• 한국미생물·생명공학회지
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• 제26권5호
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• pp.387-392
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• 1998

B. lactofermentum의 lysine 생합성에 있어서 DDH경로 및 ddh gene이 지닌 중요성을 조사하기 위하여, site-specific mutagenesis technique를 통하여 B. lactofermentum의 ddh gene을 disruption함으로서 DDH 경로를 차단시켰다. B. lactofermentum ddh mutant는 wild type 및 AEC내성 균주보다 성장이 매우 저조하였으며 lysine 생산량에서도 급격한 저하를 가져왔다. 이와 같이 B. lactofermentum이 DAP 경로만을 가졌을 때 세포의 성장 및 lysine 생산량에 있어서 극적인 저하를 가져왔기 때문에 B. lactofermentum에서의 DDH 경로는 meso-DAP 및 lysine 생합성에 있어 필수적인 경로로 작용한다는 것을 확인하였다. 그러므로 C. glutamicum과 B. lactofermentum과 같은 corynebacteria가 lysine을 많이 생산하는 것은 DDH 경로가 부가적으로 존재하기 때문이며, 이러한 DDH 경로는 metabolic flux가 증가되면 중간 대사물을 lysine으로 변화시키는 중요한 경로로 작용할 것이라 사료된다.

• Bulletin of the Korean Chemical Society
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• 제27권4호
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• pp.529-534
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• 2006

Chemical modification of the S. cerevisiae farnesyl protein transferase (FPT) with CMC, phenylglyoxal and DEPC resulted in enzyme inactivation, depending upon the reagent concentration. The peptide substrate GST-PEP-I, a GST-fused undecapeptide mimicking the C-terminus of $p21^{Ki-ras}$, protected the enzyme against inactivation by CMC which is specific to either aspartate or glutamate, while the other substrate farnesyl pyrophosphate (FPP) showed protection against phenylglyoxal which is the specific modifier of arginine residues, dependent on the substrate concentrations. Neither of the two substrates protected the enzyme against histidine inactivation by DEPC. It is suggested that there is at least one aspartate or glutamate residue at the peptide substrate binding site, and that at least one arginine residue is located at the binding site of FPP. There also seems to be at least one histidine residue which is critical for enzymic activity and is exposed toward the bulk solution, excluded from the substrate binding sites.

• BMB Reports
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• 제33권1호
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• pp.69-74
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• 2000

The Arabidopsis thaliana S-Adenosylmethionine decarboxylase (AdoMetDC) cDNA ($GenBank^{TM}$ U63633) was cloned, then the AdoMetDC protein was expressed and purified. The purified AdoMetDC was inactivated by salicylaldehyde in a pseudo first- order kinetics. The secondorder rate constant for inactivation was 126 $M^{-1}min^{-1}$ with the slope of n=0.73, suggesting that inactivation is the result of the reaction of one lysine residue in the active site of AdoMetDC. Site-specific mutagenesis was performed on the AdoMetDC to introduce mutations in conserved $lysine^{81}$ residues. These were chosen by examination of the conserved sequence and proved to be involved in enzymatic activity by chemical modification. Changing $Lys^{81}$ to alanine showed an altered optimal pH. The substrate also provided protection against inactivation by salicylaldehyde. Considering these results, we suggest that the $lysine^{81}$ residue may be involved in catalytic activity.

• BMB Reports
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• 제29권4호
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• pp.321-326
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• 1996

