• Journal of Microbiology and Biotechnology
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• 제17권8호
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• pp.1249-1253
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• 2007

A hyperthermostable endoglucanase from Pyrococcus horikoshii with the capability of hydrolyzing crystalline cellulose was analyzed. A protein engineering study was carried out to obtain a reduced-size mutant. Five amino acid residues at both the N- and C-terminus were found to be removable without any loss of activity or thermal stability. Site-directed mutagenesis was also performed on R102, N200, E201, H297, Y299, E342, and W377, residues possibly involved in the active center or in the recognition and binding of a cellulose substrate. The activity of the resulting mutants was considerably decreased, confirming that the mutated residues were all important for activity. A reduced-size enzyme, as active as the wild-type endoglucanase, was successfully obtained, plus the residues critical for its activity and specificity were confirmed. Consequently, an engineered enzyme with a reduced size was obtained, and the amino acids essential for activity were confirmed by site-directed mutagenesis and comparison with a known three-dimensional structure.

• BMB Reports
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• 제32권6호
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• pp.547-553
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• 1999

Aminoacyl-tRNA synthetases are essential enzymes catalyzing the attachment of specific amino acids to cognate tRNAs. In the present work, the substrate analogue L-methionine hydroxamate was used to identify functional residues located in the active site of the E. coli methionyl-tRNA synthetase (MetRS). This compound inhibited bacteria, yeast, and human MetRS activities to a similar degree, suggesting a conserved active site structure and mechanism between MetRSs of different phylogenetic domains. Mutants of the E. coli MetRS resistant to methionine hydroxamate were also isolated. These mutants contained a substitution either at T10, Y15, or Y94. These residues are highly conserved among the different MetRSs and the mutants showed decreased aminoacylation activity, suggesting their functional and structural significances. The putative roles of these residues are discussed on a structural basis.

• 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.

• Bulletin of the Korean Chemical Society
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• 제25권5호
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• pp.711-714
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• 2004

Peroxynitrite enters erythrocytes through band 3 anion exchanger and oxidizes cytosolic proteins therein. As a protein associated with band 3, carbonic anhydrase II may suffer from peroxynitrite-induced oxidative damages. Esterase activity of carbonic anhydrase II decreased as the concentration of peroxynitrite increased. Neither hydrogen peroxide nor hypochlorite affected the enzyme activity. Inactivation of the enzyme was in parallel with the release of zinc ion, which is a component of the enzyme's active site. SDS-PAGE of peroxynitrite-treated samples showed no indication of fragmentation but non-denaturing PAGE exhibited new bands with lower positive charges. Western analysis demonstrated that nitration of tyrosine residues increased with the peroxynitrite concentration but the sites of nitration could not be determined. Instead MALDI-TOF analysis identified tryptophan-245 as a site of nitration. Such modification of tryptophan residues is responsible for the decrease in tryptophan fluorescence. These results demonstrate that peroxynitrite nitrates tyrosine and tryptophan residues of carbonic anhydrase II without causing fragmentation or dimerization. The peroxynitrite-induced inactivation of the enzyme is primarily due to the release of zinc ion in the enzyme's active site.

• Bulletin of the Korean Chemical Society
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• 제33권9호
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• pp.2981-2984
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• 2012

Active site mapping has been done for ${\Delta}^5$-3-ketosteroid isomerase (KSI) by analyses of paramagnetic effect on $^1H-^{15}N$ HSQC spectra using 4-hydroxyl-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO) and an intermediate analog (equilenin). Our result revealed that residues in hydrophobic cavity of KSI, particularly active site region, mainly experienced a high line-broadening effect of NMR signal with HyTEMPO, while they experienced full recovery of a lineshape upon the addition of equilenin. The mapped region was very similar to the active site of KSI as described by the crystal structure. These observations indicate that a combined use of paramagnetic reagent and substrate (or analog) could rapidly identify the residues in potential active site of KSI, and can be applied to the analysis of both active site and function in unknown protein.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2003년도 제2차 연례학술대회 발표논문집
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• pp.277-287
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• 2003

