Mechanism-based inhibition of lactoperoxidase by thiocarbamide goitrogens

The irreversible inactivation of bovine lactoperoxidase by thiocarbamide goitrogens was measured, and the kinetics were consistent with a mechanism-based (suicide) mode. Sulfide ion inactivated, 2-mercaptobenzimidazole-inactivated, and 1-methyl-2-mercaptoimidazole-inactivated lactoperoxidases have different visible spectra, suggesting different products were formed. The results support a mechanism in which reactive intermediates are formed by S-oxygenation reactions catalyzed by lactoperoxidase compound II. It is proposed that the reaction of electron-deficient intermediates with the heme prosthetic group is responsible for the observed spectral changes and inactivation by thiocarbamides.     

Iodide binding and regulation of lactoperoxidase activity toward thyroid goitrogens.

The effects of the antithyroid goitrogens, methylthiouracil and methylmercaptoimidazole, on the oxidation of N-acetyltyrosylamide at pH 8.8 by lactoperoxidase have been evaluated in the presence and the absence of iodide for the purpose of elucidating the effects of iodide. At pH 8.8, iodine is not oxidized. In the absence of iodide, the two antithyroid drugs inactivate lactoperoxidase by a second order process. When iodide is added before methylthiouracil or methylmercaptoimidazole, enzyme inactivation does not occur as rapidly and both goitrogens are readily oxidized. The kinetics of the oxidation reactions have been analyzed in order to obtain the equilibrium constant of the iodide . lactoperoxidase complex. Essentially the same iodide dissociation constant, i.e. 2 x 10(-5) M, was found by studying its effects on the kinetics of oxidation of the two antithyroid drugs. A large difference absorption spectrum is observed in the Soret region between native lactoperoxidase and lactoperoxidase inactivated by methylthiouracil.     

Mechanism-based inactivation of dihydropyrimidine dehydrogenase by 5-ethynyluracil.

Uracil analogues with appropriate substituents at the 5-position inactivated dihydropyrimidine dehydrogenase (DHPDHase). The efficiency of these inactivators was highly dependent on the size of the 5-substituent. For example, 5-ethynyluracil inactivated DHPDHase with an efficiency (kinact/Ki) that was 500-fold greater than that for 5-propynyluracil. 5-Ethynyluracil inactivated DHPDHase by initially forming a reversible complex with a Ki of 1.6 +/- 0.2 microM. This initial complex yielded inactivated enzyme with a rate constant of 20 +/- 2 min-1 (kinact). Thymine competitively decreased the apparent rate constant for inactivation of DHPDHase by 5-ethynyluracil. The absorbance spectrum of 5-ethylnyluracil-inactivated DHPDHase was different from that of reduced enzyme. These optical changes were correlated with the loss of enzymatic activity. 5-Ethynyluracil inactivated DHPDHase with a stoichiometry of 0.9 mol of inactivator per mol of active site. Enzyme inactivated with [2-14C]5-ethynyluracil retained all of the radiolabel after denaturation in 8 M urea, but lost radiolabel under acidic conditions. These results suggested that inactivation was due to covalent modification of an amino acid residue and not due to modification of a noncovalently bound prosthetic group. A radiolabeled peptide was isolated from a tryptic digest of the enzyme inactivated with [2-14C]5-ethynyluracil. The sequence of this peptide was Lys-Ala-Glu-Ala-Ser-Gly-Ala-Y-Ala-Leu-Glu-Leu-Asn-Leu-Ser-X-Pro-His-Gly- Met-Gly-Glu-Arg, where X and Y were unidentified amino acids. Since the radiolabel was lost from the peptide during the first cycle on the amino acid sequenator, the position of the radiolabeled amino acid was not determined. The amino acid residue designated by X was identified as a cysteine from previous work with DHPDHase inactivated with 5-iodouracil. In contrast to 5-ethynyluracil, 5-cyanouracil was a reversible inactivator of the enzyme. 5-Cyanouracil-inactivated enzyme slowly regained activity (t1/2 = 1.8 min) after dilution into the standard assay. DHPDHases isolated from rat, mouse, and human liver had similar sensitivities to inactivation by 5-alkynyluracils.     

