Group Of pK Research Articles

A Glutamyl Residue in the Active Site of Triphosphopyridine Nucleotide-dependent Isocitrate Dehydrogenase of Pig Heart

Abstract The maximum velocity of the reaction catalyzed by the pig heart TPN-specific isocitrate dehydrogenase depends on the basic form of an enzymatic group of pK 5.7. This pK is independent of temperature from 10–30° and increases in 20% ethanol, suggesting the ionization of a carboxyl group. The enzyme is inactivated by incubation at pH 7.0 with 1 -cyclohexyl-3-(2-morpholinoethyl)-carbodiimide either alone or in the presence of glycine ethyl ester, glycinamide, or glycine. Both the dehydrogenase and decarboxylase functions of the enzyme are affected. The addition of manganous ion and isocitrate or α-ketoglutarate to the incubation mixture produces a striking decrease in the inactivation rate, implying that reaction takes place in the active site. The inactive glycinamide enzyme exhibits a decreased ability to bind manganous ion in the presence of isocitrate, which suggests that the integrity of those amino acid residues susceptible to the carbodiimide and glycinamide are important for the binding of the manganous-isocitrate complex by the enzyme. The most probable sites of reaction of the carbodiimide are tyrosine, cysteine, glutamic and aspartic acids. Inactivation is not reversed by hydroxylamine indicating that tyrosine modification is not responsible; and no significant change in the measurable sulfhydryl content is noted upon inactivation. Treatment of the enzyme with the carbodiimide in the presence of [14C]glycine ethyl ester leads to incorporation of 1 mole of radioactive compound per mole of enzyme, concomitant with inactivation. Exhaustive proteolytic digestion of the radioactive protein followed by paper chromatography and electrophoresis led to the identification of γ-glutamylglycine as the product of the modification reaction. It is concluded that a glutamyl residue is essential for the catalytic function of isocitrate dehydrogenase and may lie within the substrate binding site.

The time dependence of the activity of delta-chymotrypsin at high pH.

When δ‐chymotrypsin at a high initial pH is mixed with a specific ester substrate, the hydrolytic reaction exhibits a lag phase that can be observed from the release of both products, the alcohol or the amino acid. This lag phase is under the dependence of the ionization of a group of pK 9, at 15°C, which has been previously shown to be the α‐amino group of isoleucine‐16. This lag corresponds to the time needed for the enzyme to isomerize from its “high‐pH” to its “neutral” conformation, and fluorescence measurements have been separately used to monitor this isomerization in the presence of substrate. The initial activity observed when the enzyme is mixed with the substrate is proportional to the concentration of enzyme present in its neutral conformation and is not significantly measurable when the enzyme is in its high‐pH conformation.It is concluded that the high‐pH form of δ‐chymotrypsin is inactive and cannot be involved in the observed activity of the enzyme at high pH. Therefore the activity of δ‐chymotrypsin exhibited at high pH is linked to a substrate‐promoted isomerization of the protein, that is, to a displacement of the conformational equilibrium presented by the protein towards its active neutral conformation.

Influence of pH on the kinetics of reactions catalyzed by alkaline phosphatase.

An experimental study has been made of the kinetics of the hydrolysis of p-nitrophenyl phosphate catalyzed by chicken-intestinal alkaline phosphatase. The work was done in barbital buffer (carbonate above pH 9.6), and covered the pH range from 7.0 to 10.0. A sufficiently wide range of substrate concentration was used to allow reliable values of [Formula: see text] and [Formula: see text] to be determined. The results lead to pK values of 8.1 and 8.6 for the free enzyme, and it is concluded that the Michaelis complex and the phosphoryl intermediate ionize only on the acid side, the former also having a pK of 8.1. It is suggested that the group of pK 8.1 is probably an α-amino group and that the group of pK 8.6 probably corresponds to the ionization of a Zn(II)-coordinated water molecule.

The effect of methanol and dioxan on the rates of the beta-galactosidase-catalysed hydrolyses of some beta-D-galactrophyranosides: rate-limiting degalactosylation. The ph-dependence of galactosylation and degalactosylation.

