• 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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• 제29권1호
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• pp.173-176
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• 2008

MJ0684 from Methanococcus jannaschii is a hypothetical protein belonging to the subfamily Ig of amino acid aminotransferases. In the present study, the crystal structure of MJ0684 has been determined at 2.2 resolution. It reveals that MJ0684 has an overall structure similar to subfamily Ig aminotransferases and its active site architecture is most similar to that of kynurenine aminotransferases among several kinds of aminotransferases in the subfamily Ig?. It has two hydrophobic active site residues conserved in the kynurenine aminotransferases for recognizing hydrophobic substrates. In addition, the absence of any basic residue for recognizing the side chain carboxylic group of the aspartate in the active site rules out the possibility that MJ0684 would act as an aspartate aminotransferase. These structural observations collectively imply that MJ0684 is a novel archaeal homolog of the subfamily Ig kynurenine aminotransferase.

• KSBB Journal
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• 제6권2호
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• pp.157-166
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• 1991

The structural properties of cellulose are significantly changed with the progress of hydrolysis reaction. The effects of changes on such properties of cellulosic substrate as crystallinity, amicessibility of enzyme to the active site of cellulose surface, and particle size on the kinetics of enzymatic hydrolysis have been studied. Among those physical studies, the apparent surface active site of cellulose particle was found to have the most significant effect on the hydrolysis kinetics. Based on the experimental results, the adsorption affinity of enzyme and hydrolysis rate were mainly influenced by the surface roughness of cellulose particle. The extent of accesssible active site may be expressed as the change of particle diameter. The Langmuir isotherm was proposed in terms of enzyme activity to explain the actual action of enzyme protein.

• Molecules and Cells
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• 제39권1호
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• pp.53-59
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• 2016

Peroxiredoxins are cysteine-dependent peroxide reductases that group into 6 different, structurally discernable classes. In 2011, our research team reported the application of a bioinformatic approach called active site profiling to extract active site-proximal sequence segments from the 29 distinct, structurally-characterized peroxiredoxins available at the time. These extracted sequences were then used to create unique profiles for the six groups which were subsequently used to search GenBank(nr), allowing identification of ~3500 peroxiredoxin sequences and their respective subgroups. Summarized in this minireview are the features and phylogenetic distributions of each of these peroxiredoxin subgroups; an example is also provided illustrating the use of the web accessible, searchable database known as PREX to identify subfamily-specific peroxiredoxin sequences for the organism Vitis vinifera (grape).

• Restorative Dentistry and Endodontics
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• 제25권4호
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• pp.587-598
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• 2000

Urea in the oral cavity is hydrolyzed mainly by bacterial ureases to ammonia, which in turn, raises pH of the oral environment, maintaining oral pH homeostasis, thereby inhibiting dental caries. Streptococcus salivarius has been shown to be a major contribution to oral ureolysis. Synthesis of urease by S. salivarius appears to be constitutive, but can be greatly enhanced in the acidic environment. It has been presumed that ureolytic activity of S. salivarius strains isolated from caries-active site is greater than that of strains from caries-free site. However, no in vivo study has supported the presumption. The present study was performed to observe the ureolytic activity of S. salivarius strains isolated from different environments in the same individual, finding out whether the ureolytic activity is related to dental caries. For the purpose, S. salivarius strains were isolated from caries-active site (>C2), a caries-free site of the tooth, and the dorsum of the tongue of each of 50 patients having decayed teeth. The strains isolated from the patients who harbored S. salivarius in more than two sites were selected and then their ureolytic activities were measured. In order to examine clonal diversity of the strains, their ureC genes were amplified by polymerase chain reaction (PCR) and then restricted with EcoRV, and the protein profiles of the strains were compared by SDS-PAGE. The results were as follows: 1. Of 50 patients, 13 patients harbored S. salivarius in more than two sites; a total of 61 S. salivarius strain were isolated from the patients and selected for the study. 2. Of 17 isolates from the caries-active site of 9 patients harboring S. salivarius in more than two sites including carious lesion, 10 (58.8%) showed a high ureolytic activity (> 200 ${\mu}mol/min/mg$). While, 19 out of 44 isolates (43.2%) from the caries-free site of the teeth and the dorsum of the tongues of 13 patients were the strains with a high ureolytic activity. 3. Of 9 patients harboring S. salivarius in more than two sites including caries-active site. 6 patients were found to have the strains in the caries-active site showing a lower ureolytic activity than the strains in the other sites. 4. Of 34 isolates with ureolytic activity higher than 40 ${\mu}mol/min/mg$, 32 isolates produced 0.54-Kbp PCR products regardless of the sites of bacterial collection. In contrast, of 27 isolates with ureolytic activity lower than 40${\mu}mol/min/mg$, 26 isolates yielded 1.3-Kbp PCR products or none regardless of the sites. 5. Different clonal types of S. salivarius with relatively higher and lower ureolytic activities were found in the same individuals and even in the same sites. 6. None of strains showing different ureolytic activity appeared to be the same clonal type. The overall results suggest that ureolytic activity of the isolates does not appear to be related to differences of the environments but related to their own genetic traits.