The effects of thioureylene compounds (goitrogens) on lactoperoxidase activity.

The rates of oxidation of several goitrogens by lactoperoxidase and the rates of inactivation of lactoperoxidase by the same goitrogens have been measured. The influence of iodide on both reactions has also been evaluated. It has been shown by us that iodide acts catalytically in regulating lactoperoxidase activity at pH 8.8. The rate data have been analyzed by a computer program which solves the differential equations for the above mentioned reactions. From this computer analysis we have been able to obtain binding constants of the goitrogens and inactivation rate constants of lactoperoxidase. Iodide was shown to inhibit goitrogenic activity either by increasing the rate of drug oxidation or by reducing the rate of enzyme inactivation, or both, depending on the particular drug. Iodide had little or no effect on the goitrogen-binding constants. We have also shown that the relative rates of enzyme inactivation can be correlated with the potency of the goitrogen as an antithyroid drug.     

Mechanism-based inactivation of dopamine beta-monooxygenase by beta-chlorophenethylamine

Functionalization of the beta-carbon of phenethylamines has been shown to produce a new class of substrate/inhibitor of dopamine beta-monooxygenase. Whereas both beta-hydroxy- and beta- chlorophenethylamine are converted to alpha-aminoacetophenone at comparable rates, only the latter conversion is accompanied by concomitant enzyme inactivation ( Klinman , J. P., and Krueger , M. (1982) Biochemistry 21, 67-75). In the present study, the nature of the reactive intermediates leading to dopamine beta-monooxygenase inactivation by beta- chlorophenethylamine has been investigated employing kinetic deuterium isotope effects and oxygen- 18 labeling as tools. Mechanistically significant findings presented herein include: 1) an analysis of primary deuterium isotope effects on turnover, indicating major differences in rate-determining steps for beta-chloro- and beta- hydroxyphenethylamine hydroxylation, Dkcat = 6.1 and 1.0, respectively; 2) evidence that dehydration of the gem-diol derived from oxygen- 18-labeled beta- hydroxyphenethylamine hydroxylation occurs in a random manner, attributed to dissociation of enzyme-bound gem-diol prior to alpha-aminoacetophenone formation; 3) the observation of a deuterium isotope effect for beta- chlorophenethylamine inactivation, Dkinact = 3.7, implicating C--H bond cleavage in the inactivation process; and 4) the demonstration that alpha-aminoacetophenone can replace ascorbic acid as exogenous reductant in the hydroxylation of tyramine. As discussed, these findings support the intermediacy of enzyme-bound alpha-aminoacetophenone in beta- chlorophenethylamine inactivation, and lead us to propose an intramolecular redox reaction to generate a ketone-derived radical cation as the dopamine beta-monooxygenase-inactivating species.     

Mechanism-based inactivation of alanine racemase by 3-halovinylglycines

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 +/- 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 (lambda max = 516 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-1 s-1), the most reactive analog examined, is faster than the equivalent rate constant for D-fluoroalanine (93 M-1 s-1). 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.     

Mechanism-based inactivation of caspases by the apoptotic suppressor p35.

Caspases play a crucial role in the ability of animal cells to kill themselves by apoptosis. Caspase activity is regulated in vivo by members of three distinct protease inhibitor families, one of which--p35--has so far only been found in baculoviruses. P35 has previously been shown to rapidly form essentially irreversible complexes with its target caspases in a process that is accompanied by peptide bond cleavage. To determine the protease-inhibitory pathway utilized by this very selective protease inhibitor, we have analyzed the thermodynamic and kinetic stability of the protein. We show that the conformation of p35 is stabilized following cleavage within its reactive site loop. An inactive catalytic mutant of caspase 3 is bound by p35, but much less avidly than the wild-type enzyme, indicating that the protease catalytic nucleophile is required for stable complex formation. The inhibited protease is trapped as a covalent adduct, most likely with its catalytic Cys esterified to the carbonyl carbon of the scissile peptide bond. Together these data reveal that p35 is a mechanism-based inactivator that has adopted an inhibitory device reminiscent of the widely distributed serpin family, despite a complete lack of sequence or structural homology.     