1. The effect of methanol on the beta-galactosidase-catalysed hydrolysis of some nitrophenyl beta-d-galactopyranosides has been studied under steady-state conditions. 2. The initial fractional rate of increase of k(cat.) as a function of methanol concentration with 2,4- and 3,5-dinitrophenyl beta-d-galactopyranosides, but not with the other substrates studied, indicated that degalactosylation of the enzyme was rate-limiting. 3. The decrease in k(cat.) at high methanol concentrations for these substrates is considered to arise from causes other than galactosylation becoming rate-limiting. 4. Both galactosylation and degalactosylation of the enzyme require protonation of a group of pK(a) approx. 9.

The involvement of histidine in the action of liver carboxylesterases

Chicken and ox liver carboxylesterases are inhibited irreversibly by α-bromoacetophenone. A kinetic analysis of the process indicates that inhibition occurs at the active site by reaction with a group of pK a′ 6.15 which is reactive in the basic form. Using [ 14C]bromoacetophenone, it has been shown that loss of activity is accompanied by incorporation of one molecule of inhibitor per active site, together with some non-specific reaction. Acid hydrolysis of the inhibited enzyme leads to a number of radioactive products, including 1- and 3-phenacylhistidines. Chicken liver carboxylesterase is irreversibly inhibited by methyl p-nitrobenzenesulfonate. This inhibition is prevented by the presence of the competitive inhibitor, n-propanol.

Evidence for the His-57 sidechain acting as a general base in the “ageing” and reactivation reactions of carbonate-inhibited α-chymotrypsin

The chymotrypsin-catalyzed “ageing” reactions which follow inhibition with phosphate or carbonate esters involve an active site group of pK a near 7, implicating the imidazole of His 57, as with deacylations. With bis (p-nitrophenyl) carbonate recent evidence is in conflict over whether in the “ageing” reaction the imidazole acts as a general base or as a nucleophile. A crucial in, situ reactivation experiment has been carried out at high pH to distinguish between these cases. An ionizable enzyme group of pK 7.4 is involved in the reactivation process for at least 80% of the enzyme. This indicates that imidazole acts mainly as a general base in the ageing and reactivation reactions of labile carbonate di-esters with chymotrypsin.

On the action of carboxypeptidase a on ester substrates in alkaline solution

Summary Kinetic measurements on the pH dependency of the rate parameter k cat /K M for the Mn ++ -carboxypeptidase A δ -catalyzed hydrolysis of O -( trans -cinnamoyl)-L-β-phenyllactate have shown that an ionizing function in the enzyme with a pK a value of 9. 3 is involved in this reaction. Since experiments on the acetylation of the native γ-form of the enzyme appear to indicate that the phenolic hydroxyl groups of tyrosine residues in carboxypeptidase do not play a crucial role in the enzymatic catalysis of the hydrolysis of O -( trans -cinnamoyl)-L-β-phenyllactate, it is suggested that the group of pK a = 9. 3 is probably the active site Mn ++ -H 2 O complex.

The effect of pH on the kinetic parameters of shikimate dehydrogenase from Pisum sativum

1. 1. The pH-dependence of the kinetic parameters of shikimate dehydrogenase was examined. 2. 2. Studies of the pH-dependence of the inhibitor constants of 3 structural analogues of shikimate, which are competitive inhibitors of the enzyme with respect to shikimate, were also carried out. 3. 3. From the results obtained it is postulated that a group in the enzyme of pK 9·4, possibly an ε-NH 2 of lysine, binds the carboxylate ion, while a group of pK 8·6, possibly a sulphydryl residue, interacts with the 4-OH of shikimate and the inhibitors. 4. 4. In addition, other structural analogues of shikimate were examined as possible inhibitors of the reaction.

The specificity of penicillopepsin.

The specificity of penicillopepsin was investigated with a number of different substrates. In agreement with earlier work (1), no action was observed on di- and tripeptide and ester substrates. However, the pepsin substrate carbobenzoxyglycylglycylphenylalanylphenylalanine 3-(4-pyridyl)propyl-1 ester was hydrolyzed between the two phenylalanine residues. No cleavage of homopolymers of glycine, alanine, and glutamic acid and the random copolymer of glutamic acid and tyrosine was observed. Polymers of lysine, lysine and tyrosine (2:1), and lysine, glutamic acid, and tyrosine (13:20:1) were, however, hydrolyzed giving mainly tripeptides to pentapeptides. Polylysine hydrolysis showed a pH dependence centering about a group of pK between 3 and 4. In glucagon and the S-sulfo B-chain of insulin, penicillopepsin hydrolyzed the same peptide bonds as the other acidic proteases, including pepsin and rennin, with very few exceptions. There are, however, distinct differences between the action of penicillopepsin and that of other proteases of low specificity. Peptide bonds which have a hydrophobic amino acid in the P1′ position (as defined by Berger and Schechter, Ref. 2) are preferentially cleaved by penicillopepsin. A kinetic study of the hydrolysis of bovine serum albumin showed very high rate constants for the initial cleavages. The present study shows the requirement for an extended binding site in penicillopepsin.