• BMB Reports
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• 제28권1호
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• pp.72-78
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• 1995

Time-dependent inactivation of E. coli GTP cyclohydrolase I with a 2',3'-dialdehyde derivative of GTP (oGTP) was directed to the active site of the enzyme, and was dependent on the concentration of oGTP. The kinetics of inactivation were biphasic with a rapid reaction occurring immediately upon exposure of the enzyme to oGTP followed by a slow rate of inactivation. The $K_i$ value of oGTP for the enzyme was 0.25 mM. Inactivation was prevented by preincubation of the enzyme with GTP, the substrate of the enzyme. At 100% inactivation, 2.3 mol of [8.5'-$^3H$]oGTP were bound per each enzyme subunit, which consists of two identical polypeptides. The active site residue which reacted with the affinity label was lysine. oGTP interacted selectively with the ${\varepsilon}$-amino group of lysine in the GTP-binding site to form a morpholine-like structure which was stable without sodium borohydride treatment. However, triphosphate group was eliminated during the hydrolysis step. To identify the active site of the enzyme, [8.5'-$^3H$]oGTP-labeled enzyme was cleaved by endoproteinase Lys-C, and the $^3H$-labeled peptide was purified by HPLC. The amino acid sequence of the active site peptide was Pro-Ser-Leu-Ser-Lys, which corresponds to the aminoterminal sequence of the enzyme.

• BMB Reports
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• 제28권1호
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• pp.51-56
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• 1995

Thioredoxin is a small redox protein with an active-site disulfide/dithiol, and is ubiquitous in bacteria, plants, and animals. To investigate the structure-function relationship of thioredoxin, the genes encoding Escherichia coli thioredoxin and Corynebacterium nephridii thioredoxin C3 were fused via a common restriction site in the nucleotide sequence coding for the active site of the proteins to generate two chimeric thioredoxins, designated E-C3(N to C-terminal) and C3-E. The hybrid thioredoxin genes were put under the T7 promoter and their productions were confirmed. The two hybrid thioredoxins complemented phenotypes of a thioredoxin-deficient E. coli strain. A strain containing the C3-E hybrid thioredoxin supported growth of the T7 phage, whereas a strain expressing the E-C3 hybrid thioredoxin did not. However, both hybrids supported growth of M13 phages. The two hybrid thioredoxins were also characterized in other aspects. Differences in activity between the hybrid thioredoxins were attributed to altered interactions of the N- and C-terminal domains of the molecule, which produced changes in the three-dimensional structure of the active site region.

