Vitamin K2 (menaquinone) biosynthesis in Escherichia coli: evidence for the presence of an essential histidine residue in o-succinylbenzoyl coenzyme A synthetase.

o-Succinylbenzoyl coenzyme A (OSB-CoA) synthetase, when treated with diethylpyrocarbonate (DEP), showed a time-dependent loss of enzyme activity. The inactivation follows pseudo-first-order kinetics with a second-order rate constant of 9.2 x 10(-4) +/- 1.4 x 10(-4) microM(-1) min(-1). The difference spectrum of the modified enzyme versus the native enzyme showed an increase in A242 that is characteristic of N-carbethoxyhistidine and was reversed by treatment with hydroxylamine. Inactivation due to nonspecific secondary structural changes in the protein and modification of tyrosine, lysine, or cysteine residues was ruled out. Kinetics of enzyme inactivation and the stoichiometry of histidine modification indicate that of the eight histidine residues modified per subunit of the enzyme, a single residue is responsible for the enzyme activity. A plot of the log reciprocal of the half-time of inactivation against the log DEP concentration further suggests that one histidine residue is involved in the catalysis. Further, the enzyme was partially protected from inactivation by either o-succinylbenzoic acid (OSB), ATP, or ATP plus Mg2+ while inactivation was completely prevented by the presence of the combination of OSB, ATP, and Mg2+. Thus, it appears that a histidine residue located at or near the active site of the enzyme is essential for activity. When His341 present in the previously identified ATP binding motif was mutated to Ala, the enzyme lost 65% of its activity and the Km for ATP increased 5.4-fold. Thus, His341 of OSB-CoA synthetase plays an important role in catalysis since it is probably involved in the binding of ATP to 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.     

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.     

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.     

Evidence for an essential histidine residue in the Neurospora crassa plasma membrane H+-ATPase

The Neurospora crassa plasma membrane H+-ATPase is rapidly inactivated in the presence of diethyl pyrocarbonate (DEP). The reaction is pseudo-first-order showing time- and concentration-dependent inactivation with a second-order rate constant of 385-420 M-1.min-1 at pH 6.9 and 25 degrees C. The difference spectrum of the native and modified enzyme has a maximum near 240 nm, characteristic of N-carbethoxyhistidine. No change in the absorbance of the inhibited ATPase at 278 nm or in the number of modifiable sulfhydryl groups is observed, indicating that the inhibition is not due to tyrosine or cysteine modification, and the inhibition is irreversible, ruling out serine residues. Furthermore, pretreatment of the ATPase with pyridoxal phosphate/NaBH4 under the conditions of the DEP treatment does not inhibit the ATPase and does not alter the DEP inhibition kinetics, indicating that the inactivation by DEP is not due to amino group modification. The pH dependence of the inactivation reaction indicates that the essential residue has a pKa near 7.5, and the activity lost as a result of H+-ATPase modification by DEP is partially recovered after hydroxylamine treatment at 4 degrees C. Taken together, these results strongly indicate that the inactivation of the H+-ATPase by DEP involves histidine modification. Analyses of the inhibition kinetics and the stoichiometry of modification indicate that among eight histidines modified per enzyme molecule, only one is essential for H+-ATPase activity. Finally, ADP protects against inactivation by DEP, indicating that the essential residue modified may be located at or near the nucleotide binding site.     

Evidence for essential histidine and cysteine residues in calcium/calmodulin-sensitive cyclic nucleotide phosphodiesterase.