Mechanism-based inactivation of monoamine oxidases A and B by tetrahydropyridines and dihydropyridines.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its primary oxidation product, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), are mechanism-based inhibitors of monoamine oxidases A and B. The pseudo-first-order rate constants for inactivation were determined for various analogues of MPTP and MPDP+ and the concentrations in all redox states were measured throughout the reaction. Disproportionation was observed for all the dihydropyridiniums, but non-enzymic oxidation was insignificant. The dihydropyridiniums were poor substrates for monoamine oxidase A and, consequently, inactivated the enzyme only slowly, despite partition coefficients lower than those for the tetrahydropyridines. For monoamine oxidase B, the dihydropyridiniums were more effective inactivators than the tetrahydropyridines. Substitutions in the aromatic ring had no major effect on the inactivation of monoamine oxidase B, but the 2'-ethyl- and 3'-chloro-substituted compounds were very poor mechanism-based inactivators of monoamine oxidase A. It is clear that both oxidation steps can generate the reactive species responsible for inactivation.     

Mechanism-based inactivation of dopamine beta-hydroxylase by p-cresol and related alkylphenols

The mechanism-based inhibition of dopamine beta-hydroxylase (DBH; EC 1.14.17.1) by p-cresol (4-methylphenol) and other simple structural analogues of dopamine, which lack a basic side-chain nitrogen, is reported. p-Cresol binds DBH by a mechanism that is kinetically indistinguishable from normal dopamine substrate binding [DeWolf, W. E., Jr., & Kruse, L. I. (1985) Biochemistry 24, 3379]. Under conditions (pH 6.6) of random oxygen and phenethylamine substrate addition [Ahn, N., & Klinman, J. P. (1983) Biochemistry 22, 3096] p-cresol adds randomly, whereas at pH 4.5 or in the presence of fumarate "activator" addition of p-cresol precedes oxygen binding as is observed with phenethylamine substrate. p-Cresol is shown to be a rapid (kinact = 2.0 min-1, pH 5.0) mechanism-based inactivator of DBH. This inactivation exhibits pseudo-first-order kinetics, is irreversible, is prevented by tyramine substrate or competitive inhibitor, and is dependent upon oxygen and ascorbic acid cosubstrates. Inhibition occurs with partial covalent incorporation of p-cresol into DBH. A plot of -log kinact vs. pH shows maximal inactivation occurs at pH 5.0 with dependence upon enzymatic groups with apparent pK values of 4.51 +/- 0.06 and 5.12 +/- 0.06. p-Cresol and related alkylphenols, unlike other mechanism-based inhibitors of DBH, lack a latent electrophile. These inhibitors are postulated to covalently modify DBH by a direct insertion of an aberrant substrate-derived benzylic radical into an active site residue.     

Mechanism-based inactivation of horseradish peroxidase by sodium azide. Formation of meso-azidoprotoporphyrin IX

Catalytic turnover of sodium azide by horseradish peroxidase, which produces the azidyl radical, results in inactivation of the enzyme with KI = 1.47 mM and kinact = 0.69 min-1. Inactivation of 80% of the enzyme requires approximately 60 equiv each of NaN3 and H2O2. The enzyme is completely inactivated by higher concentrations of these two agents. meso-Azidoheme as well as some residual heme are obtained when the prosthetic group of the partially inactivated enzyme is isolated and characterized. Reconstitution of horseradish peroxidase with meso-azidoheme yields an enzyme without detectable catalytic activity even though reconstitution with heme itself gives fully active enzyme. The finding that catalytically generated nitrogen radicals add to the meso carbon of heme shows that biological meso additions are not restricted to carbon radicals. The analogous addition of oxygen radicals may trigger the normal and/or pathological degradation of heme.     

