• Archives of Pharmacal Research
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• v.25 no.5
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• pp.643-646
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• 2002

The succinic semialdehyde dehydrogenase (SSADH) inhibitory component was isolated from the EtOAc fraction of Lactuca sativa through repeated column chromatography; then, it was identified as phytol, a diterpenoid, based on the interpretation of several spectral data. Incubation of SSADH with the phytol results in a time-dependent loss of enzymatic activity, suggesting that enzyme modification is irreversible. The inactivation followed pseudo-first-order kinetics with the second-rate order constant of $6.15{\times}10^{-2}mM^{-1}min^{-1}.$ Complete protection from inactivation was afforded by the coenzyme $NAD^{+}$, whereas substrate succinic semialdehyde failed to prevent the inactivation of the enzyme; therefore, it seems likely that phytol covalently binds at or near the active site of the enzyme. It is postulated that the phytol is able to elevate the neurotransmitter GABA levels in central nervous system through its inhibitory action on one of the GABA degradative enzymes, SSADH.

• Archives of Pharmacal Research
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• v.17 no.4
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• pp.218-222
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• 1994

The natural product, spirostanol glycoside dioscin, was shown to directly inactivate human pleural fluid phospholipase $A_2{\;}(PLA_2)$ Inactivation was dose, and time dependent. The $IC_{50}$ was estimated at 18 .mu.M and virtually complete inactivation of the enzyme occurred at 50 .mu.M. Using Michaelis-Menten kinetics, dioscin inactivated the enzyme by a competitive inhibitory manner, the apparent Ki value was $6.9{\times}10_{-4}$. Reversibility was studied directly by dialysis method, the inhibition was reversible. Additioin of excess $Ca^{2+}$ concentration up to 8 mM did not antagonize the inhibitory activity of dioscin. Inactivation of several kinds of $PLA_2$ by dioscin is due to interaction with the active site of $PLA_2$ and may be a useful adjunt in the theraphy of inflammatory diseases.

• Journal of the Korean Chemical Society
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• v.20 no.5
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• pp.406-416
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• 1976

A series of $N^1$-alkylnicotinamide chlorides, $N^1$-methyl-to $N^1$-dodecylnicotinamides inclusive were studied with rabbit muscle L-${\alpha}$-glycerophosphate dehydrogenase to investigate the possibility of reversible and irreversible inactivation of the pyridine-linked dehydrogenases by the coenzyme-competitive inhibitor derivatives. The inhibition of the enzyme by $N^1$-alkylnicotinamide chlorides was demonstrated to be reversible at the dilute concentration of the inhibitors but this reversible inhibition was found to be followed by an irreversible time-dependent inactivation measuable at high concentrations of the inhibitors. The properties of this time-dependent inactivation were discussed on the basis of the denaturation of the enzyme by the binding of small micelle-like structures formed at higher concentrations of the inhibitors.

• Bulletin of the Korean Chemical Society
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• v.24 no.12
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• pp.1799-1804
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• 2003

Acetolatate synthase(ALS) catalyzes the first common step in the biosynthesis of valine, leucine, isoleucine in plants and microorganisms. ALS is the target of several classes of herbicides, including the sulfonylureas, the imidazolinones, and the triazolopyrimidines. To elucidate the roles of arginine residues in tobacco ALS, chemical modification and site-directed mutagenesis were performed. Recombinant tobacco ALS was expressed in E. coli and purified to homogeneity. The ALS was inactivated by arginine specific reagents, phenylglyoxal and 2,3-butanedione. The rate of inactivation was a function of the concentration of modifier. The inactivation by butanedione was enhanced by borate, and the inactivation was reversible on removal of excess butanedione and borate. The substrate pyruvate and competitive inhibitors fluoropyruvate and phenylpyruvate protected the enzyme against inactivation by both modifiers. The mutation of well-conserved Arg198 of the ALS by Gln abolished the enzymatic activity as well as the binding affinity for cofactor FAD. However, the mutation of R198K did not affect significantly the binding of FAD to the enzyme. Taken together, the results imply that Arg198 is essential for the catalytic activity of the ALS and involved in the binding of FAD, and that the positive charge of the Arg is crucial for the interaction with negatively charged FAD.

• Bulletin of the Korean Chemical Society
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• v.10 no.1
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• pp.107-111
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• 1989

Glucoamylase was inactivated with 1-ethyl-2-(dimethylaminopropyl)carbodiimide (EDC) at pH 5.0. Time course of inactivation of glucoamylase was at least biphasic. From the results of the titration of SH groups with Ellman's reagent and hydroxylamine treatment at pH 7.0, it was concluded that the crucial sites of modification were carboxyl groups of glucoamylase. The CD spectrum of EDC-modified glucoamylase suggested that the gross conformation of the native enzyme was retained. The inactivation of glucoamylase was reduced remarkably in the presence of maltose. The logarithm of the half-life of the inactivation of glucoamylase by EDC was a linear function of log[EDC] in each stage indicating that one carboxyl group among the modified ones was crucial for inactivation of glucoamylase. The change in the binding affinity due to modification was determined by using an affinity column. It indicates that the carboxyl group of glucoamylase seems to play a role in both, the catalysis and substrate binding in the first stage, but in the second stage the binding affinity is recovered almost up to that of native enzyme.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.74-74
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• 1995

