Modification of uridine phosphorylase from Escherichia coli by diethyl pyrocarbonate. Evidence for a histidine residue in the active site of the enzyme.

Uridine phosphorylase from Escherichia coli is inactivated by diethyl pyrocarbonate at pH 7.1 and 10 degrees C with a second-order rate constant of 840 M-1.min-1. The rate of inactivation increases with pH, suggesting participation of an amino acid residue with pK 6.6. Hydroxylamine added to the inactivated enzyme restores the activity. Three histidine residues per enzyme subunit are modified by diethyl pyrocarbonate. Kinetic and statistical analyses of the residual enzymic activity, as well as the number of modified histidine residues, indicate that, among the three modifiable residues, only one is essential for enzyme activity. The reactivity of this histidine residue exceeded 10-fold the reactivity of the other two residues. Uridine, though at high concentration, protects the enzyme against inactivation and the very reactive histidine residue against modification. Thus it may be concluded that uridine phosphorylase contains only one histidine residue in each of its six subunits that is essential for enzyme activity.     

Evidence for an essential histidine in neutral endopeptidase 24.11

Rat kidney neutral endopeptidase 24.11, "enkephalinase", was rapidly inactivated by diethyl pyrocarbonate under mildly acidic conditions. The pH dependence of inactivation revealed the modification of an essential residue with a pKa of 6.1. The reaction of the unprotonated group with diethyl pyrocarbonate exhibited a second-order rate constant of 11.6 M-1 s-1 and was accompanied by an increase in absorbance at 240 nm. Treatment of the inactivated enzyme with 50 mM hydroxylamine completely restored enzyme activity. These findings indicate histidine modification by diethyl pyrocarbonate. Comparison of the rate of inactivation with the increase in absorbance at 240 nm revealed a single histidine residue essential for catalysis. The presence of this histidine at the active site was indicated by (a) the protection of enzyme from inactivation provided by substrate and (b) the protection by the specific inhibitor phosphoramidon of one histidine residue from modification as determined spectrally. The dependence of the kinetic parameter Vmax/Km upon pH revealed two essential residues with pKa values of 5.9 and 7.3. It is proposed that the residue having a kinetic pKa of 5.9 is the histidine modified by diethyl pyrocarbonate and that this residue participates in general acid/base catalysis during substrate hydrolysis by neutral endopeptidase 24.11.     

The role of an essential histidine residue of yeast alcohol dehydrogenase.

1. Inactivation of yeast alcohol dehydrogenase for diethyl pyrocarbonate indicates that one histidine residue per enzyme subunit is necessary for enzymic activity. The inactivated enzyme regains its activity over a period of days. 2. Enzyme modified by diethyl pyrocarbonate can form the binary enzyme - NADH complex with the same maximum NADH-binding capacity as that of native enzyme. Modified enzyme cannot form normal ternary complexes of the type enzyme - NADH - acetamide and enzyme - NAD+ - pyrazole, which are characteristic of native enzyme. 3. The rate constant for the reaction of enzyme with diethyl pyrocarbonate has been determined over the pH range 5.5--9. The histidine residue involved has approximately the same pKa as free histidine, but is 10-fold more reactive than free histidine.     

Acetyl-coenzyme A:alpha-glucosaminide N-acetyltransferase. Evidence for an active site histidine residue

The lysosomal membrane enzyme acetyl-CoA:alpha-glucosaminide N-acetyltransferase catalyzes the transfer of the acetyl group from acetyl-CoA to terminal alpha-linked glucosamine residues of heparan sulfate. The reaction appears to be a transmembrane process: the enzyme is acetylated on the outside of the lysosome, and the acetyl group is transferred across the membrane to the inside of the lysosome where it is used to acetylate glucosamine. To determine the reactive site residues involved in the acetylation reaction, lysosomal membranes were treated with various amino acid modification reagents and assayed for enzyme activity. Although four thiol modification reagents were examined, only one, p-chloromercuribenzoate inactivated the N-acetyltransferase. Thiol modification by p-chloromercuribenzoate did not appear to occur at the active site since inactivation was still observed in the presence of the substrate acetyl-CoA. N-Acetyltransferase could be inactivated by N-bromosuccinimide, even after pretreatment with reagents specific for tyrosine and tryptophan, suggesting that the modified residue is a histidine. Diethyl pyrocarbonate, another histidine modification reagent, could also inactivate the enzyme; this inactivation could be reversed by incubation with hydroxylamine. N-Bromosuccinimide and diethyl pyrocarbonate modifications appear to be at the active site of the enzyme since co-incubation with acetyl-CoA protects the N-acetyltransferase from inactivation. This protection is lost if glucosamine is also present. Pre-acetylated lysosomal membranes are also able to provide protection from N-bromosuccinimide inactivation, providing further evidence for a histidine moiety at the active site and for the existence of an acetyl-enzyme intermediate.     