Mechanism-based inactivation of the flavoprotein cyclohexanone monooxygenase by S oxygenation

The FAD-dependent enzyme cyclohexanone oxygenase carries out biological Baeyer-Villiger oxidations of ketones and aldehydes with O₂ as cosubstrate. Sulfides are enzymically oxygenated to sulfoxides but thiolactones appear to be enzymically processed to acyl sulfoxides which are reactive acylating and then sulfenylating agents, inducing mechanism-based inactivation.     

Mechanism-based inhibition of dopamine beta-monooxygenase by aldehydes and amides

A mechanism for beta-chlorophenethylamine inhibition of dopamine beta-monooxygenase has been postulated in which enzyme-bound alpha-aminoacetophenone is generated, followed by an intramolecular redox reaction to yield a ketone-derived radical cation as the enzyme inhibitory species (Mangold, J. B., and Klinman, J. P. (1984) J. Biol. Chem. 259, 7772-7779). If correct, additional compounds capable of producing enzyme-bound (formula; see text) reductant should inhibit dopamine beta-monooxygenase. Phenylacetaldehyde was chosen to test this model, since beta-hydroxyphenylacetaldehyde is expected to function as a reductant in a manner analogous to alpha-aminoacetophenone. Phenylacetaldehyde exhibits the properties of a mechanism-based inhibitor. Kinetic parameters are comparable to beta-chlorophenethylamine under both initial velocity and inactivation conditions. Since phenylacetaldehyde bears little resemblance to beta-chlorophenethylamine, its analogous inhibitory action provides support for an intramolecular redox reaction (via beta-hydroxyphenylacetaldehyde oxidation to a radical cation) in dopamine beta-monooxygenase inactivation. beta-Hydroxyphenylacetaldehyde was identified as the enzymatic product of phenylacetaldehyde turnover. As predicted, this product behaves both as a time-dependent inhibitor of dopamine beta-monooxygenase and as an electron donor in enzyme-catalyzed hydroxylation of tyramine to octopamine. Phenylacetamide and p-hydroxyphenylacetamide are also found to be mechanism-based inhibitors of dopamine beta-monooxygenase. In this case the product of hydroxylation (beta-hydroxyphenylacetamide) is redox inactive and, therefore, is unable to function as either a reductant or an inhibitor. Thus, mechanism-based inhibitors are divided into two types: type I, which undergoes hydroxylation prior to inactivation, and type II, which only requires hydrogen atom abstraction. A general mechanism for dopamine beta-monooxygenase inactivation is described, in which a common mechanistic radical intermediate is formed from both pathways.     

Mechanism-based inactivation of rabbit muscle phosphoglucomutase by nojirimycin 6-phosphate

Nojirimycin 6-phosphate (N6P) was tested as a substrate and inhibitor for phosphoglucomutase (PGM). In the absence of glucose 1,6-bisphosphate (GBP), the incubation of PGM and N6P resulted in the complete inactivation of all enzyme activity. When equimolar amounts of N6P and GBP were incubated together with PGM, the GBP was quantitatively converted to glucose 6-phosphate (G6P) and phosphate. At higher ratios of GBP and N6P (greater than 100) the final concentration of G6P produced was found to be 19 times the initial N6P concentration. These results have been interpreted to suggest that the phosphorylated form of PGM catalyzes the phosphorylation of N6P at C-1. This intermediate rapidly eliminates phosphate to form an imine and the dephosphorylated enzyme. The dephosphorylated enzyme is rapidly rephosphorylated by GBP and forms G6P. The imine is nonenzymatically hydrated back to N6P. Occasionally (5%) the imine isomerizes to a compound that is not processed by PGM.     

