Chemical modification of pig kidney 3,4-dihydroxyphenylalanine decarboxylase with diethyl pyrocarbonate. Evidence for an essential histidyl residue

Diethyl pyrocarbonate inhibits pig kidney holo-3,4-dihydroxyphenylalanine decarboxylase with a second-order rate constant of 1170 M-1 min-1 at pH 6.8 and 25 degrees C, showing a concomitant increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives. Activity can be restored by hydroxylamine, and the pH curve of inactivation indicates the involvement of a residue with a pKa of 6.03. Complete inactivation of 3,4-dihydroxyphenylalanine decarboxylase requires the modification of 6 histidine residues/mol of enzyme. Statistical analysis of the residual enzyme activity and of the extent of modification shows that, among 6 modifiable residues, only one is critical for activity. Protection exerted by substrate analogues, which bind to the active site of the enzyme, suggests that the modification occurs at or near the active site. The modified inactivated 3,4-dihydroxyphenylalanine decarboxylase still retains most of its ability to bind substrates. Thus, it may be suggested that the inactivation of enzyme by diethyl pyrocarbonate is not due to nonspecific steric or conformational changes which prevent substrate binding. However, the modified enzyme fails to produce at high pH either an enzyme-substrate complex or an enzyme-product complex absorbing at 390 nm. Considerations on this peculiar feature of the modified enzyme consistent with a catalytic role for the modified histidyl residue are discussed. The overall conclusion of this study may be that the modification of only one histidyl residue of 3,4-dihydroxyphenylalanine decarboxylase inactivates the enzyme and that this residue plays an essential role in the mechanism of action of the enzyme.     

Modification of lactate oxidase with diethyl pyrocarbonate. Evidence for an active-site histidine residue

1. Diethyl pyrocarbonate inactivated l-lactate oxidase from Mycobacterium smegmatis. 2. Two histidine residues underwent ethoxycarbonylation when the enzyme was treated with sufficient reagent to abolish more than 90% of the enzyme activity, but analyses of the inactivation showed that the modification of one histidine residue was sufficient to cause the loss of enzyme activity. The rates of enzyme inactivation and histidine modification were the same. 3. Substrate and competitive inhibitors decreased the maximum extent of inactivation to a 50% loss of enzyme activity and modification was decreased from 1.9 to 0.75–1.2 histidine residues modified/molecule of FMN. 4. Treatment of the enzyme with diethyl [¹⁴C]pyrocarbonate (labelled in the carbonyl groups) confirmed that only histidine residues were modified under the conditions used and that deacylation of the ethoxycarbonylhistidine residues by hydroxylamine was concomitant with the removal of the ¹⁴C label and the re-activation of the enzyme. 5. No evidence was found for modification of tryptophan, tyrosine or cysteine residues, and no difference was detected between the conformation and subunit structure of the modified and native enzyme. 6. Modification of the enzyme with diethyl pyrocarbonate did not alter the following properties: the binding of competitive inhibitors, bisulphite and substrate or the chemical reduction of the flavin group to the semiquinone or fully reduced states. The normal reduction of the flavin by lactate was, however, abolished.     

Chemical modification of sheep-liver 6-phosphogluconate dehydrogenase by diethylpyrocarbonate. Evidence for an essential histidine residue

Sheep liver 6-phosphogluconate dehydrogenase is shown to be inactivated by diethylpyrocarbonate in a biphasic manner at pH 6.0, 25 degrees C. After allowing for the hydrolysis of the reagent, rate constants of 56 M-1 s-1 and 11.0 M-1 s-1 were estimated for the two processes. The complete reactivation of partially inactivated enzyme by neutral hydroxylamine, the elimination of the possibility that modification of cysteine or tyrosine residues are responsible for inactivation, and the magnitudes of the rate constants for inactivation relative to the experimentally determined value for the reaction of diethylpyrocarbonate with N alpha-acetylhistidine (2.2 M-1 s-1), all suggested that enzyme inactivation occurs solely by modification of histidine residues. Comparison of the experimental plot of residual fractional activity versus the number of modified histidine residues per subunit with simulated plots for three hypothetical models, each predicting biphasic kinetics, indicated that inactivation results from the modification of at most one essential histidine residue per subunit, although it appears that other (non-essential) histidines react independently. This histidine is thought to be His-242 and is present in the active site. Evidence in support of its role in catalysis is briefly discussed. Both 6-phosphogluconate and organic phosphate protect against inactivation, and a kinetic analysis of the protection indicated a dissociation constant of 2.1 X 10(-6) M for the enzyme--6-phosphogluconate complex. NADP+ also protected, but this might be due, at least in part, to a reduction in the effective concentration of diethylpyrocarbonate.     