Ca/calmodulin-sensitive cyclic nucleotide phosphodiesterase (CaM-PDE) is an important enzyme regulating cGMP levels and relaxation of vascular smooth muscle. This modification study was conducted mostly with bovine brain CaM-PDE to identify essential functional groups involved in catalysis. The effect of pH on Vmax/Km indicates two essential residues with pKa values of 6.4 and 8.2. Diethyl pyrocarbonate (DEP), a histidine-modifying agent, inhibits CaM-PDE with a second-order rate constant of 130 M-1 min-1 at pH 7.0 and 30 degrees C. Activity is restored by NH2OH. The pH dependence of inactivation reveals that the essential residue modified by DEP has an apparent pKa of 6.5. The difference spectrum of the intact and DEP-treated enzyme shows a maximum between 230 and 240 nm, suggesting formation of carbethoxy derivatives of histidine. The enzyme is also inactivated by N-ethylmaleimide (NEM) and 5,5'-dithiobis-(2-nitrobenzoic acid), both sulfhydryl-modifying agents, with the latter effect reversed by dithiothreitol, which suggests inactivation resulting from modification of cysteine residue(s). Partial inactivation of the enzyme by DEP or NEM results in an apparent decrease in the Vmax without a change in the Km or the extent of CaM stimulation. The rate of inactivation by DEP is greater in the presence than in the absence of Ca/CaM. A substrate analogue, Br-cGMP, and the competitive inhibitor 3-isobutyl-1-methylxanthine partially protect the enzyme against inactivation by DEP or NEM, suggesting that the modification of histidine and cysteine residues occurs at or near the active site. DEP also inactivated porcine brain CaM-PDE.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Identification of essential histidines in cyclodextrin glycosyltransferase isoform 1 from Paenibacillus sp. A11

The isoform 1 of cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) from Paenibacillus sp. A11 was purified by a preparative gel electrophoresis. The importance of histidine, tryptophan, tyrosine, and carboxylic amino acids for isoform 1 activity is suggested by the modification of the isoform 1 with various group-specific reagents. Activity loss, when incubated with diethylpyrocarbonate (DEP), a histidine modifying reagent, could be protected by adding 25 mM methyl-beta-cyclodextrin substrate prior to the modification. Inactivation kinetics of isoform 1 with DEP resulted in second-order rate constants (k(inactivation)) of 29.5 M(-1)s(-1). The specificity of the DEP-modified reaction for the histidine residue was shown by the correlation between the loss of isoform activity and the increase in the absorbance at 246 nm of N-carbethoxyhistidine. The number of histidines that were modified by DEP in the absence and presence of a protective substrate was estimated from the increase in the absorbance using a specific extinction coefficient of N-carbethoxyhistidine of 3,200 M(-1)cm(-1). It was discovered that methyl-beta-CD protected per mole of isoform 1, two histidine residues from the modification by DEP. To localize essential histidines, the native, the DEP-modified, and the protected forms of isoform 1 were digested by trypsin. The resulting peptides were separated by HPLC. The peptides of interest were those with R(t) 11.34 and 40.93 min. The molecular masses of the two peptides were 5,732 and 2,540 daltons, respectively. When the data from the peptide analysis were checked with the sequence of CGTase, then His-140 and His-327 were identified as essential histidines in the active site of isoform 1.     

Essential histidine residues in dextransucrase: chemical modification by diethyl pyrocarbonate and dye photo-oxidation

Treatment of Leuconostoc mesenteroides B-512F dextransucrase with diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees or photo-oxidation in the presence of Rose Bengal or Methylene Blue at pH 6.0 and 25 degrees, caused a rapid decrease of enzyme activity. Both types of inactivation followed pseudo-first-order kinetics. Enzyme partially inactivated by DEP could be completely reactivated by treatment with 100 mM hydroxylamine at pH 7 and 4 degrees. The presence of dextran partially protected the enzyme from inactivation. At pH 7 or below, DEP is relatively specific for the modification of histidine. DEP-modified enzyme showed an increased absorbance at 240 nm, indicating the presence of (ethoxyformyl)ated histidine residues. DEP modification of the sulfhydryl group of cysteine and of the phenolic group of tyrosine was ruled out by showing that native and DEP-modified enzyme had the same number of sulfhydryl and phenolic groups. DEP modification of the epsilon-amino group of lysine was ruled out by reaction at pH 6 and reactivation with hydroxylamine, which has no effect on DEP-modified epsilon-amino groups. The photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm, also indicating that histidine had been oxidized, and no decrease in the absorbance at 280 nm, indicating that tyrosine and tryptophan were not oxidized. A statistical, kinetic analysis of the data on inactivation by DEP showed that two histidine residues are essential for the enzyme activity. Previously, it was proposed that two nucleophiles at the active site attack bound sucrose, to give two covalent D-glucosyl-enzyme intermediates. We now propose that in addition, two imidazolium groups of histidine at the active site donate protons to the leaving, D-fructosyl moieties. The resulting imidazole groups then facilitate the formation of the alpha-(1----6)-glycosidic linkage by abstracting protons from the C-6-OH groups, and become reprotonated for the next series of reactions.     