Studies of a reactive histidine of dyphosphopyridine nucleotide-linked isocitrate dehydrogenase from bovine heart.

Diphosphopyridine nucleotide-linked isocitrate dehydrogenase from bovine heart was inactivated at neutral pH by bromoacetate and diethyl pyrocarbonate and by photooxidation in the presence of methylene blue or rose bengal. Inactivation by diethyl pyrocarbonate was reversed by hydroxylamine. Loss of activity by photooxidation at pH 7.07 was accompanied by progressive destruction of histidine with time; loss of 83% of the enzyme activity was accompanied by modification of 1.1 histidyl residues per enzyme subunit. The pH-rate profiles of inactivation by photooxidation and by diethyl pyrocarbonate modification showed an inflection point around pH 6.6, in accord with the pK_a for a histidyl residue of a protein. Partial protection against inactivation by photooxidation or diethyl pyrocarbonate was obtained with substrate (manganous isocitrate or magnesium isocitrate) or ADP; the combination of substrate and ADP was more effective than the components singly. As demonstrated by differential enzyme activity assays between pH 6.4 and pH 7.5 with and without 0.67 mm ADP, modification of the reactive histidyl residue of the enzyme caused a preferential loss of the positive modulation of activity by ADP. The latter was particularly apparent when substrate partially protected the enzyme against inactivation by rose bengal-induced photooxidation.     

Evidence for filaggrin as a component of the cell envelope of the newborn rat.

Diethyl pyrocarbonate inactivated D-xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus and Lactobacillus brevis with second-order rate constants of 422, 417, 99 and 92 M-1.min-1 respectively (at pH 6.0 and 25 degrees C). Activity was completely restored by the addition of neutral hydroxylamine, and total protection was afforded by the substrate analogue xylitol in the presence of either Mg2+ or Mn2+ according to the genus studied. The difference spectra of the modified enzymes revealed an absorption maximum at 237-242 nm, characteristic for N-ethoxycarbonylhistidine. In addition, the spectrum of ethoxycarbonylated D-xylose isomerase from L. xylosus showed absorption minima at both 280 and 230 nm, indicative for modification of tyrosine residues. Nitration with tetranitromethane followed by diethyl pyrocarbonate treatment eliminated the possibility that modification of tyrosine residues was responsible for inactivation, and resulted in modification of one non-essential tyrosine residue and six histidine residues. Inactivation of the other D-xylose isomerases with diethyl pyrocarbonate required the modification of one (L. brevis), two (Streptomyces sp.) and four (S. violaceoruber) histidine residues per monomer. Spectral analysis and maintenance of total enzyme activities further indicated that either xylitol Mg2+ (streptomycetes) or xylitol Mn2+ (lactobacilli) prevented the modification of one crucial histidine residue. The overall results thus provide evidence that a single active-site histidine residue is involved in the catalytic reaction mechanism of D-xylose isomerases.     

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.     

Identification of histidine residues at the active site of Megalobatrachus japonicus alkaline phosphatase by chemical modification

Alkaline phosphatase from Megalobatrachus japonicus was inactivated by diethyl pyrocarbonate (DEP). The inactivation followed pseudo-first-order kinetics with a second-order rate constant of 176 M(-1) x min(-1) at pH 6.2 and 25 degrees C. The loss of enzyme activity was accompanied with an increase in absorbance at 242 nm and the inactivated enzyme was re-activated by hydroxylamine, indicating the modification of histidine residues. This conclusion was also confirmed by the pH profiles of inactivation, which showed the involvement of a residue with pK(a) of 6.6. The presence of glycerol 3-phosphate, AMP and phosphate protected the enzyme against inactivation. The results revealed that the histidine residues modified by DEP were located at the active site. Spectrophotometric quantification of modified residues showed that modification of two histidine residues per active site led to complete inactivation, but kinetic stoichiometry indicated that one molecule of modifier reacted with one active site during inactivation, probably suggesting that two essential histidine residues per active site are necessary for complete activity whereas modification of a single histidine residue per active site is enough to result in inactivation.     