Chemical modification ofSemele solida arginase by diethyl pyrocarbonate: Evidence for a critical histidine residue

Arginase from the gills of the bivalveSemele solida was inactivated by diethyl pyrocarbonate (DEPC) in a pseudo-first-order reaction with a bimolecular rate constant of 160 M⁻¹ min⁻¹. The reaction order with respect to DEPC concentration was 1, the inactivation followed a titration curve for a residue with a pK_a of 6.4 at 25°C and the enzymatic activity was restored by hydroxylamine. It is concluded that inactivation results from the modification of a single histidine residue. Borate, a noncompetitive inhibitor with respect to arginine, protected the enzyme from inactivation by DEPC.     

Chemical modification of prostaglandin H synthase with diethyl pyrocarbonate.

The role of histidine in catalysis by prostaglandin H synthase has been investigated using chemical modification with diethyl pyrocarbonate (DEPC), an agent that has been found to rather selectively derivatize histidine residues in proteins under mild conditions. Incubation of the synthase apoprotein with DEPC at pH 7.2 resulted in a progressive loss of the capacity for both cyclooxygenase and peroxidase catalytic activities. The kinetics of inactivation of the cyclooxygenase activity were dependent on the concentration of DEPC; a second-order rate constant of 680 M-1 min-1 was estimated for reaction of the apoenzyme at pH 7.2 and 0 degrees C. The kinetics of inactivation of the cyclooxygenase by DEPC exhibited a sigmoidal dependence on the pH, indicating that deprotonation of a group with a pKa of 6.3 was required for inactivation. The presence of the heme prosthetic group slowed, but did not prevent, inactivation by DEPC. The stoichiometry of histidine modification of apoenzyme during inactivation determined from absorbance increases at 242 nm agreed well with the overall stoichiometry of derivatized residues determined with [14C]DEPC, indicating that modification by DEPC was quite selective for histidine residues on the synthase. Although modification of several histidine residues by DEPC was observed, only one of the histidine residues was essential for cyclooxygenase activity. Modification of the holoenzyme with DEPC altered the EPR signal of the hydroperoxide-induced tyrosyl free radical from the wide doublet (35 G, peak-to-trough) found with the native synthase to a narrower singlet (28 G, peak-to-trough) quite like that found in the indomethacin-synthase complex. Reaction of the indomethacin-synthase complex with DEPC was found to increase the cyclooxygenase velocity by 9 times its initial value, to about one-third of the uninhibited value, without displacement of the indomethacin; the peroxidase was significantly inactivated under the same conditions. Histidyl residues in the synthase are thus likely to have important roles not only in cyclooxygenase and peroxidase catalysis but also in the interaction of the synthase with indomethacin.     

Reaction of 5-enol-pyruvoylshikimate-3-phosphate synthase with diethyl pyrocarbonate: evidence for an essential histidine residue

5-enol-Pyruvoylshikimate-3-phosphate synthase catalyzes the reversible condensation of phosphoenolpyruvate and shikimate 3-phosphate to yield 5-enol-pyruvoylshikimate 3-phosphate and inorganic phosphate. The enzyme is a target for the nonselective herbicide glyphosate (N-phosphonomethylglycine). Diethyl pyrocarbonate inactivated this enzyme with a second-order rate constant of 220 M-1 min-1 at pH 7.0 and 0 degrees C. The rate of inactivation is pH dependent and the pH inactivation rate data show the involvement of a group with a pKa of 6.8. Almost all of the original activity was recovered by treatment of the inactivated enzyme with hydroxylamine. The difference spectrum of the inactivated and native enzyme reveals a single peak at 242 nm but no trough at around 278 nm is observed. Complete inactivation required the modification of four histidine residues per molecule of the enzyme. However, statistical analysis of the residual activity and the extent of modification shows that among the four modifiable residues, only one is critical for activity. Furthermore, this inactivation is prevented by the substrates of the enzyme. The above results indicated that one histidine is located within or very close to the active site and may play an important role in catalysis.     

