• BMB Reports
• /
• v.32 no.4
• /
• pp.326-330
• /
• 1999

An aspartase purified from Hafnia alvei was inactivated by diethylpyrocarbonate (DEP) in a pseudo-first-order inactivation. The first-order plot was biphasic. The inactivation process was not saturable and the second order rate constant was $1.3\;M^{-1}s^{-1}$. The inactivated aspartase was reactivated with NH₂OH. The difference absorption spectrum of DEP-inactivated vs native enzyme preparations revealed a marked peak around 242 nm. The pH dependence of the inactivation rate suggests that an amino acid residue having a pK value of 7.2 was involved in the inactivation. L-aspartate, fumarate (substrates), and chloride ion (inhibitor) protected the enzyme against inactivation, indicating that histidine residues for the enzyme activity are located at the active site of this aspartase. Inspection of the presence and absence of $Cl^-$ ion demonstrated that the number of essential histidine residues is less than two. Thus, one or two histidines are in or near the aspartate binding site and participate in an essential step of the catalytic reaction.

• BMB Reports
• /
• v.32 no.3
• /
• pp.254-258
• /
• 1999

The NADPH-dependent succinic semialdehyde reductase is one of the key enzymes in the brain GABA shunt, and it catalyzes the formation of the neuromodulator $\gamma$-hydroxybutyrate from succinic semi aldehyde. This enzyme was inactivated by diethylpyrocarbonate (DEP) with the second-order rate constant of $1.1{\times}10^3\;M^{-1}min^{-1}$ at pH 7.0, $25^{\circ}C$, showing a concomitant increase in absorbance at 242 nm due to the formation of N-carbethoxyhistidyl derivatives. Complete inactivation of succinic semialdehyde reductase required the modification of five histidyl residues per molecule of enzyme. However, only one residue was calculated to be essential for enzyme activity by a statistical analysis of the residual enzyme activity. The inactivation of the enzyme by DEP was prevented by preincubation of the enzyme with the coenzyme NADPH but not with the substrate succinic semialdehyde. These results suggest that an essential histidyl residue involved in the catalytic activity is located at or near the coenzyme binding site of the brain succinic semialdehyde reductase.

• BMB Reports
• /
• v.29 no.4
• /
• pp.277-285
• /
• 1996

In Pseudomonas fluorescens grown on malonate as sole carbon source, acetyl-CoA synthetase was induced, suggesting that malonate is metabolized through acetate and then acetyl-CoA. Acetyl-CoA synthetase was purified 18.6-fold in 4 steps to apparent homogeneity. The native molecular mass of the enzyme estimated by a native acrylamide gel electrophoresis was 130 kDa. The enzyme was composed of two identical subunits with a molecular mass of 67 kDa. Optimum pH was 70. The acetyl-CoA synthetase showed typical Michaelis-Menten kinetics for the substrates, acetate, ATP and CoA, whose $K_m$ values were calculated to be 33.4, 74.8, and 40.7 mM respectively. Propionate. butyrate and pentanoate were also used as substrates by the enzyme, but the rate of the formation of the CoA derivatives was decreased in the order of the increase in carbon number. The enzyme was inhibited by the group-specific reagents diethylpyro-carbonate, 2,3-butanedione, pyridoxal-5'-phosphate and N-bromosuccinimide. In the presence of substrates the inactivation rate of the enzyme, by all of the group-specific reagents mentioned above decreased, indicating the presence of catalytically essential histidine, arginine, lysine and tryptophan residues at or near the active site. Preincubation of the enzyme with ATP, $Mg^{2+}$ resulted in the increase of its susceptibility to diethylpyrocarbonate, suggesting that ATP, $Mg^{2+}$ may induce a conformational change in the active site exposing the essential histidine residue to diethylpyrocarbonate. The enzyme was acetylated in the presence of acetyl-CoA, indicating that this is one of acyl-enzyme.