Effects of di-isopropyl phosphorofluoridate on rat liver microsomal and lysosomal beta-glucuronidase

N-Bromosuccinimide completely inactivated the cellulase, and titration experiments showed that oxidation of one tryptophan residue per cellulase molecule coincided with 100% inactivation. CM-cellulose protected the enzyme from inactivation by N-bromosuccinimide. The cellulase was inhibited by active benzyl halides, and reaction with 2-hydroxy-5-nitrobenzyl bromide resulted in the incorporation of 2.3 hydroxy-5-nitrobenzyl groups per enzyme molecule; one tryptophan residue was shown to be essential for activity. Diazocarbonyl compounds in the presence of Cu2+ ions inhibited the enzyme. The pH-dependence of inactivation was consistent with the reaction occurring with a protonated carboxyl group. Carbodi-imide inhibited the cellulase, and kinetic analysis indicated that there was an average of 1 mol of carbodi-imide binding to the cellulase during inactivation. Treatment of the cellulase with diethyl pyrocarbonate resulted in the modification of two out of the four histidine residues present in the cellulase. The modified enzyme retained 40% of its original activity. Inhibition of cellulase activity by the metal ions Ag+ and Hg2+ was ascribed to interaction with tryptophan residues, rather than with thiol groups.     

Identification of active-site residues of sheep liver serine hydroxymethyltransferase.

Chemical modification of amino acid residues with phenylglyoxal, N-ethylmaleimide and diethyl pyrocarbonate indicated that at least one residue each of arginine, cysteine and histidine were essential for the activity of sheep liver serine hydroxymethyltransferase. The second-order rate constants for inactivation were calculated to be 0.016 mM-1 X min-1 for phenylglyoxal, 0.52 mM-1 X min-1 for N-ethylmaleimide and 0.06 mM-1 X min-1 for diethyl pyrocarbonate. Different rates of modification of these residues in the presence and in the absence of substrates and the cofactor pyridoxal 5'-phosphate as well as the spectra of the modified protein suggested that these residues might occur at the active site of the enzyme.     

Comparison of aspartate transcarbamoylases from wheat germ and Escherichia coli: functionally identical histidines in nonhomologous local sequences

Aspartate transcarbamoylase (ATCase) from wheat germ and the catalytic subunit of the enzyme from Escherichia coli are trimers of similar size. The former is a regulatory enzyme in its trimeric state, while the latter is a component of a complex regulatory dodecamer. In a comparison of the two enzymes, reaction with diethyl pyrocarbonate revealed a highly active, essential histidine residue in each case. The two histidines (i.e., one in each enzyme) behaved nearly identically with respect to the following functional properties: kinetics of acylation (ethoxyformylation) and concomitant inactivation; kinetics of deacylation by hydroxylamine and concomitant reactivation; hyperbolic dependence of the apparent first-order rate constant (kapp) on diethyl pyrocarbonate concentration; pH dependence of kapp; failure of active-center ligands to protect the residue against diethyl pyrocarbonate, producing instead near-identical increases in the inactivation rate. These similarities point to an essential, highly conserved histidine in each enzyme, in a functional microenvironment that has changed relatively little since the divergence of plants and bacteria. Ethoxyformylated peptides were isolated from tryptic digests of the two inactivated enzymes. Sequencing of the major labeled peptide in each case showed the wheat and E. coli histidines embedded in nonhomologous primary segments, suggesting that, contrary to expectation, these segments are not part of the conserved microenvironment. In the case of the E. coli enzyme, the essential residue was identified as His-134 in the known sequence, which has a potential catalytic role on crystallographic evidence [Krause, K. L., Volz, K. W., & Lipscomb, W. N. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1643-1647]. A second, much less reactive histidine was identified as His-64.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the presence of a critical histidine residue at the active site in glyceraldehyde-3-phosphate dehydrogenase of Ehrlich ascites carcinoma cells

Ehrlich ascites carcinoma (EAC) cell glyceraldehyde-3-phosphate dehydrogenase (GA3PD) (EC. 1.2.1.12) was completely inactivated by diethyl pyrocarbonate (DEPC), a fairly specific reagent for histidine residues in the pH range of 6.0-7.5. The rate of inactivation was dependent on pH and followed pseudo-first order reaction kinetics. The difference spectrum of the inactivated and native enzymes showed an increase in the absorption maximum at 242 nm, indicating the modification of histidine residues. Statistical analysis of the residual enzyme activity and the extent of modification indicated modification of one essential histidine residue to be responsible for loss of the catalytic activity of EAC cell GA3PD. DEPC inactivation was protected by substrates, D-glyceraldehyde-3-phosphate and NAD, indicating the presence of essential histidine residue at the substrate-binding region of the active site. Double inhibition studies also provide evidence for the presence of histidine residue at the active site.     