Interaction of adenosine deaminase with inhibitors. Chemical modification by diethyl pyrocarbonate

The interaction of adenosine deaminase (adenosine aminohydrolase, ADA) from bovine spleen with inhibitors— erythro-9-(2-hydroxy-3-nonyl)adenine, erythro-9-(2-hydroxy-3-nonyl)-3-deazaadenine, and 1-deazaadenosine—was investigated. Using selective chemical modification by diethyl pyrocarbonate (DEP), the possible involvement of His residues in this interaction was studied. The graphical method of Tsou indicates that of six His residues modified in the presence of DEP, only one is essential for ADA activity. Inactivation of the enzyme, though with low rate, in complex with any of the inhibitors suggests that the adenine moiety of the inhibitors (and consequently, of the substrate) does not bind with the essential His to prevent its modification. The absence of noticeable changes in the dissociation constants of any of the enzyme–inhibitor complexes for the DEP-modified and control enzyme indicates that at least the most available His residues modified in our experiments do not participate in binding the inhibitors—derivatives of adenosine or erythro-9-(2-hydroxy-3-nonyl)adenine.     

The reaction of a histidine residue in glutamate dehydrogenase with diethyl pyrocarbonate

1. One mol of diethyl pyrocarbonate will react with one mol of glutamate dehydrogenase polypeptide chains to form one mol of N(1)-carbethoxyhistidine. Reaction is prevented by NADH. 2. The 1:1 complex has an increased specific activity (1.4-2.0-fold). 3. The reason for the activation is discussed. The results are not consistent with NADH dissociation from the enzyme-glutamate-NADH complex being rate-limiting in the steady state measured. 4. The effects of modification on the properties of the enzyme were investigated. The effects of GTP and NAD(+) on the enzyme activity are unaltered by activation. NADH binding is unaltered and there is no apparent change in the molecular weight. However, the activated enzyme can still be further activated by ADP. K(s) for ADP is decreased fivefold.     

Chemical modification of chloroperoxidase with diethylpyrocarbonate. Evidence for the presence of an essential histidine residue

Chloroperoxidase from Caldariomyces fumago is well documented as an extremely versatile catalyst, and studies are currently being conducted to delineate the fine structural features that allow the enzyme to possess chemical and physical similarities to the peroxidases, catalases, and P-450 cytochromes. Earlier investigations of ligand binding to the heme iron of chloroperoxidase, along with the presence of an invariant distal histidine residue in the active site of peroxidases and catalases, have led to the hypothesis that chloroperoxidase also possesses an essential histidine residue that may participate in catalysis. To address this in a more direct fashion, chemical modification studies were initiated with diethylpyrocarbonate. Incubation of chloroperoxidase with this reagent resulted in a time-dependent inactivation of enzyme. Kinetic analysis revealed that the inactivation was due to a simple bimolecular reaction. The rate of inactivation exhibited a pH dependence, indicating that modification of a titratable residue with a pKa value of 6.91 was responsible for inactivation; this data provided strong evidence for histidine derivatization by diethylpyrocarbonate. To further support these results, inactivation due to cysteine, tyrosine, or lysine modification was ruled out. The stoichiometry of histidine modification was estimated by the increase in absorption at 246 nm, and it was found that more than 1 histidine residue was derivatized when chloroperoxidase was inactivated with diethylpyrocarbonate. However, it was shown that the rates of modification and inactivation were not equivalent. This was interpreted to reflect that both essential and nonessential histidine residues were modified by diethylpyrocarbonate. Kinetic analysis indicated that modification of a single essential histidine residue was responsible for inactivation of the enzyme. Studies with [14C]diethylpyrocarbonate provided stoichiometric support that derivatization of a single histidine inactivated chloroperoxidase. Based on sequence homology with cytochrome c peroxidase, histidine 38 was identified as a likely candidate for the distal residue. Molecular modeling, based on secondary structure predictions, allows for the construction of an active site peptide, and implicates a number of other residues that may participate in catalysis.     

