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.     

Inactivation of Saccharomyces cerevisiae glucose-6-phosphate dehydrogenase by diethylpyrocarbonate

Glucose-6-phosphate dehydrogenase purified from Saccharomyces cerevisiae is rapidly inactivated by diethylpyrocarbonate at pH 6.8 and 30 degrees C with a concomitant increase in absorbance at 242 nm. The second-order rate constant for inactivation was calculated to be 487.8 M-1 min-1. The pH dependence of inactivation suggests the involvement of an amino acid residue having a pKa of 6.77. These results indicate that the inactivation is due to the modification of a histidine residue(s). In the presence of substrate, glucose-6-phosphate or NADP+, the rate of inactivation is decreased, indicating that the essential histidine residue(s) is located at the active site, possibly at the region of overlap of substrates at the binding site.     

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.     

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.     

Histidine 268 in 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase plays the same role as histidine 202 in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase

The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase (Phe) is inactivated by diethyl pyrocarbonate (DEPC). The inactivation is first order with respect to enzyme and DEPC concentrations with a pseudo-second order rate constant of inactivation by DEPC of 4.9 +/- 0.8 m(-1) s(-1) at pH 6.8 and 4 degrees C. The dependence of inactivation on pH and the spectral features of enzyme modified at specific pH values imply that both histidine and cysteine residues are modified, which is confirmed by site-directed mutagenesis. Analysis of the chemical modification data indicates that one histidine is essential for activity. DAH 7-P synthase (Phe) is protected against DEPC inactivation by phosphoenolpyruvate, whereas d-erythrose 4-phosphate offers only minimal protection. The conserved residues H-172, H-207, H-268, and H-304 were individually mutated to glycine. The H304G and H207G mutants retain some level of activity, whereas the H268G and H172G mutants are virtually inactive. A comparison of the circular dichroism spectra of wild-type enzyme and the various mutants demonstrates that H-172 may play a structural role. Comparison of the UV spectra of the H268G and wild-type enzymes saturated with Cu(2+) indicates that the metal-binding site of the H268G mutant resembles that of the wild-type enzyme. The residue H-268 may play a catalytic role based on the site-directed mutagenesis and spectroscopic studies. Cysteine 61 appears to influence the pK(a) of H-268 in the wild-type enzyme. The pK(a) of H-268 increases from 6.0 to 7.0 following mutation of C-61 to glycine.     

Essential histidine residue in 3-ketosteroid-delta 1-dehydrogenase.

The variation with pH of kinetic parameters was examined for 3-ketosteroid-delta 1-dehydrogenase from Nocardia corallina. The Vmax/Km profile for 4-androstenedione indicates that activity is lost upon protonation of a cationic acid-type group with a pK value of 7.7. The enzyme was inactivated by diethylpyrocarbonate at pH 7.4 and the inactivation was substantially prevented by androstadienedione. Analyses of reactivation with neutral hydroxylamine, pH variation, and spectral changes of the inactivated enzyme revealed that the inactivation arises from modification of a histidine residue. Studies with [14C]diethylpyrocarbonate provided support for the idea that the 1-2 essential histidine residues are essential for the catalytic activity of the enzyme. Dye-sensitized photooxidation led to 50% inactivation of the enzyme with the decomposition of two histidine residues. This inactivation was also prevented by androstadienedione. Dancyl chloride caused a loss of the enzyme activity. Modifiers of glutamic acid, aspartic acid, cysteine, and lysine did not affect the enzyme activity. Butanedione and phenylglyoxal in the presence of borate rapidly inactivated the enzyme, indicating that arginine residues also have a crucial function in the active site. The data described support the previously proposed mechanism of beta-oxidation of 3-ketosteroid.     

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.     

Original Research Report: Structure-Function Analysis of the ArsA ATPase: Contribution of Histidine Residues

The ArsA ATPase is the catalytic subunit of the ArsAB oxyanion pump in Escherichia coli that is responsible for extruding arsenite or antimonite from inside the cell, thereby conferring resistance. Either antimonite or arsenite stimulates ArsA ATPase activity. In this study, the role of histidine residues in ArsA activity was investigated. Treatment of ArsA with diethyl pyrocarbonate (DEPC) resulted in complete loss of catalytic activity. The inactivation could be reversed upon subsequent incubation with hydroxylamine, suggesting specific modification of histidine residues. ATP and oxyanions afforded significant protection against DEPC inactivation, indicating that the histidines are located at the active site. ArsA has 13 histidine residues located at position 138, 148, 219, 327, 359, 368, 388, 397, 453, 465, 477, 520, and 558. Each histidine was individually altered to alanine by site-directed mutagenesis. Cells expressing the altered ArsA proteins were resistant to both arsenite and antimonite. The results indicate that no single histidine residue plays a direct role in catalysis, and the inhibition by DEPC may be caused by steric hindrance from the carbethoxy group.     