Spinach glycolate oxidase was subjected to a series of chemical modifications aimed at identifying amino acid residues essential for catalytic activity. The oxidase was reversibly inactivated by treatment with pyridoxal 5'-phosphate (PLP). The inactivation by PLP was accompanied by the appearance of an absorption peak of around 430 nm, which was shifted to 325 nm upon reduction with $NaBH_4$. After reduction, the PLP-treated oxidase showed a fluorescence spectrum with a maximum of around 395 nm by exciting at 325 nm. The substrate-competitive inhibitors oxalate and oxaloacetate provided protection against inactivation of the oxidase by PLP. These results suggest that PLP inactivates the enzyme by fonning a Schiff base with lysyl residue(s) at an active site of the oxidase. The enzyme was also inactivated by tryptophan-specific reagent N-bromosuccinimide (NBS). However, competitive inhibitors oxalate and oxaloacetate could not protect the oxidase significantly against inactivation of the enzyme by NBS. The results implicate that the inactivation of the oxidase by NBS is not directly related to modification of the tryptophanyl residue at an active site of the enzyme. Treatments of the oxidase with cysteine-specific reagents iodoacetate, silver nitrate, and 5,5'-dithiobis-2-nitrobenzoic acid did not affect significantly the activity of the enzyme.

• 한국미생물·생명공학회지
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• 제25권2호
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• pp.173-177
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• 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.

• BMB Reports
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• 제31권2호
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• pp.139-143
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• 1998

The catabolic ${\alpha}-acetolactate$ synthase purified from Serratia marcescens ATCC 25419 was rapidly inactivated by the tryptophane-specific reagent, N -bromosuccinimide, and the arginine-specific reagent, phenylglyoxal. The enzyme was inactivated slowly by the cysteine-specific reagent N-ethylmaleimide. The second-order rate constants for the inactivation by N-bromosuccinimide, phenylglyoxal. and N -ethylmaleimide were $114,749M^{-1}min^{-1}$, $304.3M^{-1}min^{-1}$, and $5.1M^{-1}min^{-1}$, respectively. The reaction order with respect to N-bromosuccinimide, phenylglyoxal, and N-ethylmaleimide were 1.5,0.71, and 0.86, respectively. The inactivation of the catabolic aacetolactate synthase by these modifying reagents was protected by pyruvate. These results suggest that essential tryptophane, arginine, and cysteine residues are located at or near the active site of the catabolic ${\alpha}-acetolactate$ synthase.

• BMB Reports
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• 제28권1호
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• pp.40-45
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• 1995

Acetolactate synthase purified from Serratia marcescens ATCC 25419 was rapidly inactivated by the thiol specific reagent p-chloromercuribenzoate (PCMB), the tryptophan specific reagent N-bromosuccinimide (NBS), and the arginine modifying reagent phenylglyoxal (PGO). Inactivation by PCMB was prevented by both ${\alpha}$-ketobutyrate and pyruvate, and the second order rate constant for the inactivation was $2480\;M^{-1}{\cdot}min^{-1}$. The reaction order with respect to PCMB was 0.94. The inactivation of the enzyme by NBS was also substantially reduced by both ${\alpha}$-ketobutyrate and pyruvate. The second order rate constant for inactivation by NBS was $15,000\;M^{-1}{\cdot}min^{-1}$, and the reaction order was 2.0. On the other hand, inactivation by PGO was partially prevented by ${\alpha}$-ketobutyrate, but not by pyruvate. The second order rate constant for the inactivation was $1480\;M^{-1}{\cdot}min^{-1}$ and the order of reaction with respect to PGO was 0.75. These results suggest that essential cysteine, tryptophan and arginine are located at or near the substrate binding site.

• Bulletin of the Korean Chemical Society
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• 제24권12호
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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.

• BMB Reports
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• 제30권4호
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• pp.280-284
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• 1997

Phenylglyoxal diethyl pyrocarbonate (DEPC), and 1-cyclohexyl-3-[2-morpholinoethyl]-carbodiimide metho-p-toluenesulfonate (CMC) are modifying reagents specific for arginine, histidine, and aspartate or glutamate, respectively. They were found to inactivate S. cerevisiae farnesyl protein transferase (FPTase). The peptide substrate protected the enzyme against inactivation by CMC and the other substrate farnesyl pyrophosphate showed protection against inactivation by phenylglyoxal. while neither of the two substrates protected the enzyme against DEPC inactivation. These results suggest the presence of aspartate/glutamate, arginine and histidine residues at the active site of this enzyme.

• BMB Reports
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• 제28권2호
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• pp.124-128
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• 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.