The interaction of alpha-chymotrypsin with phenylalanine derivatives containing a free alpha-amino group.

The action of α-chymotrypsin on L- and D-phenylalanine ethyl esters (PEE), L- and D-phenylalanine p-nitrobenzyl esters (PNBE), L-phenylalanine methyl and isopropyl esters, and N-methyl-L-phenylalamne methyl ester has been studied using a pH-stat. The D-esters were not hydrolyzed but acted as competitive inhibitors of the hydrolysis of the L-isomers. The N-methyl ester was very slowly hydrolyzed due to its low Kcat. For L-PEE (pK 7.23) and L-PNBE (pK 6.93), the activity of α-chymotrypsin is displaced to a more acid region relative to that for the N-acyl amino acid esters. The Km increases sharply below pH 6.5 while the kcat and kcat/Km show maxima at pH 6 and 7.6, respectively. On the acid side kcat is controlled by a basic group of pK 4.86 for L-PNBE and pK 5.1 for L-PEE, and kcat/Km by a basic group of pK 6.4 for L-PNBE and pK 6.6 for L-PEE. It is proposed that (i) deacylation is rate-limiting, (ii) in the catalytically active entities of the enzyme–substrate complex and acyl enzyme, the α-amino group of the substrate is protonated, (iii) the pK of the basic group on the acyl enzyme is considerably lowered by the presence of the [Formula: see text] of the substrate, and (iv) the increase in Km and Ki below pH 6.8 is due to the development of unfavorable charge interactions.

Modification of trypsin by methyl [formula omitted]-guanidiniumbenzenesulfonate fluoroborate

Methyl meta -guanidiniumbenzenesulfonate fluoroborate has been found to irreversibly inhibit the activity of trypsin. The rate of inhibition was found to be dependent on the basic form of an ionizable group of pK a about 7. The presence of a competitive inhibitor was found to protect trypsin against modification by the meta -ester. Methyl para -guanidiniumbenzenesulfonate fluoroborate was found not to irreversibly inhibit trypsin.

The inhibition of chymotrypsin A4 with a homologous series of chloromethyl ketone reagents.

The inactivation of chymotrypsin A4 (CHT-A4) by a homologous phenylalkyl series of bifunctional reagents, viz. phenyl chloromethyl ketone (PCK), benzyl chloromethyl ketone (BCK), and β-phenylethyl chloromethyl ketone (βPECK) (C6H5 [CH2]n COCH2Cl where n = 0, 1, or 2), has been investigated. The inactivation of CHT-A4 by PCK has been shown to be essentially pH independent whereas inactivation by BCK and βPECK appears to be dependent on a basic group of pK 6.0–6.3. PCK has been shown to inhibit CHT-A4 by virtue of the complete S-alkylation of methionine-192. BCK and βPECK, on the other hand, inactivate CHT-A4 through a combination of partial S-alkylation of methionine-192 (0.2 and 0.3 residue, respectively) and partial alkylation of histidine-57 (0.2 and 0.4 residue, respectively). Identification of the particular residue alkylated was obtained through the isolation and analysis of alkylated and oxidized peptic peptides obtained from the alkylated CHT-A4 by the diagonal peptide ionophoresis technique.The initial site of alkylation of BCK and βPECK on the imidazole ring of histidine-57 has not been identified. However, a unique histidine derivative has been isolated from the acid hydrolysate of oxidized histidine-57 peptide from CHT-A4–βPECK and is suggested to be 2(or 4)-hydroxymethylhistidine.On the basis of the present studies on the phenylalkyl series and on studies reported earlier on a phenylalkylamido halomethyl ketone series of bifunctional reagents which alkylates only methionine in CHT-A4, it has been suggested that alkylation of histidine by members of the phenylalkyl series is primarily a result of binding to the hydrophobic binding site of CHT-A4 rather than binding to the acylamido binding site as is suggested to be the case for the phenylalkylamido series.