Evidence for an essential histidine residue in the ascorbate-binding site of cytochrome b561

Cytochrome b(561) mediates equilibration of the ascorbate/semidehydroascorbate redox couple across the membranes of secretory vesicles. The cytochrome is reduced by ascorbic acid and oxidized by semidehydroascorbate on either side of the membrane. Treatment with diethyl pyrocarbonate (DEPC) inhibits reduction of the cytochrome by ascorbate, but this activity can be restored by subsequent treatment with hydroxylamine, suggesting the involvement of an essential histidine residue. Moreover, DEPC inactivates cytochrome b(561) more rapidly at alkaline pH, consistent with modification of a histidine residue. DEPC does not affect the absorption spectrum of cytochrome b(561) nor does it change the midpoint reduction potential, confirming that histidine modification does not affect the heme. Ascorbate protects the cytochrome from inactivation by DEPC, indicating that the essential histidine is in the ascorbate-binding site. Further evidence for this is that DEPC treatment inhibits oxidation of the cytochrome by semidehydroascorbate but not by ferricyanide. This supports a reaction mechanism in which ascorbate loses a hydrogen atom by donating a proton to histidine and transferring an electron to the heme.     

Reaction of Ascaris suum phosphofructokinase with diethylpyrocarbonate. Inactivation and desensitization to allosteric modulation

Reaction of the phosphofructokinase from Ascaris suum with the reagent, diethylpyrocarbonate (DEPC), results in the loss of enzymatic activity. Treatment of the inactivated enzyme with hydroxylamine brings about the recovery of almost 80% of the original activity suggesting that the modified residues are histidines. Further evidence for the modification of histidines is that concomitant with the loss of activity, there is a change in A242 nm that corresponds to the derivatization of 5-6 histidines per subunit. There is no change in A278 nm during the derivatization process, thereby ruling out the modification of tyrosines by DEPC. Analyses of the first order inactivation rate constant for DEPC derivatization at different pH values resulted in the determination of a pKa of 6.4 +/- 0.1 for the group on the enzyme that reacts with DEPC. Derivatization of the enzyme with DEPC in the presence of fructose 6-phosphate (Fru-6-P) protected the enzyme against inactivation by 80%. ATP or MgATP gave no protection against DEPC inactivation. When the Fru-6-P-protected enzyme was further reacted with DEPC in the absence of Fru-6-P, a total of 2 histidines were modified per subunit, and the derivatization of one of these could be correlated with activity loss. When the phosphofructokinase that had been derivatized by DEPC in the presence of Fru-6-P was assayed, it was found that it no longer exhibited allosteric properties and appeared to be desensitized to ATP inhibition. This loss of ATP inhibition could be correlated with the modification of 2 histidines per subunit by DEPC. The first order rate constant for desensitization was determined at different pH values and a pKa value of 7.0 +/- 0.2 was obtained for the group(s) responsible for the desensitization. Regulatory studies with the desensitized enzyme revealed that the enzyme was not stimulated by AMP, NH4+, K+, phosphate, sulfate, or hexose bisphosphates. It is concluded that histidine may be involved both in the active site and the ATP inhibitory site of the ascarid phosphofructokinase.     

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.     

Photooxidation and carbethoxylation of a minor ribonuclease from Aspergillus saitoi.

In order to investigate the nature of amino acid residues involved in the active in the active site of a ribonuclease from Aspergillus saitoi, the pH dependence of the rates of inactivation of RNase Ms by photooxidation and modification with diethylpyrocarbonate were studied. (1) RNase Ms was inactivated by illumination in the presence of methylene blue at various pH's. The pH dependence of the rate of photooxidative inactivation of RNase Ms indicated that at least one functional group having pKa 7.2 was involved in the active site. (2) Amino acid analyses of photooxidized RNase Ms at various stages of photooxidative inactivation at pH's 4.0 and 6.0 indicated that one histidine residue was related to the activity of RNase Ms, but that no tryptophan residue was involved in the active site. (3) 2',(3')-AMP prevented the photooxidative inactivation of RNase Ms. The results also indicated the presence of a histidine residue in the active site. (4) Modification of RNase Ms with diethylpyrocarbonate was studied at various pH's. The results indicated that a functional group having pKa 7.1 was involved in the active site of RNase Ms.     

The effect of histidine modification on the activity of myo-inositol monophosphatase from bovine brain.