Mechanism-based inhibition of Sir2 deacetylases by thioacetyl-lysine peptide

Sir2 protein deacetylases (or sirtuins) catalyze NAD+-dependent conversion of epsilon-amino-acetylated lysine residues to deacetylated lysine, nicotinamide, and 2'-O-acetyl-ADP-ribose. Small-molecule modulation of sirtuin activity might treat age-associated diseases, such as type II diabetes, obesity, and neurodegenerative disorders. Here, we have evaluated the mechanisms of sirtuin inhibition of histone peptides containing thioacetyl or mono-, di-, and trifluoroacetyl groups at the epsilon-amino of lysine. Although all substituted peptides yielded inhibition of the deacetylation reaction, the thioacetyl-lysine peptide exhibited exceptionally potent inhibition of sirtuins Sirt1, Sirt2, Sirt3, and Hst2. Using Hst2 as a representative sirtuin, the trifluoroacetyl-lysine peptide displayed competitive inhibition with acetyl-lysine substrate and yielded an inhibition constant (Kis) of 4.8 microM, similar to its Kd value of 3.3 microM. In contrast, inhibition by thioacetyl-lysine peptide yielded an inhibition constant (Kis) of 0.017 microM, 280-fold lower than its Kd value of 4.7 microM. Examination of thioacetyl-lysine peptide as an alternative sirtuin substrate revealed conserved production of deacetylated peptide and 1'-SH-2'-O-acetyl-ADP-ribose. Pre-steady-state and steady-state analysis of the thioacetyl-lysine peptide showed rapid nicotinamide formation (4.5 s-1) but slow overall turnover (0.0024 s-1), indicating that the reaction stalled at an intermediate after nicotinamide formation. Mass spectral analysis yielded a novel species (m/z 1754.3) that is consistent with an ADP-ribose-peptidyl adduct (1'-S-alkylamidate) as the stalled intermediate. Additional experiments involving solvent isotope effects, general base mutational analysis, and density functional calculations are consistent with impaired 2'-hydroxyl attack on the ADP-ribose-peptidyl intermediate. These results have implications for the development of mechanism-based inhibitors of Sir2 deacetylases.     

Mechanism-based inactivation of thioredoxin reductase from Plasmodium falciparum by Mannich bases. Implication for cytotoxicity

Thioredoxin reductase (TrxR) is the homodimeric flavoenzyme that catalyzes reduction of thioredoxin disulfide (Trx). For Plasmodium falciparum, a causative agent of tropical malaria, TrxR is an essential protein which has been validated as a drug target. The high-throughput screening of 350000 compounds has identified Mannich bases as a new class of TrxR mechanism-based inhibitors. During catalysis, TrxR conducts reducing equivalents from the NADPH-reduced flavin to Trx via the two redox-active cysteine pairs, Cys88-Cys93 and Cys535'-Cys540', referred to as N-terminal and C-terminal cysteine pairs. The structures of unsaturated Mannich bases suggested that they could act as bisalkylating agents leading to a macrocycle that involves both C-terminal cysteines of TrxR. To confirm this hypothesis, different Mannich bases possessing one or two electrophilic centers were synthesized and first studied in detail using glutathione as a model thiol. Michael addition of glutathione to the double bond of an unsaturated Mannich base (3a) occurs readily at physiological pH. Elimination of the amino group, promoted by base-catalyzed enolization of the ketone, is followed by addition of a second nucleophile. The intermediate formed in this reaction is an alpha,beta-unsaturated ketone that can react rapidly with a second thiol. When studying TrxR as a target of Mannich bases, we took advantage of the fact that the charge-transfer complex formed between the thiolate of Cys88 and the flavin in the reduced enzyme can be observed spectroscopically. The data show that it is the C-terminal Cys 535'-Cys540' pair rather than the N-terminal Cys88-Cys93 pair that is modified by the inhibitor. Although alkylated TrxR is unable to turn over its natural substrate Trx, it can reduce low M(r) electron acceptors such as methyl methanethiolsulfonate by using its unmodified N-terminal thiols. On the basis of results with chemically distinct Mannich bases, a detailed mechanism for the inactivation of TrxR is proposed.     

Mechanism-based partial inactivation of glutathione S-transferases by nitroglycerin: tyrosine nitration vs sulfhydryl oxidation.