Substrate-decreased modification by diethyl pyrocarbonate of two histidines in isocitrate lyase from Escherichia coli

The inactivation of tetrameric 188-kDa isocitrate lyase from Escherichia coli at pH 6.8 (37 degrees C) by diethyl pyrocarbonate, exhibiting saturation kinetics, is accompanied by modification of histidine residues 266 and 306. Substrates isocitrate, glyoxylate, or glyoxylate plus succinate protect the enzyme from inactivation, but succinate alone does not. Removal of the carbethoxy groups from inactivated enzyme by treatment with hydroxylamine restores activity of isocitrate lyase. The present results suggest that the group-specific modifying reagent diethyl pyrocarbonate may be generally useful in determining the position of active site histidine residues in enzymes.     

Evidence for an essential histidine residue in 4S-limonene synthase and other terpene cyclases.

(4S)-Limonene synthase, isolated from glandular trichome secretory cell preparations of Mentha x piperita (peppermint) leaves, catalyzes the metal ion-dependent cyclization of geranyl pyrophosphate, via 3S-linalyl pyrophosphate, to (-)-(4S)-limonene as the principal product. Treatment of this terpene cyclase with the histidine-directed reagent diethyl pyrocarbonate at a concentration of 0.25 mM resulted in 50% loss of enzyme activity, and this activity could be completely restored by treatment of the preparation with 5 mM hydroxylamine. Inhibition with diethyl pyrocarbonate was distinguished from inhibition with thiol-directed reagents by protection studies with histidine and cysteine carried out at varying pH. Inactivation of the cyclase by dye-sensitized photooxidation in the presence of rose bengal gave further indication of the presence of a readily modified histidine residue. Protection of the enzyme against inhibition with diethyl pyrocarbonate was afforded by the substrate geranyl pyrophosphate in the presence of Mn2+, and by the sulfonium ion analog of the linalyl carbocation intermediate of the reaction in the presence of inorganic pyrophosphate plus Mn2+, suggesting that an essential histidine residue is located at or near the active site. Similar studies on the inhibition of other monoterpene and sesquiterpene cyclases with diethyl pyrocarbonate suggest that a histidine residue (or residues) may play an important role in catalysis by this class of enzymes.     

Chemical and kinetic evidence for an essential histidine in the phosphotriesterase from Pseudomonas diminuta

The pH rate profile for the hydrolysis of diethyl-p-nitrophenyl phosphate catalyzed by the phosphotriesterase from Pseudomonas diminuta shows a requirement for the deprotonation of an ionizable group for full catalytic activity. This functional group has an apparent pKa of 6.1 +/- 0.1 at 25 degrees C, delta Hion of 7.9 kcal/mol, and delta Sion of -1.4 cal/K.mol. The enzyme is not inactivated in the presence of the chemical modification reagents dithiobis-(2-nitrobenzoate), methyl methane thiosulfonate, carbodiimide, pyridoxal, butanedione, or iodoacetic acid and thus cysteine, asparate, glutamate, lysine, and arginine do not appear to be critical for catalytic activity. However, the phosphotriesterase is inactivated completely with methylene blue, Rose Bengal, or diethyl pyrocarbonate. The enzyme is not inactivated by diethyl pyrocarbonate in the presence of bound substrate analogs, and inactivation with diethyl pyrocarbonate is reversible upon addition of neutralized hydroxylamine. The modification of a single histidine residue by diethyl pyrocarbonate, as shown by spectrophotometric analysis, is responsible for the loss of catalytic activity. The pKinact for diethyl pyrocarbonate modification is 6.1 +/- 0.1 at 25 degrees C. These results have been interpreted to suggest that a histidine residue at the active site of phosphotriesterase is facilitating the reaction by general base catalysis.     

Identification of two essential histidine residues of ribonuclease T2 from Aspergillus oryzae

Ribonuclease (RNase) T2 from Aspergillus oryzae was modified by diethyl pyrocarbonate and iodoacetic acid. RNase T2 was rapidly inactivated by diethyl pyrocarbonate above pH 6.0 and by incorporation of a carboxymethyl group. No inactivation occurred in the presence of 3'AMP. 1H-NMR titration and photo-chemically induced dynamic nuclear polarization experiments demonstrated that two histidine residues were involved in the active site of RNase T2. Furthermore, analysis of inactive carboxymethylated RNase T2 showed that both His53 and His115 were partially modified to yield a total of one mole of N tau-carboxymethylhistidine/mole enzyme. The results indicate that the two histidine residues in the active site of RNase T2 are essential for catalysis and that modification of either His53 or His115 inactivates the enzyme.     