Kinetics of inhibition of papain by TLCK and TPCK in the presence of BAEE as substrate

The inhibition of papain by TPCK and TLCK was first reported by Shaw, Guia and Cohen [ 11. Subsequently Husain and Lowe [2] demonstrated that TGCK reacted with papain to give an enzymically inactive product in which the sulphydryl group of cysteine-25 was substituted. Bender and Brubacher [3] were able to show a similar reaction of papain with TPCK by following the loss of the cysteine content of the enzyme after reaction with the inhibitor. The rate of inhibition increased withincreasing pH, being dependent on a single prototropic group of pK 8.29 (25O), suggesting that a thiol anion was the reactive species. Similar results were obtained by Whitaker and Perez-VillaseAor [4] for the reaction with TLCK. This inhibitor reacts with cysteins25 much faster than does TPCK, due to the fact that papain preferentially hydrolyses derivatives of arginine and lysine. The effect of substrates on the inhibition rates of TPCK and TLCK is unknown. We therefore undertook a study to determine whether TPCK and TLCK have substrate-like properties. If these inhibitors behave like substrates, it would be expected that substrates should compete in the inhibition reaction. The results suggest that TLCK has substrate-like properties and so could be used in X-ray diffraction studies of papain [S] as a model for an enzymesubstrate complex.

The inhibition of chymotrypsin A4 and B with chloromethyl ketone reagents.

The inactivation of chymotrypsin A4 and B by the bifunctional reagents L-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone (L-TPCK) and phenoxymethyl chloromethyl ketone (PMCK) has been investigated. The rate of inactivation of chymotrypsin A4 with both reagents as a function of pH has been shown to be dependent on a basic group of pK = 6.3–6.5. Chymotrypsin B inactivation appears to be dependent on a basic group with a somewhat lower pK. For each enzyme the reaction with both reagents is associated with the loss of a single histidine residue. By the isolation and identification of 3-carboxymethylhistidine from the alkylated and oxidized peptic histidine-57 peptides, it has been concluded that both enzymes are alkylated at the nitrogen-3 position of histidine-57 by L-TPCK and PMCK. Evidence for the submolar alkylation of methionine-192 of chymotrypsin A4 by L-TPCK, PMCK, D-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone (D-TPCK), and N-methyl-L-TPCK is presented. There is no alkylation of the histidine of chymotrypsin A4 by D-TPCK or.N-methyl-L-TPCK.From a comparison of the structures of a number of reagents known to alkylate chymotrypsin A4, it has been concluded that the alkylation of methionine-192 is nonspecific and relatively independent of any defined stereochemistry of the reagent employed. To date the alkylation of histidine-57 has been shown to occur only with haloketones, and is dependent on the distance between the haloketone and the aromatic ring when the latter is present. Although the presence of an asymmetric α-carbon and acylamido group in straight-chain reagents is unnecessary for histidine alkylation, these must be of the L configuration if present.

Effect of Modification of a Methionyl Residue on the Kinetic and Molecular Properties of Isocitrate Dehydrogenase