Kinetics of inactivation of phytase (phy A) during modification of histidine residue by IAA and DEP

Chemical probing of histidine residues using specific modifiers, iodoacetic acid (IAA) and diethylpyrocarbonate (DEP) resulted in the inactivation of phytase (phy A). The kinetic theory of the substrate reaction during the modification of enzyme activity was applied to a study of the kinetics of the course of inactivation of phytase by IAA and DEP. The results suggested that histidine residues are involved in the active site of the enzyme. They also indicated that inactivation of the enzyme by IAA was via a complexing type inhibition, while the inhibition by DEP reaction involved a conformational change step before inactivation. The dissociation constant of the enzyme-inhibitor complex of IAA, the constant of the conformational change of DEP and the microscopic rate constants of two inhibitors were obtained.     

Structural role of histidine residues in NAD(P)-glutamate dehydrogenase from the bovine liver

Photooxidation of bovine liver glutamate dehydrogenase (GDH, EC 1.4.1.3) in the presence of methylene blue at a low light intensity occurs in two stages. At the first stage, the duration of which depends on temperature and dye concentration, a slight activation is observed simultaneously with the oxidation of two histidine residues. At the second stage, the inactivation is concomitant with the oxidation of three histidine and one tryptophan residues. The inactivation is a first order reaction (k = 3,22 X 10(-2) min-1) and is correlated with changes in the circular dichroism spectra. These data testify to the structural role of histidine residues in the GDH molecule. The kinetic behaviour of GDH during its modification with diethylpyrocarbonate (DEP) depends on pH and the reagent concentration. Four histidine residues undergo carbethoxylation at pH 6.0 and 7.5, but the modification rate is much higher at pH 7.5. At low DEP concentrations, a remarkable activation is observed with a simultaneous modification of one histidine residue, which is independent of pH. At high DEP concentrations, a rapid inactivation takes place at pH 7.5. Treatment of the carbethoxylated inactive enzyme with hydroxylamine results in the deacylation of histidine residues without any noticeable reactivation. The data on the combined effect of DEP and pyridoxal-5'-phosphate suggest that GDH inactivation by DEP at pH 7.5 is a result of modification of an essential epsilon-NH2 group of lysine-126.     

Modification of pig liver dimeric dihydrodiol dehydrogenase with diethylpyrocarbonate and by rose bengal-sensitized photooxidation: evidence for an active-site histidine residue.

Dihydrodiol dehydrogenase from pig liver was inactivated by diethylpyrocarbonate (DEP) and by rose bengal-sensitized photooxidation. The DEP inactivation was reversed by hydroxylamine and the absorption spectrum of the inactivated enzyme indicated that both histidine and tyrosine residues were carbethoxylated. The rates of inactivation by DEP and by photooxidation were dependent on pH, showing the involvement of a group with a pKa of 6.4. The kinetics of inactivation and spectrophotometric quantification of the modified residues suggested that complete inactivation was caused by modification of one histidine residue per active site. The inactivation by the two modifications was partially prevented by either NADP(H) or the combination of NADP+ and substrate, and completely prevented in the presence of both NADP+ and a competitive inhibitor which binds to the enzyme-NADP+ binary complex. The DEP-modified enzyme caused the same blue shift and enhancement of NADPH fluorescence as did the native enzyme, suggesting that the modified histidine is not in the coenzyme-binding site of the enzyme. The results suggest the presence of essential histidine residues in the catalytic region of the active site of pig liver dihydrodiol dehydrogenase.     