Aspartokinase-homoserine dehydrogenase I from Escherichia coli: pH and chemical modification studies of the kinase activity

The pH variation of the kinetic parameters was examined for the kinase activity of the bifunctional enzyme aspartokinase--homoserine dehydrogenase I isolated from Escherichia coli. The V/K profile for L-aspartic acid indicates the loss of activity upon protonation of a cationic acid type group with a pK value near neutrality. Incubation of the enzyme with diethyl pyrocarbonate at pH 6.0 results in a loss of enzymic activity. The reversal of this reaction by neutral hydroxylamine, the appearance of a peak at 242 nm for the inactivated enzyme, and the observation of a pK value of 7.0 obtained from variation of the inactivation rate with pH all suggest that enzyme inactivation occurs by modification of histidine residues. The substrate L-aspartic acid protects one residue against inactivation, which implies that this histidine may participate in substrate binding or catalysis. Activity loss was also observed at high pH due to the ionization of a neutral acid group with a pK value of 9.8. The reactions of AK-HSD I with N-acetylimidazole and tetranitromethane have been investigated to obtain information about the functional role of tyrosyl residues in the enzyme. The acylation of tyrosines leads to inactivation of the enzyme, which can then be fully reversed by treatment with hydroxylamine. Incubation of the enzyme with tetranitromethane at pH 9.5 also leads to rapid inactivation, and the substrates of the kinase reaction provide substantial protection against inactivation. However, three tyrosines are protected by substrates, implying a structural role for these amino acids.     

A catalytic role for histidine 237 in rat mammary gland thioesterase II

The involvement of a histidyl residue in the catalytic mechanism of thioesterase II, a serine active-site enzyme that catalyzes the chain terminating reaction in de novo fatty acid synthesis, has been inferred from studies with the inhibitor diethyl pyrocarbonate. Its likely location has been predicted by identification of conserved residues in related thioesterases and ultimately confirmed by site-directed mutagenesis. Diethyl pyrocarbonate inactivated the enzyme with a second-order rate constant of 49 M-1 s-1 at pH 6, 10 degrees C. Data analysis indicated that although several residues reacted with the reagent, modification of a single residue was responsible for the inactivation. Removal of a single ethoxycarbonyl moiety by treatment with neutral hydroxylamine completely restored enzyme activity. Prior ethoxycarbonylation of the histidyl residue blocked the ability of the active-site serine to react with phenylmethanesulfonyl fluoride. Comparison of the amino acid sequences of five structurally related proteins indicated that only 1 histidine has been completely conserved. Replacement of this residue in rat thioesterase II (His-237) with arginine and leucine by mutagenesis reduced the catalytic activity by 2-3 orders of magnitude. The activity of the mutant thioesterases, unlike that of the wild-type enzyme, was relatively insensitive to inhibition by diethyl pyrocarbonate and phenylmethylsulfonyl fluoride. These studies provide strong evidence that His-237 is involved directly in catalysis and suggest that its role is to increase the nucleophilic character of the active-site Ser-101 by acting as a proton acceptor thus facilitating acylation of the seryl residue. The mechanism appears to share certain common features with the charge-relay system characteristic of other esterases.     

Identification of the essential histidine residue at the active site of Escherichia coli dehydroquinase.

The shikimate pathway enzyme 3-dehydroquinase is very susceptible to inactivation by the group-specific reagent diethyl pyrocarbonate (DEP). Inactivation follows pseudo first-order kinetics and exhibits a second-order rate constant of 148.5 M-1 min-1. An equilibrium mixture of substrate and product substantially protects against inactivation by DEP, suggesting that residues within the active site are being modified. Complete inactivation of the enzyme correlates with the modification of 6 histidine residues/subunit as determined by difference spectroscopy at 240 nm. Enzymic activity can be restored by hydroxylamine treatment, which is also consistent with the modification occurring at histidine residues. Using the kinetic method of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1558), it was shown that modification of a single histidine residue leads to inactivation. Ligand protection experiments also indicated that 1 histidine residue was protected from DEP modification. pH studies show that the pKa for this inactivation is 6.18, which is identical to the single pKa determined from the pH/log Vmax profile for the enzyme. A single active site peptide was identified by differential peptide mapping in the presence and absence of ligand. This peptide was found to comprise residues 141-158; of the 2 histidines in this peptide (His-143 and His-146), only one, His-143, is conserved among all type I dehydroquinases. We propose that His-143 is the active site histidine responsible for DEP-mediated inactivation of dehydroquinase and is a good candidate for the general base that has been postulated to participate in the mechanism of this enzyme.     

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.