• The Korean Journal of Mycology
• /
• v.24 no.4 s.79
• /
• pp.255-264
• /
• 1996

The hydrolysis products formed from birchwood xylan by the action of an alkaline xylanase (CX-III) from alkalophilic Cephaloxporium sp. RYM-202 were xylobiose and xylooligosaccharides polymerized with more than 4 sugar molecules. This enzyme was not active on xylobiose but readily attacked xylotriose accumulating xylobiose as a major product. The predominant end-products from xylotetraose by CX-III were xylobiose and xylotriose. These results indicate that the enzyme is typically endo-type xylanase possessing transglycosidase activity. Chemical modification of CX-III with N-bromosuccinimide revealed that two tryptophan residues per molecule of CX-III were essential for its catalytic activity on xylan. On the other hand, iodoacetamide and diethylpyrocarbonate did not influence the activity of the enzyme, suggesting that cysteine and histidine residues are not involved in the active site of this alkaline xylanase.

• BMB Reports
• /
• v.28 no.2
• /
• pp.94-99
• /
• 1995

The properties of binding sites in the active site of $Zn^{2+}$-glycerophosphocholine cholinephosphodiesterase were examined using substrates and inhibitors of the enzyme. Phosphodiesterase hydrolyzed p-nitrophenylphosphocholine, p-aminophenylphosphocholine, and glycerophosphocholine, but did not hydrolyze either acylated glycerophosphocholine or bis (p-nitrophenyl)phosphate, suggesting a size limitation for interaction with a glyceryl moiety-binding subsite. The hydrolysis of p-nitrophenylphosphocholine was competitively inhibited by glycerophosphocholine and p-aminophenylphosphocholine, while glycerophosphoethanolamine was a weak inhibitor. The enzyme was also inhibited by choline, but not by ethanolamine. Thiocholine, a much more potent inhibitor than choline, was more inhibitory than cysteamine, suggesting a strict specificity of an anionic subsite adjacent to a $Zn^{2+}$ subsite. Of all oxyanions tested, the tellurite ion was found to strongly inhibit the enzyme by binding to a $Zn^{2+}$ subsite. The inhibitory role of tellurite was synergistically enhanced by tetraalkylammonium salts, but not by glycerol. Deactivation of the enzyme by diethylpyrocarbonate was partially protected by choline, but not by glycerophosphate. It is suggested that the active site of phosphodiesterase contains three binding subsites.

• Microbiology and Biotechnology Letters
• /
• v.25 no.2
• /
• pp.173-177
• /
• 1997

Glycerol-3-phosphate cytidylyltransferase from Bacillus subtilis was modified with various chemical modifiers to determine the active sites of the enzyme. Treatment of the enzyme with group-specific reagents diethylpyrocarbonate, N-bromosuccinimide, or carbodiimide resulted in complete loss of enzyme activity, which shows histidine, tryptophan, and glutamic acid or aspartic acid residues are at or near the active site. In each case, inactivation followed pseudo first-order kinetics. Inclusion of glycerol-3-phosphate and/or CTP prevented the inactivation, indicating the presence of tryptophan and glutamic acid or aspartic acid residues at the substrate binding site. Analysis of kinetics of inactivation showed that the loss of enzyme activity was due to modification of a two histidine residues, single tryptophan residue, and two glutamic acid or aspartic acid residues.

• The Korean Journal of Food And Nutrition
• /
• v.7 no.1
• /
• pp.1-7
• /
• 1994

To investigate the characteristics of active sites of the D-xylanase and $\beta$-xylosidase purified from Penicillium verruculosum, effects of various chemicals on the enzyme activity were analyzed. The D-xylanase was activated by Cua), however it was inhibited by metal ions, Hg2+ and Mna+, by chemicals, N-bromosuccinimide, iodine, diethylpyrocarbonate, and 2,3-butanedione. These results suggested that the D-xylanase from Penicillium verruculosum contained tyrosine, histidine, arginine and tryptophan at the active center. The $\beta$-xylosidase was inhibited by Hg2+, N-bromosuccinimide and sodium dodecyl sulfate, however it was not effected by Mn2+ and Cu2). It was suggested that the enzyme contained tryptophan at the active center.

