- 1 microM. Derivatives that do not involve either the o-chlorophenyl substituent, the free tertiary amine function, or the thiophene ring of ticlopidine did not lead to such spectral interactions and failed to inhibit CYP 2C19. Ticlopidine is oxidized by CYP 2C19 with formation of two major metabolites, the keto tautomer of 2-hydroxyticlopidine (1) and the dimers of ticlopidine S-oxide (TSOD) (V(max) = 13 +/- 2 and 0.4 +/- 0.1 min(-1)). During this oxidation, CYP 2C19 was inactivated; the rate of its inactivation was time and ticlopidine concentration dependent. This process meets the chemical and kinetic criteria generally accepted for mechanism-based enzyme inactivation. It occurs in parralel with CYP 2C19-catalyzed oxidation of ticlopidine, is inhibited by an alternative well-known substrate of CYP 2C19, omeprazole, and correlates with the covalent binding of ticlopidine metabolite(s) to proteins. Moreover, CYP 2C19 inactivation is not inhibited by the presence of 5 mM glutathione, suggesting that it is due to an alkylation occurring inside the CYP 2C19 active site. The effects of ticlopidine on CYP 2C19 are very analogous with those previously described for the inactivation of CYP 2C9 by tienilic acid. This suggests that a similar electrophilic intermediate, possibly a thiophene S-oxide, is involved in the inactivation of CYP 2C19 and CYP 2C9 by ticlopidine and tienilic acid, respectively. The kinetic parameters calculated for ticlopidine-dependent inactivation of CYP 2C19, i.e., t(1/2max) = 3.4 min, k(inact) = 3.2 10(-3) s(-1), K(I) = 87 microM, k(inact)/K(I) = 37 L.mol(-1).s(-1), and r (partition ratio) = 26 (in relation with formation of 1 + TSOD), classify ticlopidine as an efficient mechanism-based inhibitor although somewhat less efficient than tienilic acid for CYP 2C9. Importantly, ticlopidine is the first selective mechanism-based inhibitor of human liver CYP 2C19 and should be a new interesting tool for studying the topology of the active site of CYP 2C19.     

An acetylenic mechanism-based inhibitor of dopamine beta-hydroxylase

The catalytic action of dopamine beta-hydroxylase on 1-phenyl-1-propyne results in concomitant loss of enzyme activity. At pH 5.5 and 25 degrees C, 1-phenyl-1-propyne inactivates dopamine beta-hydroxylase in a mechanism-based fashion. The inactivation rate is first-order, follows saturation kinetics, and is strictly dependent on catalysis (oxygen and ascorbate are essential). The inactivation rate of saturating 1-phenyl-1-propyne (kinact) increases from 0.08 to 0.22 min-1 when the oxygen saturation increases from 21 to 100%, respectively. Inactivation also requires a copper-containing catalytically competent enzyme. Tyramine and norepinephrine (respectively, substrate and product of the normal catalytic reaction) protect against inactivation, and no regain of enzyme activity occurs after prolonged dialysis. Experiments with ether-extracted incubation solutions (+/- enzyme) showed no difference in their gas chromatography-mass spectral patterns implying that inactivation of dopamine beta-hydroxylase by 1-phenyl-1-propyne occurs through a kinetic process with a partition ratio (kcat/kinact) equal to or near 1. Thus, this acetylenic substrate analog appears to be a very efficient mechanism-based inhibitor of dopamine beta-hydroxylase. We propose that inactivation of this enzyme by 1-phenyl-1-propyne proceeds by formation of a reactive intermediate that occurs prior to product formation and that alkylates an amino acid residue at the active site of the enzyme.     

Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor

The mechanism-based inactivations of a number of serine proteases, including human leukocyte (HL) elastase, cathepsin G, rat mast cell proteases I and II, several human and bovine blood coagulation proteases, and human factor D by substituted isocoumarins and phthalides which contain masked acyl chloride or anhydride moieties, are reported. 3,4-Dichloroisocoumarin, the most potent inhibitor investigated here, inactivated all the serine proteases tested but did not inhibit papain, leucine aminopeptidase, or beta-lactamase. 3,4-Dichloroisocoumarin was fairly selective toward HL elastase (kobsd/[I] = 8920 M-1 s-1); the inhibited enzyme was quite stable to reactivation (kdeacyl = 2 X 10(-5) s-1), while enzymes inhibited by 3-acetoxyisocoumarin and 3,3-dichlorophthalide regained full activity upon standing. The rate of inactivation was decreased dramatically in the presence of reversible inhibitors or substrates, and ultraviolet spectral measurements indicate that the isocoumarin ring structure is lost upon inactivation. Chymotrypsin A gamma is totally inactivated by 1.2 equiv of 3-chloroisocoumarin or 3,4-dichloroisocoumarin, and approximately 1 equiv of protons is released upon inactivation. These results indicate that these compounds react with serine proteases to release a reactive acyl chloride moiety which can acylate another active site residue. These are the first mechanism-based inhibitors reported for many of the enzymes tested, and 3,4-dichloroisocoumarin should find wide applicability as a general serine protease inhibitor.