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 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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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 of Escherichia coli RNA polymerase by diethyl pyrocarbonate: evidence of histidine requirement for enzyme activity and intrinsic zinc binding

RNA polymerase (RPase) from Escherichia coli contains five subunits (alpha 2 beta beta' sigma) and two intrinsic Zn ions located in the beta and beta' subunits. This enzyme was rapidly inactivated by diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees C. The difference spectrum of the DEP-inactivated and native RPases showed a single peak at 240 nm indicating the formation of N-carbethoxyhistidines. No decrease in absorbance at 278 nm, due to O-carbethoxytyrosine, or modification of amino and sulfhydryl groups was observed. Inactivated RPase with six to nine histidines being modified could be fully reactivated by incubation with 0.5 M hydroxylamine at pH 6.0 and room temperature for 1 h. No structural difference was detected between the native and modified enzymes as evidenced by UV/visible and fluorescence spectra, sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern, or gel filtration properties. Substrate ATP at 0.11 and 1.14 mM concentrations provided, respectively, 25% and 90% protection against DEP inactivation, while template DNA did not. These results suggest that one or more histidine residues is/are in close proximity to the substrate binding site. The pH dependence of the DEP inactivation of RPase suggested the modification of histidine at the active site with a pK value of 6.9. The inactivation of RPase by DEP and the formation of N-carbethoxyhistidine displayed a similar second-order rate constant of approximately 0.9 mM-1 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 Corynebacterium sarcosine oxidase: role of sulfhydryl and histidyl groups

Sarcosine oxidase [sarcosine: oxygen oxidoreductase (demethylating) EC 1.5.3.1] from Corynebacterium contained 8 sulfhydryl groups per mol of enzyme as determined with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of 0.2% SDS and by titration with p-chloromercuribenzoate (PMB). Among them, 2 groups were easily modified by iodoacetamide (IAA) and the modification resulted in complete loss of enzymatic activity. The inactivation by IAA followed first-order kinetics with respect to IAA concentration. The presence of acetate, a competitive inhibitor (I), protected the enzyme from inactivation by IAA. However, the protection was only approximately 50%. The enzyme was also inactivated by PMB, but in this case, there was practically no recovery of activity after treatment with thiol compounds. The enzyme was also rapidly inactivated by incubation with diethylpyrocarbonate (DEP). The absorbance change accompanying the inactivation showed that a single histidyl residue was modified by DEP, resulting in a complete loss of enzymatic activity. In the presence of acetate, the enzyme was completely protected from DEP-inactivation. Furthermore, DEP-inactivated enzyme recovered its enzymatic activity on treatment with hydroxylamine. These observations seem to imply that the modified histidine is essential for enzyme activity. In addition, modification by DEP changed the absorption spectrum in the visible region. This strongly suggests that the modified histidyl residue is present in the vicinity of the flavin moiety of the enzyme molecule.     

Chemical modification of bovine heart mitochondrial malate dehydrogenase. Selective modification of cysteine and histidine.

Bovine mitochondrial malate dehydrogenase (EC 1.1.1.37) was inactivated by the specific modifications of a single histidine residue upon reaction with iodoacetamide. NADH protected against this loss of activity and reaction with the histidine residue, suggesting that the histidine is at the NADH binding site. N-Ethylmaleimide also modified the enzyme by reacting with 1 sulfhydryl residue. The reaction rate with N-ethylmaleimide was increased by decreasing the pH from neutrality or by the addition of urea. NADH protected against the modification of the sulfhydryl group under all the conditions tested, again suggesting active site specificity for this inactivation. This enzyme has a subunit weight of 33,000 and is a dimer. The native malate dehydrogenase will bind only 1 mol of NADH and it is thus assumed that there is only a single active site per dimer.     

Chemical modification of cyclomaltodextrin glucanotransferase from Bacillus circulans var. alkalophilus.