Abstract Pig heart isocitrate dehydrogenase, which catalyzes the TPN-dependent dehydrogenation of isocitrate as well as the decarboxylation of oxalosuccinate, is inactivated by treatment with iodoacetate at pH 5.5 and 30°. Approximately 1 mole of 14C-labeled reagent is incorporated per mole of protein. The effect of alkylation on the isocitrate dehydrogenase activity is greater than that on the oxalosuccinate decarboxylase activity of the enzyme, and therefore the active sites for these two catalytic functions are distinguishable. Inactivation can be prevented by the inclusion of isocitrate in the reaction mixture, suggesting that iodoacetate causes a specific modification in the functional region of the enzyme. Vmax for both the native and the partially modified enzyme depends on the unprotonated form of an ionizable group of pK 5.4, which shifts to pK 6.3 when measured in 20% ethanol as solvent. A requirement for the dissociable form of a carboxyl group is suggested. The Michaelis constants of partially alkylated enzymes are unchanged from those of the native enzyme. It is concluded that iodoacetate alters an amino acid residue essential for the dehydrogenase reaction. Iodoacetate treatment does not produce marked structural alterations in the enzyme, since no differences in molecular weight or in the ultraviolet absorption or optical rotatory dispersion spectra were detected. The pseudo-first order rate constant for inactivation of the enzyme by iodoacetate decreases from pH 7.8 to 7.2, is almost constant from pH 7.2 to 6.1, and increases markedly below pH 6.0. Enzyme alkylated at pH 5.6 exhibits the same sulfhydryl content as the native enzyme, and an unaltered histidine, lysine, and tyrosine composition. Amino acid analysis indicates that the methionine content of the alkylated enzyme is decreased by 1 residue as compared to the native enzyme, and a new peak, that of carboxymethylhomocysteine, is observed. Paper chromatography of proteolytic digests of 14C-labeled alkylated enzyme confirms the conclusion that alkylation of 1 methionine residue is responsible for the altered activity of isocitrate dehydrogenase. The rate of inactivation at pH 5.6 is independent of ionic strength. Modified enzyme exhibits a mobility identical with that of native enzyme in cellulose acetate electrophoresis from pH 6.2 to 9.5. These observations are consistent with attack of iodoacetate on an uncharged methionyl residue to yield dipolar carboxymethylmethionine sulfonium salt.

Investigations of the Chymotrypsin-catalyzed Hydrolysis of Specific Substrates: I. THE pH DEPENDENCE OF THE CATALYTIC HYDROLYSIS OF N-ACETYL-l-TRYPTOPHANAMIDE BY THREE FORMS OF THE ENZYME AT ALKALINE pH

Abstract The pH dependence of the steady state kinetic parameters Km (app) and kcat of the δ-, acetylated δ-, and α-chymotrypsin-catalyzed hydrolysis of the specific amide substrate N-acetyl-l-tryptophanamide, has been investigated in the neutral and alkaline pH regions. A completely automatic technique for measuring the hydrolysis of amides was used. New results and important aspects of the chymotrypsin-catalyzed hydrolysis of a specific amide substrate which have emerged from this investigation are the following. The pH dependence of the catalytic reaction at alkaline pH is adequately accounted for by the pH dependence of Km(app). In earlier experiments we have shown that in amide hydrolysis the steady state kinetic parameter Km(app) is a measure of an over-all enzyme-substrate dissociation constant (K's). (a) Km(app) was observed to be pH-dependent in the hydrolysis of N-acetyl-l-tryptophanamide as catalyzed by all three forms of the enzyme. Analysis of this pH dependence shows that an ionizing group with pK(app) ∼ 8.5 is involved; an ionizing group of the enzyme with this pK(app) has been implicated in all chymotrypsin-catalyzed reactions that have been studied. (b) The catalytic rate constant kcat was found to be pH-independent in the pH region 8 to 10 for the δ-chymotrypsin-catalyzed reaction and in the pH region 8 to 9.2 for the α-chymotrypsin-catalyzed reaction. (c) The pH dependence of the steady state kinetic parameters kcat and Km(app) in the chymotrypsin-catalyzed hydrolysis ofN-acetyl-l-tryptophanamide is consistent with the findings of our earlier studies of the individual steps in the reaction between chymotrypsin and diisopropyl fluorophosphate and in the chymotrypsin-catalyzed hydrolysis of N-acetyl-l-phenylalaninamide: studies which indicated that the pH dependence of chymotrypsin-catalyzed hydrolysis above pH 8 is due to the effect of pH on the formation of chymotrypsin complexes and not on the bond-breaking step. (d) The experiments reported here are not in agreement with earlier studies of Bender and his co-workers, which indicated that the pH dependence of chymotrypsin-catalyzed reactions above pH 8 is due to the effect of pH on the bond-breaking step, the formation of chymotrypsin-substrate complexes being pH-independent. The δ form of chymotrypsin is a more efficient catalyst than the α form in the hydrolysis of N-acetyl-l-tryptophanamide, as indicated by both the observed maximum rate of reaction and the binding of substrate to enzyme. Both the values and the pH dependencies of the steady state kinetic parameters are different for these two forms of the enzyme, but are the same for δ- and acetylated δ-chymotrypsin. Only limited and qualified information regarding differences in the catalytic properties of α- and δ-chymotrypsin has been previously available. These and previous experiments are consistent with the hypothesis that the pH dependence of chymotrypsin-catalyzed reactions at alkaline pH is due to a pH-dependent equilibrium between two major conformations of the enzyme, with the same ionizing group of pK (app) ∼ 8.5 controlling both the equilibrium between enzyme conformations and the decrease in catalytic properties of the enzyme. This hypothesis leads to several predictions, discussed in this paper, which can be tested experimentally.