GABA transaminase is inactivated by preincubation with p$^1$, p$^2$-bis(5'-pyridoxal) diphosphate at pH 7.0. The inactivation under pseudo-first order conditions proceeds at a slow rate (K$\_$obs/=0.035 min$\^$-1/). The degree of labeling of the enzyme by p$^1$, p$^2$-bis(5'-pyridoxal) diphosphate was determined by absorption spectroscopy, The blocking of 2 lysyl residues/dimer is needed for inactivation of the transaminase. The time course of the reaction is significantly affected by the substrate ${\alpha}$-ketoglutarate, which afforded complete protection against the loss of the catalytic activity. Whereas cofator pyridoxal phosphate failed to prevent the inactivation of the enzyme. Therefore, it is postulated that binding of ${\alpha}$-ketoglutarate tn lysyl residues is the major factor contributing to stabilization of the catalytic site and bifuctional reagent p$^1$, p$^2$bis(5'-pyridoxal) diphosphate blocks lysyl residues other than those involved in the binding of the cofactor.

• Microbiology and Biotechnology Letters
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• v.22 no.6
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• pp.636-642
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• 1994

Essential amino acids involving in the catalytic mechanism of the $\beta$-D-xylosidase of Bacillus stearothermophilus were determined by chemical modification studies. Among various che- mical modifiers tested N-bromosuccinimide (NBS), $\rho$-hydroxymercurybenzoate (PHMB), N-ethylma- leimide, 1-[3-(di-ethylamino)-propyl]$-3-ethylcarbodi-imide (EDC), and Woodward's Reagent K(WRK)inactivated the enzyme, resulting in the residual activity of less than 20%. WRK reduced the enzyme activity by modifying carboxylic amino acids, and the inactivation reacion proceeded in the form of pseudo-first-order kinetics. The double-lagarithmic plot of the observed pseudo-first- order rate constant against the modifier concentration yielded a reaction order of 2, indicating that two carboxylic amino acids were essential for the enzyme activity. The $\beta$-D-xylosidase was also inactivated by N-ethylmaleimide which specifically modified a cysteine residue with a reaction order of 1, implying that one cysteine residue was important for the enzyme activity. Xylobiose protected the enzyme against inactivation by WRK and N-ethylmaleimide, revealing that carboxylic amino acids and a cysteine residue were present at the substrate-binding site of the enzyme molecule.

• Animal cells and systems
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• v.1 no.4
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• pp.583-587
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• 1997

Incubation of an NADPH-dependent succinic semialdehyde reductase from bovine brain with N-bromosuccinimide (NBS) resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first-order kinetics with the second-order rate constant of $6.8\times{10}^3$ $M^-1$ $min^{-1}$. The inactivation was prevented by preincubation of the enzyme with substrate succinic semialdehyde, but not with coenzyme NADPH. There was a linear relation-ship between oxindole formation and the loss of enzyme activity. Spectro-photometric studies indicated that about one oxindole group per molecule of the enzyme was formed following complete loss of enzymatic activity. It is suggested that the catalytic function of succinic semialdehyde reductase is modulated by binding of NBS to a specific tryptophan residue at or near the substrate binding site of the enzyme.

• BMB Reports
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• v.38 no.1
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• pp.58-64
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• 2005

Myo-inositol monophosphate phosphatase (IMPP) is a key enzyme in the phosphoinositide cell-signaling system. This study found that incubating the IMPP from a porcine brain with pyridoxal-5'-phosphate (PLP) resulted in a time-dependent enzymatic inactivation. Spectral evidence showed that the inactivation proceeds via the formation of a Schiff's base with the amino groups of the enzyme. After the sodium borohydride reduction of the inactivated enzyme, it was observed that 1.8 mol phosphopyridoxyl residues per mole of the enzyme dimer were incorporated. The substrate, myo-inositol-1-phosphate, protected the enzyme against inactivation by PLP. After tryptic digestion of the enzyme modified with PLP, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. Amino acid sequencing of the peptide identified a portion of the PLP-binding site as being the region containing the sequence L-Q-V-S-Q-Q-E-D-I-T-X, where X indicates that phenylthiohydantoin amino acid could not be assigned. However, the result of amino acid composition of the peptide indicated that the missing residue could be designated as a phosphopyridoxyl lysine. This suggests that the catalytic function of IMPP is modulated by the binding of PLP to a specific lysyl residue at or near its substrate-binding site of the protein.

• Korean Journal of Environmental Biology
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• v.19 no.3
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• pp.211-217
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• 2001

Salicylate is involved in the induction of pathogen-related proteins and plant defense response. The effects of salicylate on the activity isoperoxidase $A_3$ from tobacco callus (Nicotiana tabacum L.) and the protection against the enzyme inactivation by salicylate in the presence of $Fe^{2+}$ were examined. About 20% and 85% activity losses of peroxidase occurred at 0.48 mM and 0.6 mM salicylate, respectively, showing that isoperoxidase $A_3$ was inactivated by salicylate. The inactivation occurred depending on pH and showed noncompetitive inhibition mode. Moreover, inactivation of the enzyme by salicylate was completely protected in the presence of $Fe^{2+}$. Apoperoxidase without heme moiety was constructed and the effects of various metal ions on the recovery of enzyme activities were investigated. More than 80% of the activity was reconstituted by the addition of $Fe^{2+}$ or hemin. However, the enzyme activity was not recovered by $Cu^{2+},\;Zn^{2+},\;Co^{2+},\;or\;Mn^{2+}$.