• BMB Reports
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• v.33 no.1
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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.

• The KIPS Transactions:PartD
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• v.14D no.7
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• pp.743-752
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• 2007

Proteins combine with other materials to achieve their function and have similar function if their active sites are similar. Thus we can infer the function of protein by identifying the binding area of proteins. This paper suggests the novel method to select binding area of protein using MCL (Markov Cluster) algorithm. We construct the distance matrix from surface residues distance on protein. Then this distance matrix is transformed to connectivity matrix for applying MCL process. We adopted Catalytic Site Atlas (CSA) data to evaluate the proposed method. In the experimental result using CSA data (94 selected single chain proteins), our algorithm detects the 91 (97%) binding area near by active site of each protein. We introduced a new geometrical features and this mainly contributes to reduce the time to analyze the protein by selecting the residues near by active site.

• BMB Reports
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• v.41 no.12
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• pp.881-885
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• 2008

Protein phosphatase 1 consists of a secondary structure arrangement, conserved in the serine/threonine protein phosphatase gene family, flanked by nonconserved N-terminal and C-terminal domains. The deletion mutant of PP1 with the 8 nonconserved N-terminal residues removed was designated PP1-(9-330). PP1-(9-330) had a higher activity and affinity than PP1 when assayed against four different substrates, and it also demonstrated a 6-fold higher sensitivity to the inhibitor okadaic acid. This suggested that the N-terminal domain suppresed the activity of PP1 and interfered with its inhibition by okadaic acid. The ANS fluorescence intensity of PP1-(9-330) was greater than that of PP1, which implies that the hydrophobic groove running from active site in the truncated PP1 was more hydrophobic than in PP1. Our findings provide evidence that the nonconserved N-terminus of PP1 functions as an important regulatory domain that influences the active site and its pertinent properties.

• Bulletin of the Korean Chemical Society
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• v.24 no.6
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• pp.817-823
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• 2003

We have performed ab initio calculation on the active site of HIV-1 protease. The FEP method was used to determine the binding free energy of four different of protonated states of HIV-1 protease with inhibitor. The structure of the active site and hole structure was taken from the X-ray crystallographic coordinates of the C₂ symmetric inhibitor A74704 protease bound. The active site was modeled with the fragment molecules of binding pocket, acetic acid/ acetate anion (Asp25, Asp125), formamide (amide bond of Thr26/Gly27, Thr126/ Gly127), and methanol as inhibitor fragment. All possibly protonated states of the active site were considered, which were diprotonated state (0, 0), monoprotonated (-1, 0),(0, -1) and diunprotonated state (-1, -1). Once the binding energy Debind, of each model was calculated, more probabilistic protonated states can be proposed from binding energy. From ab-initio results, the FEP simulations were performed for the three following mutations: Ⅰ) Asp25 … Asp125 → AspH25 … Asp125, ⅱ) Asp25 … Asp125 → Asp25 … AspH125, ⅲ) AspH25 … Asp125 → AspH25 … AspH125. The free energy difference between the four states gives the information of the more realistic protonated state of active site aspartic acid. These results provide a theoretical prediction of the protonation state of the catalytic aspartic residues for A74707 complex, and may be useful for the evaluation of potential therapeutic targets.

• BMB Reports
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• v.32 no.4
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• pp.326-330
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• 1999

An aspartase purified from Hafnia alvei was inactivated by diethylpyrocarbonate (DEP) in a pseudo-first-order inactivation. The first-order plot was biphasic. The inactivation process was not saturable and the second order rate constant was $1.3\;M^{-1}s^{-1}$. The inactivated aspartase was reactivated with NH₂OH. The difference absorption spectrum of DEP-inactivated vs native enzyme preparations revealed a marked peak around 242 nm. The pH dependence of the inactivation rate suggests that an amino acid residue having a pK value of 7.2 was involved in the inactivation. L-aspartate, fumarate (substrates), and chloride ion (inhibitor) protected the enzyme against inactivation, indicating that histidine residues for the enzyme activity are located at the active site of this aspartase. Inspection of the presence and absence of $Cl^-$ ion demonstrated that the number of essential histidine residues is less than two. Thus, one or two histidines are in or near the aspartate binding site and participate in an essential step of the catalytic reaction.

• BMB Reports
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• v.33 no.1
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• pp.43-48
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• 2000

4-Aminobutyrate aminotransferase is a key enzyme of the 4-aminobutyric acid shunt. It converts the neurotransmitter 4-aminobutyric acid to succinic semialdehyde. In order to study the structural and functional aspects of catalytically active Cys residues of pig brain 4-aminobutyrate aminotransferase, we purified the active form in E. coli by coproduction of thioredoxin. The structural arrangement for functional requirements of a dimeric protein using a bifunctional sultbydryl reagent was then characterized, and the spatial proximity between the essential SH groups and a cofactor (pyridoxal-5'-phosphate) binding site was determined. The bifunctional sultbydryl reagent DMDS reacted with the enzyme at the ratio of one molecule per enzyme dimer. This resulted in an approximately 50% loss of enzymatic activity. The spatial proximity of the distance between the essential SH groups and the cofactor-binding site was determined by the energy transfer measurement technique. The result (approximate 20 ${\AA}$) suggested that cross-linking of two sulfhydryl groups with DMDS is not near a PLP binding site.