Acetolactate synthase (ALS, EC 4.1.3.18; also referred to as acetohydroxy acid synthase) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine in microorganisms and plants. Recently X-ray structure of yeast ALS was available. Pair-wise alignment of yeast and tobacco ALS sequences revealed 63% sequence similarity. Using Deep View and automatic modeling on Swiss model server, we have generated reliable models of tobacco ALS based on yeast ALS template with a calculated pair-wise RMSD of 0.86 Angstrom. Functional roles of four residues located on the subunit interface (H142, El43, M350, and R376) were examined by site-directed mutagenesis. Seven mutants were generated and purified, of which three mutants (H142T, M350V, and R376F) were found to be inactivated under various assay conditions. The H142k mutant showed moderately altered kinetic properties. The E143A mutant increased 10-fold in K$_m$ value while other parameters remained unchanged. The M350C mutant was strongly resistant to three tested herbicides, while the R376k mutant can bind with herbicide carder at similar affinity to that of wild type enzyme, as determined by tryptophan quenching study. Except M350V mutant, all other mutants were ate to bind with cofactor FAD. Taken together, it is likely that residues H142 and E143 are located at the active site, while residues M350 and R376 are possibly located at the overlapping region of active site and herbicide binding site of the enzyme. Our data also allows us to hypothesize that the interaction between side chains of residues M350 and R376 are probably essential for the correct conformation of the active site. It remains to be elucidated that, whether the herbicide, upon binding with enzyme, inactivates the enzyme by causing change in the active site allosterically, which is unfavorable for catalytic activity.

• Journal of Microbiology and Biotechnology
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• 제9권4호
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• pp.381-385
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• 1999

Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in a polymerase chain reaction. Taq DNA polymerase has a domain at the amino terminus (residues 1 to 290) that has 5'-3' exonuclease activity and a domain at the C-terminus that catalyzes the polymerase reaction. Taq DNA polymerase is classified into the Pol I family, which is represented by E. coli DNA polymerase I. The alignment of amino acid sequences for the 5'-3' exonuclease domains of the Pol I family DNA polymerases shows ten highly conserved carboxylic amino acids. Crystallographic studies suggested that six of the carboxylic amino acids are clustered within a 7 $\AA$ radius by chelating three metal ions in the active site. Those six carboxylic residues are mutagenized to alanines in order to better understand their function. All six carboxylic residues, Asp l8, Glu1l7, Asp1l9, Asp120, Asp142, and Aspl44, are crucial for catalysis of 5'-3' exonuclease.

• Bulletin of the Korean Chemical Society
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• 제33권12호
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• pp.4169-4172
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• 2012

To elucidate the roles of cysteine residues in rice Phi-class GST F3, in this study, all three cysteine residues were replaced with alanine by site-directed mutagenesis in order to obtain mutants C22A, C73A and C77A. Three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The substitutions of Cys73 and Cys77 residues in OsGSTF3 with alanine did not affect the glutathione conjugation activities, showing non-essentiality of these residues. On the other hand, the substitution of Cys22 residue with alanine resulted in approximately a 60% loss of specific activity toward ethacrynic acid. Moreover, the ${K_m}^{CDNB}$ value of the mutant C22A was approximately 2.2 fold larger than that of the wild type. From these results, the evolutionally conserved cysteine 22 residue seems to participate rather in the structural stability of the active site in OsGSTF3 by stabilizing the electrophilic substrates-binding site's conformation than in the substrate binding directly.

• BMB Reports
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• 제31권5호
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• pp.498-502
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• 1998

HBV polymerase shares several regions of amino acid homology with other DNA-directed and RNA-directed polymerases. The amino acid residues $Asp^{429}$, $Gly^{518}$, $Asp^{551}$, $Lys^{585}$, and $Gly^{641}$ in the conserved motifs A, B', C, D, and E in the polymerase domain of HBV polymerase were mutated to alanine or histidine by in vitro site-directed mutagenesis. Those mutants were overexpressed, purified, and analyzed against DNA-dependent DNA polymerase activity and affinity for DNA binding. All those mutants did not show DNA-dependent DNA polymerase activities indicating that those five amino acid residues are all critical in DNA polymerase activity. South-Western analysis shows that amino acid residues $ASp^{429}$ and $ASp^{551}$ are essential to DNA binding, and $Gly^{318}$ and $Gly^{585}$ also affect DNA binding to a certain extent.

• 한국미생물·생명공학회지
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• 제34권2호
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• pp.121-128
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• 2006

Bacillus alcalophilus AX2000으로부터 xylanase를 정제한 후 효소의 활성부위를 조사하기 위하여 여러 가지 화학수식제를 사용하여 효소활성의 저해도를 측정하였다. 여러 가지 화학 수식제 중에서 carbodiimide와 N-bromosuccinimide가 효소 활성을 완전히 저해시켜 glutamic acid또는 aspartic acid 잔기와 tryptophan 잔기가 효소의 활성부위에 관여하리라 추측되었다. 각각의 경우에 효소 실활은 수식제의 첨가농도에 따라 pseudo first-order kinetics 양식을 보여주었으며, carbodiimide와 N-bromosuccinimide는 각각 비경쟁적 저해와 경쟁적 저해방식을 나타내었다. 기질첨가에 의한 효소활성 보호실험을 통하여 tryptophan 잔기가 기질결합부위라 판단 되었다. 효소 실활속도의 분석에 의해 효소활성에는 2개의 glutamic acid 또는 aspartic acid 잔기와 1개의 tryptophan 잔기가 관여하는 것으로 나타났다.