• 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.

• Bulletin of the Korean Chemical Society
• /
• v.21 no.6
• /
• pp.567-570
• /
• 2000

Bovine liver catalase was exposed to cysteine, as a natural inactivator metabolize, causing autoxidation-generating $H_2O_2$ continuously. The catalase species concentrations and activity measurement were done by spectrophotometry in phosphate buffer 10mM, pH 6.5, and 27 $^{\circ}C$. The activity of catalase decreased continuously due to the conversion of active ferricatalase species, E-Fe (III), to an inactive enzyme species, E-Fe (IV). This conversion is related to the slow production of $H_2O_2generated$ by autoxidation of cysteine. The free SH-group of cysteine has an essential role in production of $H_2O_2$ and hence inactivation of catalase. NADPH can protect catalase against inactivation due to the conversion of inactive form of E-Fe (IV) to ferricatalase species, E-Fe (III).

• Korean Journal of Microbiology
• /
• v.29 no.5
• /
• pp.301-306
• /
• 1991

Extracellular fructosyl transferase from Aureobasidium pullulans C-23 was characterized. The molecular weight of the isolated enzyme was determined to be approximately 170,000 by SDS polyacrylamide gel electrophoresis. The enzyme has the pI value of about 3.7. The enzyme was almost completely inhibited by 5mM $Hg^{2+}$ , but was not significantly affected by other cations tested. The enzyme was inactivated by treatment of tryptophan-specific reagent N-bromo- succinimide and tyrosine-specific reagent iodine. The substrate sucrose showed protective effect on the inactivation of the enzyme by the both reagents. These results suggest that tryptophan and tyrosine residues are probably located at or near active site of the enzyme.

• BMB Reports
• /
• v.33 no.2
• /
• pp.172-178
• /
• 2000

The lipoxygenase was purified 35 fold to homogeneity from the Korean red potato by an ammonium sulfate precipitation and DEAE-cellulose column chromatography. The simple purification method is useful for the preparation of pure lipoxygenase. The molecular weight of the enzyme was estimated to be 38,000 by SDS-polyacrylamide gel electrophoreses and Sepharose 6B column chromatography. The purified enzyme with 2 M $(NH_4)_2SO_4$ in a potassium phosphate buffer, pH 7.0, was very stable for 5 months at $-20^{\circ}C$. Because the purified lipoxygenase is very stable, it could be useful for the screening of a lipoxygenase inhibitor. The optimal pH and temperature for lipoxygenase purified from the red potato were found to be pH 9.0. and $30^{\circ}C$, respectively. The Km and Vmax values for linoleic acid of the lipoxygenase purified from the red potato were $48\;{\mu}M$ and $0.03\;{\mu}M$ per minute per milligram of protein, respectively. The enzyme was insensitive to the metal chelating agents tested (2 mM KCN, 1 and 10mM EDTA, and 1 mM $NaN_3$), but was inhibited by several divalent cations, such as $Cu^{++}$, $Co^{++}$ and $Ni^{++}$. The essential amino acids that were involved in the catalytic mechanism of the 5-lipoxygenase from the Korean red potato were determined by chemical modification studies. The catalytic activity of lipoxygenase from the red potato was seriously reduced after treatment with a diethylpyrocarbonate (DEPC) modifying histidine residue and Woodward's reagent (WRK) modifying aspartic/glutamic acid. The inactivation reaction of DEPC (WRK) processed in the form of pseudo-first-order kinetics. The double-logarithmic plot of the observed pseudo-first-order rate constant against the modifier concentration yielded a reaction order 2, indicating that two histidine residues (carboxylic acids) were essential for the lipoxygenase activity from the red potato. The linoleic acid protected the enzyme against inactivation by DEPC(WRK), revealing that histidine and carboxylic amino acids residues were present at the substrate binding site of the enzyme molecules.

• Journal of Ginseng Research
• /
• v.13 no.1
• /
• pp.92-97
• /
• 1989

This study investigated the effects of high light intensity (100 KLw) and high temperature (45 ℃, dark) on enzyme (glucose-6-phosphate dehydrogenase, acid phosphatase, catalase, peroxidase, and proteinase) activities and characteristics of Panax ginseng C.A. Meyer leaves. Enzyme activity and protein content decreased rapidly under treatment with high light intensity In P ginseng the thermal stabilities of catalase and peroxidase were high (above 70%), and the coagulation rates of soluble proteins were low (below 17%). Therefore, the decrease in enzyme activity and protein content was not caused by increase in leaf temperature due to the high light intensity, but by increase in proteolytic activities. The photochemical formation rate of superoxide radical (O-2) was higher in the P ginseng leaf extracts than in Solanum nigmm, and was accelerated by addition of crude saponin to the buffer extracts.