Chemical modification of chalcone isomerase by diethyl pyrocarbonate: histidine residues are not essential for catalysis

Chalcone isomerase form soybean is inactivated by treatment with diethyl pyrocarbonate (DEP). The competitive inhibitor 4',4-dihydroxychalcone provides kinetic protection against inactivation by DEP with a binding constant at the site of protection in agreement with its binding constant at the active site. Very high concentrations of the competitive inhibitors 4',4-dihydroxychalcone or morin hydrate offer a 10- to 40-fold maximal protection, suggesting a second slower mechanism for inactivation which cannot be prevented by blockage of the active site. Blockage of the only cysteine residue in chalcone isomerase with p-mercuribenzoate does not affect the rate constant for DEP-dependent inactivation and indicates that the modification of the cysteine residue is not responsible for the activity loss observed in the presence of DEP. Treatment of inactivated enzyme with hydroxylamine does not restore catalytic activity, indicating that the modification of histidine or tyrosine residues is not responsible for the activity loss. All five histidines of chalcone isomerase are modified by DEP at pH 5.7 and ionic strength 1.0 M. The rate constant for the modification of the histidine residues of chalcone isomerase is close to that for the reaction of N-acetyl histidine with DEP, indicating that the histidine residues are quite accessible to the modifying reagent. The rate of histidine modification is the same in native enzyme, in urea-denatured enzyme, and in the presence of a competitive inhibitor. In the presence of the competitive inhibitor morin hydrate, all of the histidine residues of chalcone isomerase can be modified without significant loss in catalytic activity. These results demonstrate that the histidine residues of chalcone isomerase are not essential for catalysis and therefore cannot function as nucleophilic catalysts as previously proposed.     

20 beta-hydroxysteroid dehydrogenase of neonatal pig testis: 3 alpha/beta-hydroxysteroid dehydrogenase activities catalyzed by highly purified enzyme

Pig testicular 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) has also 3 alpha- and 3 beta-HSD (3 alpha/beta-HSD) activities. The purified 20 beta-HSD preparation from neonatal pig testes could catalyze the conversion of 5 alpha-dihydrotestosterone (5 alpha-DHT) in the presence of beta-NADPH to 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol at the ratio of 4:3, and the specific 3 alpha/beta-HSD activity of 20 beta-HSD for 5 alpha-DHT was about 10 or 15 times larger than the 20 beta-HSD activities for 17 alpha-hydroxypregn-4-ene-3,20-dione (17 alpha-hydroxyprogesterone) or progesterone, respectively. The result indicates that the testicular 20 beta-HSD has high 3 alpha(axial, 3R)- and 3 beta(equatorial, 3S)-HSD activity. The testicular 20 beta-HSD could catalyze the reversible conversion of various 5 alpha- or 5 beta-dihydrosteroids which have a 3-carbonyl or 3-hydroxyl group with beta-NADP(H) as the preferred cofactor. The enzyme transferred the 4-proS hydrogen of NADPH to the 5 alpha-DHT for both 3 alpha- and 3 beta-hydroxylation and it was the same as the 20 beta-hydroxylation of 17 alpha-hydroxyprogesterone. Although the 3 alpha/beta-HSD activity has been known to be present in 3 alpha,20 beta-HSD of Streptomyces hydrogenans, the enzymological properties for 3 alpha/beta-HSD activity catalyzed by testicular 20 beta-HSD were different from the properties for 3 alpha/beta-HSD activity catalyzed by prokaryotic 3 alpha, 20 beta-HSD with respect to the specificity of the catalytic reaction and the cofactor requirement.     

Crystals of active tetramers of 3 alpha, 20 beta-hydroxysteroid dehydrogenase

The NADH-dependent steroid metabolizing enzyme 3 alpha, 20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53), from Streptomyces hydrogenans, has been crystallized in the active tetrameric form. Single crystals (approximately 0.75 X 0.40 X 0.40 mm) of square bipyramid shape have been grown reproducibly at room temperature in the presence of excess NADH. Diffraction experiments have been performed at the Cornell High Energy Synchrotron Source. The space group is P43212 or its enantiomorph, and the cell dimensions are a = 106.0(5) A and c = 204(1) A. The asymmetric unit is a tetramer of identical subunits of approximately 25,000 daltons each. The specific volume is 2.8 A3/dalton. A native data set at 2.5-A resolution has been collected. Two potential heavy atom derivatives, with K2Pt(CN)4 and KAu(CN)2, have been identified from the diffraction photographs.     