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.     

Studies of histidine residues in soybean (Glycine max) urease

Soybean urease has been investigated extensively to reveal the presence of histidine residue (s) in the active site and their potential role in the catalysis. The spectrophotometric studies using diethylpyrocarbonate (DEP) showed the modification of 11.76 ± 0.1 histidine residues per mole of native urease. Therefore, the results are indicative of the presence of twelve histidine residues per urease molecule. It is presumed that the soybean urease, being a hexameric protein possess two histidine residues per subunit. Correlation plot showed that the complete inactivation of soybean urease corresponds to the modification of 1.97 histidine residues per subunit. Further, double logarithmic plot of kapp versus DEP concentration has resulted in a linear correlation and thereby demonstrating that only one of the two histidine residues per subunit is catalytically essential. Significant protection has been observed against inactivation when urea or acetohydroxamate (AHA) is incubated with DEP treated urease. The studies have demonstrated the presence of one histidine residue at the active site of soybean urease and its significance in catalysis.     

Studies on the active site of rat glutathione S-transferase isoenzyme 4-4. Chemical modification by tetrachloro-1,4-benzoquinone and its glutathione conjugate

The active site of glutathione S-transferase isoenzyme 4-4, purified from rat liver, was studied by chemical modification. Tetrachloro-1,4-benzoquinone, a compound previously shown to inactivate glutathione S-transferases very efficiently by covalent binding in or close to the active site, completely prevented the alkylation of the enzyme by iodoacetamide, indicating that the reaction had taken place with cysteine residues. Both from radioactive labeling and spectral quantification experiments, evidence was obtained for the covalent binding of three benzoquinone molecules per subunit, i.e. equivalent to the number of cysteine residues present. This threefold binding was achieved with a fourfold molar excess of the benzoquinone, illustrating the high reactivity of this compound. Comparison of the number of amino acid residues modified by tetrachloro-1,4-benzoquinone with the decrease of catalytic activity revealed an almost complete inhibition after modification of one cysteine residue. Chemical modification studies with diethylpyrocarbonate indicated that all four histidine residues of the subunit are ethoxyformylated in an at least partially sequential manner. Modification of the second histidine residue resulted in complete loss of catalytic activity. Preincubation of the transferase with the glutathione conjugate of tetrachloro-1,4-benzoquinone resulted in 78% protection against this modification. However, glutathione itself hardly protected against the reaction with diethylpyrocarbonate. The intrinsic fluorescence properties of the enzyme were affected by covalent binding of tetrachloro-1,4-benzoquinone. The concentration dependency of the fluorescence quenching is strongly correlated with the inactivation of the enzyme, indicating that covalent binding of the benzoquinone occurs in the vicinity of at least one tryptophan residue. Finally, the binding of bilirubin, as measured by means of circular dichroism, was inhibited by preincubation of the enzyme with tetrachloro-1,4-benzoquinone in a manner which strongly correlated with the loss of enzymatic activity, the protection against inactivation by diethylpyrocarbonate, and the fluorescence quenching. All processes showed a 70-80% decrease after incubation of the enzyme with an equimolar amount of the benzoquinone. Thus, evidence is presented for the presence of a cysteine, a histidine and a tryptophan residue in, or in the vicinity of, the active site of the glutathione S-transferase 4 subunit.     