Counting of integral numbers of cysteine residues of the reduced and denaturated form of cyclomaltodextrin glucanotransferase (CGTase) from Bacillus circulans var. alkalophilus (ATCC 21783) showed two cysteine residues per enzyme molecule. Titrations of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) led to the same result. No free SH-group was detected in denatured form of CGTase, indicating that the two cysteine residues are linked by one disulfide bridge. Cyclizing activity of the GdmCl-denaturated and reduced enzyme was 13% of that of the native one. Incubation of CGTase with diethylpyrocarbonate (DEP) showed a pseudo-first-order inhibition with second-order rate constant of 3.2 M-1 s-1. Reaction with hydroxylamine and spectroscopic studies implied that inactivation of CGTase by DEP is due to modification of one histidine residue concomitantly with a 50% decrease in the cyclizing activity (t1/2 = 10.8 min). The inhibition was partially reversible. CGTase was protected against inactivation by alpha- and beta-cyclodextrins suggesting that the modified histidine residue is at or near the active site. Conversion of starch with DEP-modified enzyme resulted in a decreased formation of cyclodextrins while the relative amount of reducing sugars increased. Preliminary results on modification of CGTase with other reagents, e.g., Woodward's reagent K, 2,3-butanedione and carbodiimide are included.     

Chemical modifications of D-amino acid oxidase. Evidence for active site histidine, tyrosine, and arginine residues

1. D-amino acid oxidase is inactivated by reaction with a low molar excess of dansyl chloride at pH 6.6, with complete inactivation accompanied by incorporation of 1.7 dansyl residues per mol of enzyme-bound flavin. The presence of benzoate, a potent competitive inhibitor, protects substantially against inactivation. Evidence is presented that the inactivation is due to dansylation of an active site histidine residue. Reactivation may be obtained by incubation with hydroxylamine. Diethylpyrocarbonate also inactivates the enzyme and modifies the labeling pattern with dansyl chloride. 2. Butanedione in the presence of borate reacts rapidly to inactivate D-amino acid oxidase. Reactivation is obtained spontaneously on removal of borate, implicating reaction of butanedione with an active site arginine residue. 3. Fluorodinitrobenzene appears to behave as an active site-directed reagent when mixed with D-amino acid oxidase at pH 7.4. Complete inactivation is obtained with incorporation of 2.0 dinitrophenyl residues per mol of enzyme-bound flavin. Again benzoate protects against inactivation; only one dinitrophenyl residue is incorporated in the presence of benzoate. The active site residue attacked by fluorodinitrobenzene has been identified as tyrosine.     

Chemical modification of 3 alpha,20 beta-hydroxysteroid dehydrogenase with diethyl pyrocarbonate. Evidence for an essential, highly reactive, lysyl residue

Diethyl pyrocarbonate inactivated the tetrameric 3 alpha,20 beta-hydroxysteroid dehydrogenase with second-order rate constants of 1.63 M-1 s-1 at pH 6 and 25 degrees C or 190 M-1 s-1 at pH 9.4 and 25 degrees C. The activity was slowly and partially restored by incubation with hydroxylamine (81% reactivation after 28 h with 0.1 M hydroxylamine, pH 9, 25 degrees C). NADH protected the enzyme against inactivation with a Kd (10 microM) very close to the Km (7 microM) for the coenzyme. The ultraviolet difference spectrum of inactivated vs. native enzyme indicated that a single histidyl residue per enzyme subunit was modified by diethyl pyrocarbonate, with a second-order rate constant of 1.8 M-1 s-1 at pH 6 and 25 degrees C. The histidyl residue, however, was not essential for activity because in the presence of NADH it was modified without enzyme inactivation and modification of inactivated enzyme was rapidly reversed by hydroxylamine without concomitant reactivation. Progesterone, in the presence of NAD+, protected the histidyl residue against modification, and this suggests that the residue is located in or near the steroid binding site of the enzyme. Diethyl pyrocarbonate also modified, with unusually high reaction rate, one lysyl residue per enzyme subunit, as demonstrated by dinitrophenylation experiments carried out on the treated enzyme. The correlation between inactivation and modification of lysyl residues at different pHs and the protection by NADH against both inactivation and modification of lysyl residues indicate that this residue is essential for activity and is located in or near the NADH binding site of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)