Inhibition of Streptomyces hydrogenans 3 alpha,20 beta-hydroxysteroid dehydrogenase by licorice-derived compounds and crystallization of an enzyme-cofactor-inhibitor complex.

Streptomyces hydrogenans 3 alpha,20 beta-hydroxysteroid dehydrogenase reduces the C20 ketone on glucocorticoids and progestins. We find that two licorice-derived compounds, glycyrrhizic acid and carbenoxolone, inhibit this enzyme with microM Kis. Inhibition is competitive, indicating that these compounds are binding at or close to the catalytic site. Carbenoxolone's high aqueous solubility and affinity for 3 alpha,20 beta-hydroxysteroid dehydrogenase enabled us to prepare crystals of a carbenoxolone-NADH-enzyme ternary complex, which preliminary X-ray analysis indicates has a crystal structure that is significantly different from that of the 3 alpha,20 beta-hydroxysteroid dehydrogenase-NADH complex. A comparison of the tertiary structures of these two complexes should prove useful in understanding this enzyme's catalytic mechanism, as well as those of two homologous enzymes, mammalian 11 beta-hydroxysteroid dehydrogenase and 15-hydroxyprostaglandin dehydrogenase that also are inhibited by carbenoxolone.     

Chemical modification of Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase by diethyl pyrocarbonate.

Diethyl pyrocarbonate inactivates Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17] by a simple bimolecular reaction. The inactivation is not reversed by hydroxylamine. The pH curve of inactivation indicates the involvement of a residue with a pK of 8.8. Several lines of evidence show that the inactivation is due to the modification of epsilon-amino groups of lysyl residues. Although histidyl residue is also modified, this is not directly correlated to the inactivation. No cysteinyl, tyrosyl, or tryptophyl residue or alpha-amino group is significantly modified. The modification of three lysyl residues per enzyme subunit results in the complete loss of aldolase activity toward various 4-hydroxy-2-oxo acid substrates, whereas oxaloacetate beta-decarboxylase activity associated with the enzyme is not inhibited by this modification. Statistical analysis suggests that only one of the three lysyl residues is essential for activity. l-4-Carboxy-4-hydroxy-2-oxoadipate, a physiological substrate for the enzyme, strongly protects the enzyme against inactivation. Pi as an activator of the enzyme shows no specific protection. The molecular weight of the enzyme, Km for substrate or Mg2+, and activation constant for Pi are virtually unaltered after modification. These results suggest that the modification occurs at or near the active site and that the essential lysyl residue is involved in interaction with the hydroxyl group but not with the oxal group of the substrate.     

Crystallization and preliminary crystallographic study of 3 alpha, 20 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans

3 alpha, 20 beta-Hydroxysteroid dehydrogenase, an NADH-dependent oxidoreductase isolated from Streptomyces hydrogenans , is a tetramer containing four subunits each of Mr 25,000. The enzyme has been crystallized by the vapor diffusion technique using either phosphate or borate buffered ammonium sulfate (pH between 6.0 and 8.7) as the precipitant. The crystals are hexagonal bipyramids ; they have the symmetry of space group P6(4)22 (or P6(2)22), with unit cell dimensions a = 127.3 A, c = 112.2 A. Volume and density considerations imply that the crystallographic asymmetric unit contains two monomers, and therefore that the tetramer possesses a 2-fold axis of symmetry that is coincident with a crystallographic 2-fold symmetry element.     

Chemical modification of glucosamine-6-phosphate synthase by diethyl pyrocarbonate: evidence of histidine requirement for enzymatic activity.

Glucosamine-6-phosphate synthase from Escherichia coli was inactivated by diethylpyrocarbonate at pH 7.3 and 4 degrees C with a second-order rate constant of 1220 M-1 min-1. The difference spectrum of inactivated vs native enzyme had a maximum absorption at 242 nm, which is characteristic of N-carbethoxyhistidine. No trough at around 280 nm due to O-carbethoxytyrosine was observed and the sulfhydryl content of the enzyme was unchanged. Studies with [14C]diethylpyrocarbonate provided evidence that derivatization of a single histidine residue of the amino-terminal glutamine-binding domain inactivated glucosamine-6P synthase. These results are consistent with the participation of an histidine residue in a catalytic triad, Cys/His/Asp, necessary to generate ammonia from glutamine.     