Chemical modification of dopamine beta-hydroxylase

Dopamine beta-hydroxylase (3,4- dihydroxyphenylethylamine ,ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) is the terminal enzyme in the biosynthetic pathway of norepinephrine. Chemical modification studies of this enzyme were executed to investigate contributions of specific amino-acid side-chains to catalytic activity. Sulfhydryl reagents were precluded, since no free cysteine residue was detected upon titration of the denatured or native protein with 2-chloromercuri-4-nitrophenol. Incubation of enzyme with diazonium tetrazole caused inactivation of the protein coupled with extensive reaction of lysine and tyrosine residues. Reaction with iodoacetamide resulted in complete loss of enzymatic activity with reaction of approximately three histidine residues; methionine reaction was also observed. Modification of the enzyme using diethylpyrocarbonate resulted in complete inactivation of the enzyme, and analysis of the reacted protein indicated a loss of approx. 1.7 histidine residues per protein monomer with no tyrosine or lysine modification observed. The correlation of activity loss with histidine modification supports the view that this residue participates in the catalytic function of dopamine beta-hydroxylase.     

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.     

Isolation and characterisation of the major outer membrane protein of Erwinia carotovora

Treatment of the tonoplast H(+)-ATPase from mung bean seedlings (Vigna radiata L.) with histidine-specific modifier, diethyl pyrocarbonate (DEP), caused a marked loss of the ATP hydrolysis activity and the proton translocation in a concentration-dependent manner. The reaction order of inhibition was calculated to be 0.98, suggesting that at least one histidine residue of vacuolar H(+)-ATPase was modified by DEP. The absorbance of the vacuolar H(+)-ATPase at 240 nm was progressively increased after incubation with DEP, suggesting that N-carbethoxyhistidine had been formed. Hydroxylamine, which could break N-carbethoxyhistidine, reversed the absorbance change and partially restored the enzymic activity. The pK(a) of modified residues of vacuolar H(+)-ATPase was kinetically determined to be 6.73, a value close to that of histidine. Thus, it is assuredly concluded that histidine residues of the vacuolar H(+)-ATPase were modified by DEP. Kinetic analysis showed that V(max) but not K(m) of vacuolar H(+)-ATPase was decreased by DEP. This result is interpreted as that the residual activity after DEP inhibition was primarily due to the unmodified enzyme molecules. Moreover, simultaneous presence of DEP and DCCD (N,N'-dicyclohexyl-carbodiimide), an inhibitor modified at proteolipid subunit of vacuolar H(+)-ATPase, did not induce synergistic inhibition, indicating their independent effects. The stoichiometry studies further demonstrate that only one out of four histidine residues modified was involved in the inhibition of vacuolar H(+)-ATPase by DEP. Mg(2+)-ATP, the physiological substrate of vacuolar H(+)-ATPase, but not its analogs, exerted preferentially partial protection against DEP, indicating that the histidine residue involved in the inhibition of enzymatic activity may locate at/or near the active site and directly participate in the binding of the substrate.     

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.     

Inactivation of Acinetobacter calcoaceticus acetate kinase by diethylpyrocarbonate

Acetate kinase purified from Acinetobacter calcoaceticus was inhibited by diethylpyrocarbonate with a second-order rate constant of 620 M-1.min-1 at pH 7.4 at 30 degrees C and showed a concomitant increase in absorbance at 240 nm due to the formation of N-carbethoxyhistidyl derivative. Activity could be restored by hydroxylamine and the pH curve of inactivation indicates the involvement of a residue with a pKa of 6.64. Complete inactivation of acetate kinase required the modification of seven residues per molecule of enzyme. Statistical analysis showed that among the seven modifiable residues, only one is essential for activity. 5,5'-dithiobis(2-nitrobenzoic acid), p-chloromercuryphenylsulfonate, N-ethylmaleimide and phenylglyoxal did not affect the enzyme activity. These results suggest that the inactivation is due to the modification of one histidine residue. The substrates, acetate and ATP, protected the enzyme against inactivation, indicating that the modified histidine residue is located at or near the active site.