Transglutaminase: mechanistic features of the active site as determined by kinetic and inhibitor studies.

Abstract 1. 1. The K m values for the transglutaminase-catalyzed (amine: R-glutamine transferase (hydrolyzing) hydrolysis and hydroxylamine incorporation reactions and the K n values for inhibition by carbobenzoxy-(CBZ)-amino acids and CBZ-peptides, all obtained as functions of pH, reveal an enzyme group of pK 7.5 to 8.5. Inhibitor specificity studies indicate that this group is involved in binding substrate at the peptide bond formed through the α-amino group of the glutamine residue. 2. 2. Inhibitor constants for chloroacetyl amino acids show no variation with pH. It is suggested that these inhibitors, as well as chloroacetic acid, are bound to an enzyme active center group through the carbonyl carbon of their chloroacetyl position. The change in K i values for chloroacetic acid at about pH 6.5 has been attributed to an induced shift in pK of another group which is in the close structural vicinity. 3. 3. This group, tentatively identified as an-SH on the basis of its sensitivity to thiol reagents, has an apparent pK of around 6.5, as judged from activity titration versus p H with chloroacetamide, decrease in v max . with pH and induced shift in its pK with chloroacetic acid. 4. 4. The efficient protection against thiol reagent inhibition by substrate supports a supposition that this -SH group forms a covalent bond with substrate during the catalytic reaction.

Purification and properties of erythro-β-hydroxyasparate dehydratase from Micrococcus denitrificans

1. The novel enzyme, erythro-beta-hydroxyaspartate dehydratase, a key enzyme of the beta-hydroxyaspartate pathway (Kornberg & Morris, 1963, 1965), has been purified 30-fold from extracts of glycollate-grown Micrococcus denitrificans. The purified preparation was devoid of erythro-beta-hydroxyaspartate-aldolase activity, and free from enzymes that act on oxaloacetate. 2. Properties of the purified dehydratase were studied by direct assay of the enzymic formation of oxaloacetate and ammonia from added erythro-beta-hydroxyaspartate. 3. The enzyme was highly substrate-specific, utilizing only the l-isomer of erythro-beta-hydroxyaspartate (K(m), 0.43mm, and V(max.), 99mumoles of oxaloacetate formed/min./mg. of protein at pH9.15 and 30 degrees ). Of many compounds tested, only maleate was a competitive inhibitor (K(i), 32mm at pH7.6). 4. The optimum pH for activity was about 9.5. The K(m) varied with pH, showing a marked optimum at pH7.8. The V(max.) also varied with pH in a manner suggesting the presence in the enzyme-substrate complex of a dissociable group of pK'(a) about 8.5. 5. Carbonyl reagents were inhibitory, but of three thiol reagents tested only p-chloromercuribenzoate was inhibitory. 6. A partially resolved preparation of the enzyme was activated four-fold by the addition of pyridoxal phosphate and thereby restored to half activity. 7. EDTA (0.1mm) was almost completely inhibitory, activity being restored by bivalent cations (Mg(2+), Ca(2+) and Mn(2+)); no activation by univalent cations was observed. 8. The findings are discussed in the light of reported properties of related hydroxyamino acid dehydratases.

Liberation of phenol from diphenylphosphoryl chymotrypsin

The chymotrypsin-catalyzed “ageing” reactions which follow inhibition with phosphate or carbonate esters involve an active site group of pKa near 7, implicating the imidazole of His 57, as with deacylations. With bis (p-nitrophenyl) carbonate recent evidence is in conflict over whether in the “ageing” reaction the imidazole acts as a general base or as a nucleophile. A crucial in, situ reactivation experiment has been carried out at high pH to distinguish between these cases. An ionizable enzyme group of pK 7.4 is involved in the reactivation process for at least 80% of the enzyme. This indicates that imidazole acts mainly as a general base in the ageing and reactivation reactions of labile carbonate di-esters with chymotrypsin.