Liver glutathione-S-transferases (GSTs) are responsible for the detoxification of electrophiles, and specifically for the metabolism of orally administered organic nitrates such as nitroglycerin (NTG). Recent studies showed that reactive nitrogen species produced by tetranitromethane (TNM), peroxynitrite, or the myeloperoxidase/H2O2/nitrite system can inactivate GST. It is not known whether NTG can similarly inactivate liver GSTs, and if shown, by what mechanism(s). We incubated purified GSTs with NTG, S-nitroso-N-acetylpenicillamine (SNAP), TNM, or vehicle (5% dextrose, D5W), followed by determination of GST activity. Incubation of GST with NTG and TNM caused significant decreases in GST activity whereas no changes were observed with SNAP or D5W. The relative GST activity (vs preincubation) was 73+/-14% for NTG, 37+/-8% for TNM, 98+/-13% for SNAP, and 98+/-9% for D5W, respectively. Exogenous glutathione (GSH) prevented both NTG- and TNM-induced changes in GST activity, consistent with the observed oxidative modification of GST, such as -SH oxidation and dimerization of oxidized GST. In contrast, NTG and TNM exhibited substantial differences in their ability to nitrate tyrosine (TYR) sites in GST. These results demonstrated that NTG can reduce the activity of its own metabolizing enzyme such as GST and this inhibitory effect of NTG was unlikely to be mediated through NO, as such, since SNAP had no effect on GST activity. The partial inactivation of GST by NTG appeared to involve -SH oxidation, but not TYR nitration. These findings provided the first evidence of mechanism-based protein inactivation by NTG, and may lend insight into the hepatic metabolism of NTG and other organic nitrates after repeated oral exposure.     

Mechanism-based in vivo inactivation of lauric acid hydroxylases

The hepatic cytochrome P-450 isozymes that catalyze omega- and (omega - 1)-hydroxylation of lauric acid are specifically inactivated in vitro but not in vivo by 10-undecynoic acid. The lack of in vivo activity may result from rapid degradation of the inhibitor by beta-oxidation. Strategies for the construction of fatty acid analogues that retain the ability to inactivate fatty acid hydroxylases but are resistant to metabolic degradation have therefore been sought. Fatty acid analogues in which the carboxylic acid group is replaced by a sulfate moiety, or in which two methyl groups are placed vicinal to the carboxylic acid group, have been found to inactivate lauric acid hydroxylases in vitro and in vivo without causing time-dependent inhibition of ethoxycoumarin O-deethylation or N-methyl-p-chloroaniline N-demethylation.     

Mechanism-based inactivation of cytochrome P450 3A4 by L-754,394

Mechanism-based inactivation of human liver P450 3A4 by L-754,394, a Merck compound synthesized as a potential HIV protease inhibitor, was investigated using recombinant P450 3A4. Enzyme inactivation was characterized by a small partition ratio (3.4 or 4.3 +/- 0.4), i.e., the total number of metabolic events undergone by the inhibitor divided by the number of enzyme inactivating events, lack of reversibility upon extensive dialysis, no decrease in the characteristic 450-nm species relative to control, and covalent modification of the apoprotein. The major and minor products formed during the inactivation of P450 3A4 were the monohydroxylated and the dihydrodiol metabolites of L-754,394, respectively. L-754,394 that had been adducted to P450 3A4 was hydrolyzed under the conditions used for SDS-PAGE, Ni(2+) affinity chromatography, and proteolytic digestion. In addition, the modification was not stable to the acidic conditions of HPLC separation and CNBr digestion. The labile nature of the peptide adduct and the nonstoichiometric binding of the inactivating species to P450 3A4 precluded the direct identification of a covalently modified amino acid residue or the peptide to which it was attached. However, Tricine SDS-PAGE in combination with MALDI-TOF-MS and homology modeling, allowed I257-M317 to be tentatively identified as an active site peptide, while prior knowledge of the stability of N-, O-, and S-linked conjugates of activated furans implicates Glu307 as the active site amino acid that is labeled by L-754, 394.     

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.