• BMB Reports
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• v.32 no.5
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• pp.515-520
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• 1999

Incubation of two types of glutamate dehydrogenase isoproteins from bovine brain with the arginine-specific dicarbonyl reagent phenylglyoxal resulted in a biphasic loss of enzyme activity. Reaction of the glutamate dehydrogenase isoproteins with phenylglyoxal caused a rapid loss of 53~62% of the enzyme activities and modification of two residues of arginine per enzyme subunit. Prolonged incubation of the glutamate dehydrogenase isoproteins with phenylglyoxal resulted in the modification of an additional four residues of arginine per enzyme subunit without further loss of the residual activities. Partial protection against inactivation was provided by the coenzyme NADH or substrate 2-oxoglutarate. The most marked decrease in the rate of inactivation was observed by the combined addition of NADH and 2-oxoglutarate, suggesting that the first two modified arginine residues are in the vicinity of the catalytic site. However, inactivation of the glutamate dehydrogenase isoproteins by phenylglyoxal appears to be partial with approximately 40% activity remained after an extended reaction time with excess reagent, suggesting that the modified arginine residues may not be directly involved in catalysis. The lack of complete protection by substrates also suggest the possibility that the modified arginine residues are not directly involved at the active site, and the partial loss of activity by the modification of arginine residues may be due to a conformational change. There were no significant differences between the two glutamate dehydrogenase isoproteins in sensitivities to inactivation by phenylglyoxal, indicating that the microenvironmental structures of the glutamate dehydrogenase isoproteins are very similar to each other.

• BMB Reports
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• v.49 no.6
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• pp.349-354
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• 2016

The archaeon Sulfolobus solfataricus P1 carboxylesterase is a thermostable enzyme with a molecular mass of 33.5 kDa belonging to the mammalian hormone-sensitive lipase (HSL) family. In our previous study, we purified the enzyme and suggested the expected amino acids related to its catalysis by chemical modification and a sequence homology search. For further validating these amino acids in this study, we modified them using site-directed mutagenesis and examined the activity of the mutant enzymes using spectrophotometric analysis and then estimated by homology modeling and fluorescence analysis. As a result, it was identified that Ser151, Asp244, and His274 consist of a catalytic triad, and Gly80, Gly81, and Ala152 compose an oxyanion hole of the enzyme. In addition, it was also determined that the cysteine residues are located near the active site or at the positions inducing any conformational changes of the enzyme by their replacement with serine residues.

• Journal of Integrative Natural Science
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• v.11 no.2
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• pp.121-129
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• 2018

CXCR6 is a major target in drug design as it is a determinant receptor in many diseases like AIDS, Type I Diabetes, some cancer types, atherosclerosis, tumor formation, liver disease and steatohepatitis. In this study, we propose the active site residues of CXCR6 molecule. We employed homology modelling and molecular docking approach to generate the 3D structure for CXCR6 and to explore its interaction between the antagonists and agonists. 3D models were generated using 14 different templates having high sequence identity with CXCR6. Surflex docking studies using pyridine and pyrimidine derivatives enabled the analysis of the binding site and finding of the important residues involved in binding. 3D structure of CXCL16, a natural ligand for CXCR6, was modelled using PHYRE and protein - protein docking was performed using ClusPro. The residues which were found to be crucial in interaction with the ligand are THR110, PHE113, TYR114, GLN160, GLN195, CYS251 and SER255. This study can be used as a guide for therapeutic studies of human CXCR6.

• Bulletin of the Korean Chemical Society
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• v.22 no.1
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• pp.77-83
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• 2001

To gain further insight into the relationship between structure and function of glutathione S-transferase (GST), the four cysteine mutants, C14S, C47S, C101S and C169S, of human GST P1-1 were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized glutathione (GSH). The catalytic activities of the four mutant enzymes were characterized with five different substrates as well as by their binding to four different inhibitors. Cys14 seems to participate in the catalytic reaction of GST by stabilizing the conformation of the active-site loop, not in the GSH binding directly. The substitution of Cys47 with serine significantly reduces the affinity of GSH binding, although it does not prevent GSH binding. On the other hand, the substitution of Cys101 with serine appears to change the binding affinity of electrophilic substrate by inducing a conformational change of the $\alpha-helix$ D. Cys169 seems to be important for maintaining the stable conformation of the enzyme. In addition, all four cysteine residues are not needed for the steroid isomerase activity of human glutathione S-transferase P1-1.