Ontogeny of testicular steroid dehydrogenase enzymes in pig (3 alpha/beta-, 20 alpha- and 20 beta-): evidence for two forms of 3 alpha/beta-hydroxysteroid dehydrogenase.

Three enzymatic activities (3 alpha/beta-hydroxysteroid dehydrogenase, 20 beta- and 20 alpha-hydroxysteroid dehydrogenases) were measured in testes of pigs as a function of age. Earlier studies reported a highly purified 20 beta-hydroxysteroid dehydrogenase from neonatal pig testes that also showed strong 3 alpha/beta-hydroxysteroid dehydrogenase activity [Ohno et al., J. Steroid Biochem. Molec. Biol. 38 (1991) 787-794]. We report here that neonatal pigs testis is rich in 3 alpha/beta- and 20 beta-hydroxysteroid dehydrogenase activities, both of which fall to low levels (measured as specific activity) at 60 days. Thereafter the activity of 3 alpha/beta-reduction rises to high levels whereas 20 beta-reduction remains low. Activity of 20 alpha-reduction is of intermediate level in the neonate, falls to a nadir at 60 days and rises to high levels in the mature animal. Western blots of cytosolic proteins show that the bifunctional enzyme (3 alpha/beta-plus 20 beta-hydroxysteroid dehydrogenase) is high in neonatal testes and falls to low levels at maturity. It is proposed that the neonatal testis possesses the bifunctional enzyme which is replaced by a second enzyme at maturity, that is a 3 alpha/beta-hydroxysteroid dehydrogenase without 20 beta-reductase activity. The possible functional significance of these changes is considered.     

Chemical modification ofPseudomonas fluorescens malonyl-CoA synthetase by diethylpyrocarbonate: Kinetic evidence for an essential histidyl residue on alpha subunit

Malonyl-CoA synthetase fromPseudomonas fluorescens was inactivated by diethylpyrocarbonate (DEP) with the second-order rate constant of 775 M^?1 min^?1 atpH 7.0, 25°C, showing a concomitant increase in absorbance at 242 nm due to the formation of N-carbethoxyhistidyl derivatives. The inactivated enzyme at low concentration of DEP (<0.2 mM) could be completely reactivated by hydroxylamine but not completely reactivated at high concentration (>0.5 mM), indicating that there may be another functional group modified by DEP. Complete inactivation of malonyl-CoA synthetase required the modification of seven residues per molecule of enzyme; however, only one is calculated to be essential for enzyme activity by a statistical analysis of the residual enzyme activity.pH dependence of inactivation indicated the involvement of a residue with apK _a of 6.7, which is closely related to that of histidyl residue of proteins. Whena subunit treated with DEP was mixed with β subunits complex, the enzyme activity completely disappeared, whereas when β subunit complex treated with the reagent was mixed witha subunit, the activity remained. Inactivation of the enzyme by the reagent was protected by the presence of malonate and ATP. These results indicate that a catalytically essential histidyl residue is located at or near the malonate and ATP binding region ona subunit of the enzyme.     

Chemical modification by diethylpyrocarbonate of an essential histidine residue in 3-ketovalidoxylamine A C-N lyase

3-Ketovalidoxylamine A C-N lyase of Flavobacterium saccharophilum is a monomeric protein with a molecular weight of 36,000. Amino acid analysis revealed that the enzyme contains 5 histidine residues and no cysteine residue. The enzyme was inactivated by diethylpyrocarbonate (DEP) following pseudo-first order kinetics. Upon treatment of the inactivated enzyme with hydroxylamine, the enzyme activity was completely restored. The difference absorption spectrum of the modified versus native enzyme exhibited a prominent peak around 240 nm, but there was no absorbance change above 270 nm. The pH-dependence of inactivation suggested the involvement of an amino acid residue having a pKa of 6.8. These results indicate that the inactivation is due to the modification of histidine residues. Substrates of the lyase, p-nitrophenyl-3-ketovalidamine, p-nitrophenyl-alpha-D-3-ketoglucoside, and methyl-alpha-D-3-ketoglucoside, protected the enzyme against the inactivation, suggesting that the modification occurred at or near the active site. Although several histidine residues were modified by DEP, a plot of log (reciprocal of the half-time of inactivation) versus log (concentration of DEP) suggested that one histidine residue has an essential role in catalysis.