• Archives of Pharmacal Research
• /
• v.15 no.3
• /
• pp.229-233
• /
• 1992

The pKa value of histidine-51 residue was determined by the pH dependency of contents of NADH bound to the active site in the orse liver alcohol dehydrogenase and % inactivation with diethyl pyrocarbonate treatment of the enzyme. The pKa for His-51 was -7.15 in the ternary complex and -6.7 in the enzyme itself.

• Journal of Microbiology
• /
• v.34 no.3
• /
• pp.270-273
• /
• 1996

Micrococcus luteus purine nucleoside phosphorylase (PNP) has been purified and characterized. The physical and kinetic properties have been described previously. Chemical modification of the enzyme was attempted to gain insight on the active site. The enzyme was inactivated in a time-dependent manner by the arginine- specific modifying reagent phenylglyoxal. There was a linear relationship between the observed rate of inactivation and the phenylglyoxal concentration. At 30 $^{\circ}C$ the bimolecular rate constant for the modification was 0.015 $min^{-1}mM^{-1}$ in 50 mM $NaHCO_3$ buffer, pH 7.5. The plot of logk versus log phenylglyoxal concentration was a strainght line with a slope value of 0.9, indicating that modification of one arginine residue was needed to inactivate the enzyme. Preincubation with saturated solutions of substrates protected the enzyme from inhibition of phenylglyoxal, indicating that reactions with phenylglyoxal were directed at arginyl residues essential for the catalytic functioning of the enzyme.

• The Korean Journal of Mycology
• /
• v.13 no.2
• /
• pp.111-114
• /
• 1985

The production of lac case by the funguson various media was studied. The characteristics of the enzyme were also studied regarding to the optimum pH, stability, Km value, and inactivation. The maximum activity of laccase reached the 40 days of incubation and the barley straw extract appeared to be a strong inducer for laccase. The enzyme showed stability at wide range of pH with optimum pH of 6.6. Temperature stability of the enzyme was high. Laccase was not inactivated by the organic solvents used for the precipitation. The enzyme, how­ever, was completely inactivated by trichloroacetic acid and sodium azide.

• Korean Journal of Microbiology
• /
• v.32 no.3
• /
• pp.222-231
• /
• 1994

Kinetic analysis was done to elucidate the reaction mechanism of purine nucleoside phosphorylase (PNP) in Saccharomyces cerevisiae. The binary complexes of PNP${\cdot}$phosphate and PNP${\cdot}$ribose 1-phosphate were involved in the reaction mechanism. The initial velocity and product inhibition studies demonstrated were consistent with the predominant mechanism of the reaction being an ordered bi, bi reaction. The phosphate bound to the enzyme first, followed by nucleoside and base were the first product to leave, followed by ribose 1-phosphate. The kinetically suggested mechanism of PNP in S. cerevisiae was in agreement with the results of protection studies against the inactivation of the enzyme by sulfhydryl reagents, p-chloromercuribenzoate (PCMB) and 5,5'-dithiobisnitrobenzoate (DTNB). PNP was protected by ribose 1-phosphate and phosphate, but not by nucleoside or base, supporting the reaction order of ordered bi, bi mechanism. PCMB or DTNB-inactivated PNP was totally reactivated by dithiothreitol (DTT) and the activity was returned to the level of 77% by 2-mercaptoethanol, indicating that inactivation was reversible. The kinetic behavior of the PCMB-inactivated enzyme had been changed with higher $K_m$ value of inosine and lower $V_m$, and was restored by DTT. Inactivation of enzyme by DTNB showed similar pattern of K sub(m) value with that by PCMB, but had not changed the $V_m$ value, significantly. Negative cooperativity was not found with PCMB or DTNB treated PNP at high concentration of phosphate.

• Proceedings of the Korean Society for Food Science of Animal Resources Conference
• /
• 2001.11a
• /
• pp.141-141
• /
• 2001