• Bulletin of the Korean Chemical Society
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• v.27 no.4
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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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• v.31 no.2
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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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• v.29 no.4
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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.

• Journal of Pharmaceutical Investigation
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• v.30 no.2
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• pp.73-83
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• 2000

Polyethylene glycol (PEG) is a water soluble, biocompatible, non-toxic polymer and PEGylation is a well established technique for the modification of therapeutic proteins and peptides. PEG-protein drugs have been extensively studies in relation to therapies for various diseases: cancer, inflammation and others. The covalent attachment of PEG to proteins and peptides prolonged plasma half-life, reduced antigenicity and immunogenicity, increased thermal and mechanical stability, and prevented degradation by enzymes. Several chemical groups for general and site specific conjugation have been exploited to activate PEG for amino group, carboxyl group, and cysteine groups. PEGylation of many proteins and peptides have been studied to enhance their properties for the potential uses. Also, the different positional isomers in several PEG-proteins have shown the difference in vivo stability and biological indicating that the site of PEG molecule attachment is one of the important factor to develop PEG-proteins as potential therapeutic agents.

• 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.

• BMB Reports
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• v.30 no.4
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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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• v.28 no.2
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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.

• BMB Reports
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• v.37 no.2
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• pp.260-267
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• 2004

Transducin (T), the heterotrimeric guanine nucleotide binding protein in rod outer segments, serves as an intermediary between the receptor protein, rhodopsin, and the effector protein, cGMP phosphodiesterase. Labeling of T with dansyl chloride (DnsCl) inhibited its light-dependent guanine nucleotide binding activity. Conversely, DnsCl had no effect on the functionality of rhodopsin. Approximately 2-3 mol of DnsCl were incorporated per mole of T. Since fluoroaluminate was capable of activating DnsCl-modified T, this lysine-specific labeling compound did not affect the guanine nucleotide-binding pocket of T. However, the labeling of T with DnsCl hindered its binding to photoexcited rhodopsin, as shown by sedimentation experiments. Additionally, rhodopsin completely protected against the DnsCl inactivation of T. These results demonstrated the existence of functional lysines on T that are located in the proximity of the interaction site with the photoreceptor protein.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.55-55
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• 1993

Farnesyl Protein transferase(FPT)는 발암유전자 ras의 단백질 산물인 p$^{21}$의 post-translational modification의 첫 단계인 ras-farnesylation에 관여하는 효소로 본 연구에서는 정제된 FPT와 E. coli에서의 발현 system을 이용하여 FPT의 구조와 기능을 밝히고 이를 FPT 방해제의 설계에 이용하고자 한다. Bovine testis에 존재하는 FPT를 30%-50%의 Ammonium sulfate로 fractionation하고, DEAE-Sephacel, Sephacryl S-300 column을 통과시킨 후 peptide(KKCVIM) affinity column을 이용하여 순수 정제하였다. 정제된 효소의 분자량은 gel-filtration에 의해 100KDa으로 추정되었고 SDS-PAGE 결과 49KDa과 46KDa의 두 subunit로 구성되었음이 확인되었다. 효소활성에는 $Mg^{2＋}$ 와 $Zn^{2＋}$가 필수적이며 최적 pH는 7.0이었다. Yeast의 FPT의 두 subunit 유전자는 Yeast genomic DNA를 template로 사용하고 각 subunit에 specific한 합성된 primer들과 vent polymerase를 이용하여 Polymerase chain reaction을 통하여 얻었다. 두 유전자를 pBluescriptII SK＋ vector를 변형시킨 두 vector, pBSK＋4와 pBChl＋4에 재조합 시킨 후 E.coli에 transformation시켜 발현시켰다. 현재 정제된 Bovine FPT와 E. coli에서 발현된 Yeast FPT의 chemical modification과 site-directed mutagenesis를 통하여 FPT의 active site와 substrate binding site에 관한 연구를 진행시키고 있다.

• Journal of Microbiology and Biotechnology
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• v.9 no.1
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• pp.62-69
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• 1999

In order to investigate the critical amino acid residues involved in the catalytic activities of $\beta$-cyclodextrin glucanotransferase ($\beta$-CGTase) excreted by Bacillus firmus var. alkalophilus, the amino acid residues in $\beta$-CGTase were modified by various site-specific amino acid modifying reagents. The cyclizing and amylolytic activities of $\beta$-CGTase were all seriously reduced after treatment with Woodward's reagent K (WRK) modifying aspartic/glutamic acid, N-bromosuccinimde (NBS) modifying tryptophan, and diethylpyrocarbonate (DEPC) modifying histidine residues. The roles of tryptophan and histidine residues in $\beta$-CGTase were further investigated by measuring the protection effect of various substrates during chemical modification, comparing protein mobility in native and affinity polyacrylamide gel electrophoresis containing soluble starch, and comparing the $K_m$ and $V_{max}$ values of native and modified enzymes. Tryptophan residues were identified as affecting substrate-binding ability rather than influencing catalytic activities. On the other hand, histidine residues influenced catalytic ability rather than substrate-binding ability, plus histidine modification had an effect on shifting the optimum pH and pH stability.