• Archives of Pharmacal Research
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• 제29권11호
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• pp.1006-1017
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• 2006

A series of 3-(substituted-benylidene)-1, 3-dihydro- indolin-2-one, 3-(substituted-benylidene)-1, 3-dihydro- indolin-2-thione and 2, 2'-dithiobis 3-(substituted-benylidene)-1, 3-dihydro-indole derivatives was investigated as inhibitor of $p60^{c-Src}$tyrosine kinase by performing receptor docking studies and inhibitory activity toward tyrosine phosphorylation. Some compounds were shown to be docked at the site, where the selective inhibitor PP1 [1-tert-Butyl-3-p-tolyl-1H-pyrazolo[3,4-d]pyrimidine-4-yl-amine] was embedded at the enzyme active site. Evaluation of all compounds for the interactions with the parameters of lowest binding energy levels, capability of hydrogen bond formations and superimposibility on enzyme active site by docking studies, it can be assumed that 3-(substituted-benzylidene)-1, 3-dihydro-indolin-2-one and thione derivatives have better interaction with enzyme active site then 2, 2'-dithiobis 3-(substituted-benzylidene)-1, 3-dihydro indole derivatives. The test results for the inhibitory activity against tyrosine kinase by Elisa method revealed that 3-(substituted-benylidene)-1, 3-dihydro- indolin-2-thione derivatives have more activity then 3-(substituted-benylidene)-1, 3-dihydro- indolin-2-one derivatives.

• Toxicological Research
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• 제15권1호
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• pp.95-101
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• 1999

Cytochrome P450 (CYP) 3A4 metabolizes aflatoxin B1 (AFB1) to AFB1-exo-8,9-epoxide (8,9-epoxidation) and aflatoxin Q1 (AFQ1; 3$\alpha$-hydroxylation) simultaneously. We investigated whether each metabolite was formed via its own binding site of CAP3A4 active site. Kinetics of the formation of the two metabolites were sigmoidal and consistent with the kinetics of substrate activation. The HIll model predicted that two substrate binding wites are involved in the oxidationof AFB1 by CYP3A4. Dehydronifedipine, a metabolite of nifedipine generated by CYP3A4, inhibited the formation of AFQ1 without any inhibition in the formation of AFB1-exo-8,9-epoxidation. Dehydronifedipine was found to act as a reversible competitive inhibitor against 3$\alpha$-hydroxylation of AFB1. Vmax and S0.5 of the 8,9-epoxidation were not changed in the presence of 0, 50, or 100 $\mu\textrm{M}$ dehydronifedipine. S0.5 of 3$\alpha$-hydroxylation was increased from 58$\pm$4 $\mu\textrm{M}$ to 111$\pm$8 $\mu\textrm{M}$ in the presence of 100 $\mu\textrm{M}$ nifedipine whereas Vmax was not changed. These results suggest that there exist two independent binding sites in the active site of CAP3A4 . One binding site is responsible for AFB1-exo-8,9-epoxidation and the other is involved in 3$\alpha$-hydroxylation of AFB1. Dehydronifedipine might selectively bind to the site which is responsible for the formation of AFQ1 in the active site of CYP3A4.

• BMB Reports
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• 제35권4호
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• pp.437-441
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• 2002

Dihydrolipoamide dehydrogenase (E3) is a member of the pyridine nucleotide-disulfide oxidoreductase family. Thr residues are highly conserved. They are at the active site disulfide-bond regions of most E3s and other oxidoreductases,. The crystal structure of Azotobacter vinelandii E3 suggests that the hydroxyl group of Thr that are involved in the FAD binding interact with the adenosine phosphate of FAD. However, several prokaryotic E3s have Val instead of Thr. To investigate the meaning and importance of the Thr conservation in many E3s, the corresponding residue, Thr-44, in human E3 was substituted to Val by site-directed mutagenesis. The mutant’s E3 activity showed about a 2.2-fold decrease. Its UV-visible and fluorescence spectra indicated that the mutant might have a slightly different microenvironment at the FAD-binding region.