• Animal cells and systems
• /
• v.2 no.2
• /
• pp.243-248
• /
• 1998

We have partially sequenced glutaminyl cyclases from several mammalian and one avian species and found that the two cysteine residues of the human glutaminyl cyclase are completely conserved. The mammalian glutaminyl cyclase has been reported to possess reactive thiols (Busby, Jr, et aI., 1987, J BioI Chern 262, 8532-8536). Mutagenesis of these cysteine residues, however, resulted in only a slight decrease in enzyme activity. Likewise, the recombinant human enzyme was completely resistant to attempted chemical modification of the putative reactive thiols. Although the human glutaminyl cyclase did not appear to have reactive thiols, it was sensitive to diethylpyrocarbonate and acetylimidazole, indicating the presence of functionally important histidine and tyrosine residues which could act as acid/base catalysts. Almost identical deuterium solvent isotope effect (1.2 vs 1.3) upon the reaction by the human and papaya enzymes, respectively, provides an evidence both animal and plant glutaminyl cyclases catalyze pyroglutamyl-peptide formation by intramolecular cyclization.

• BMB Reports
• /
• v.28 no.2
• /
• pp.124-128
• /
• 1995

Biodegradative threonine dehydratase purified from Serratia marcescens ATCC 25419 was inactivated by the arginine specific modification reagent, phenylglyoxal (PGO) and the lysine modification reagent, pyridoxal 5'-phosphate (PLP). The inactivation by PGO was protected by L-threonine and L-serine. The second order rate constant for the inactivation of the enzyme by PGO was calculated to be 136 $M^{-1}min^{-1}$. The reaction order with respect to PGO was 0.83. The inactivation of the enzyme by PGO was reversed upon addition of excess hydroxylamine. The inactivation of the enzyme by PLP was protected by L-threonine, L-serine, and a-aminobutyrate. The second order rate constant for the inactivation of the enzyme by PLP was 157 $M^{-1}min^{-1}$ and the order of reaction with respect to PLP was 1.0. The inactivation of the enzyme by PLP was reversed upon addition of excess acetic anhydride. Other chemical modification reagents such as N-ethylmaleimide, 5,5'-dithiobis (2-nitrobenzoate), iodoacetamide, sodium azide, phenylmethyl sulfonylfluoride and diethylpyrocarbonate had no effect on the enzyme activity. These results suggest that essential arginine and lysine residues may be located at or near the active site.

• BMB Reports
• /
• v.34 no.3
• /
• pp.230-236
• /
• 2001

dTDP-rhamnose is synthesized from dTTP and glucose-1-phosphate by four enzymatic steps in the gram-negative bacteria. By using a homologous PCR product, a gene cluster encoding four genes (rfbA, rfbB, rfbC, rfbD) involved in L-rhamnose biosynthesis by Acinetobacter calcoaceticus was isolated and sequenced. The four genes were clustered on the biosynthetic operon in the order of rfbB, D, A, C. A gene, rfbA, encoding glucose-l-phosphate thymidylyltransferase (RfbA), was cloned from A. calcoaceticus pathogenic and encapsulated in the gram-negative bacterium. This enzyme catalyzes the formation of dTDP-D-glucose From $\alpha$-D-glucose-1-phosphate and dTTP.RfbA was amplified by PCR and inserted into the $T_7$ expression system. The activity of RfbA was determined by the capillary electrophoresis. The $K_m$ values for dTTP and $\alpha$-D-glucose-1-phosphate were calculated to be 1.27 mM and 0.80 mM, respectively by using the Line-Weaver Burk plot. RfbA is inactivated by diethylpyrocarbonate.