The pH dependence of myo-inositol monophosphatase may indicate a role for histidine residues in the catalytic mechanism (Ganzhorn, A. J., and Chanal, M.-C. (1990) Biochemistry 29, 6065-6071). This possibility was investigated by chemical modification. At pH 6.0 and 25 degrees C, the enzyme was inactivated by diethylpyrocarbonate in a pseudo-first order reaction with a bimolecular rate constant of 0.37 M-1 s-1. Two histidines were modified rapidly with no effect on enzyme activity, while 3 residues were modified at a slower rate corresponding to the rate of inactivation. No noticeable changes in the secondary structure of the enzyme were observed by comparison of circular dichroic spectra before and after modification. Treatment of myo-inositol monophosphatase with diethylpyrocarbonate in the presence of inositol 1-phosphate, Mg2+, and Li+ protected 2 residues from modification and decreased the inactivation rate by about 5-fold. Spectrophotometric analysis, the restoration of enzyme activity by hydroxylamine, and the lack of any inhibitory effect with alkylating agents suggest that inactivation is due solely to modification of histidine. We conclude that a histidine residue is essential for activity and may act as a base catalyst during hydrolysis of the substrate.     

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.     

米曲霉GX0011β-果糖基转移酶的化学修饰及活性中心研究

用化学修饰法及其修饰动力学对米曲霉GX0011β-果糖基转移酶的活性中心结构进行了研究。结果表明：NBS、PMSF、EDC能显著抑制酶的活性,底物对这些抑制有明显的保护作用,且残留酶活与修饰剂的浓度相关,抑制均符合拟一级动力学规律,进一步动力学分析,初步认定该酶活性中心包括至少一个丝氨酸（或苏氨酸）、一个色氨酸和一个天冬氨酸(或谷氨酸)残基。pCMB、TNBS能显著抑制酶的活性,但底物对抑制无明显保护作用,推断半胱氨酸和赖氨酸残基可能与维系酶活性中心构象有关,但不是酶活性中心基团。DEPC、AA和NAI对酶的活性抑制作用不明显,排除了组氨酸、精氨酸和酪氨酸残基是该酶活性中心必需基团的可能。     

Chemical and kinetic evidence for an essential histidine in horseradish peroxidase for iodide oxidation.

Horseradish peroxidase (HRP), when incubated with diethylpyrocarbonate (DEPC), shows a time-dependent loss of iodide oxidation activity. The inactivation follows pseudo-first order kinetics with a second order rate constant of 0.43 min-1 M-1 at 30 degrees C and is reversed by neutralized hydroxylamine. The difference absorption spectrum of the modified versus native enzyme shows a peak at 244 nm, characteristic of N-carbethoxyhistidine, which is diminished by treatment with hydroxylamine. Correlation between the stoichiometry of histidine modification and the extent of inactivation indicates that out of 2 histidine residues modified, one is responsible for inactivation. A plot of the log of the reciprocal half-time of inactivation against log DEPC concentration further suggests that only 1 histidine is involved in catalysis. The rate of inactivation shows a pH dependence with an inflection point at 6.2, indicating histidine derivatization by DEPC. Inactivation due to modification of tyrosine, lysine, or cysteine has been excluded. CD studies reveal no significant change in the protein or heme conformation following DEPC modification. We suggest that a unique histidine residue is required for maximal catalytic activity of HRP for iodide oxidation.     

Identification of a critical lysine residue at the active site in glyceraldehyde-3-phosphate dehydrogenase of Ehrlich ascites carcinoma cell. Comparison with the rabbit muscle enzyme.

The involvement of the lysine residue present at the active site of Ehrlich ascites carcinoma (EAC) cell glyceraldehyde-3-phosphate dehydrogenase (Gra3PDH) was investigated by using the lysine specific reagents trinitrobenzenesulfonic acid (TNBS) and pyridoxal phosphate (PP). Both TNBS and PP inactivated EAC cell Gra3PDH with pseudo-first-order kinetics with the rate dependent on modifier concentration. Kinetic analysis, including a Tsou plot, indicated that both TNBS and PP apparently react with one lysine residue per enzyme molecule. Two of the substrates, d-glyceraldehyde-3-phosphate and NAD, and also NADH, the product and competitive inhibitor, almost completely protected the enzyme from inactivation by TNBS. A comparative study of Gra3PDH of EAC cell and rabbit muscle indicates that the nature of active site of the enzyme is significantly different in these two cells. A double inhibition study using 5,5'-dithiobis(2-nitrobenzoic acid) and TNBS and subsequent reactivation of only the rabbit muscle enzyme by dithiothreitol suggested that a cysteine residue of this enzyme possibly reacts with TNBS. These studies on the other hand, confirm that an essential lysine residue is involved in the catalytic activity of the EAC cell enzyme. This difference in the nature of the active site of EAC cell Gra3PDH that may be related to the high glycolysis of malignant cells has been discussed.     

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.     

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.     

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.