• 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.

• Journal of Microbiology and Biotechnology
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• v.18 no.3
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• pp.465-471
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• 2008

Highly active, stable, and magnetically separable immobilized enzymes were developed using carboxymethyl cellulose (CMC) and diethylaminoethyl cellulose DEAE-C; hereafter designated "DEAE" as supporting materials. Iron oxide nanoparticles penetrated the micropores of the supporting materials, rendering them magnetically separable. Lipase (LP) was immobilized on the surface of the supporting materials by using cross-linked enzyme aggregation (CLEA) by glutaraldehyde. The activity of enzyme aggregates coated on DEAE was approximately 2 times higher than that of enzyme aggregates coated on CMC. This is explained by the fact that enzyme aggregates with amine residues are more efficient than those with carboxyl residues. After a 96-h enantioselective ibuprofen esterification reaction, 6% ibuprofen propyl ester was produced from the racemic mixture of ibuprofen by using DEAE-LP, and 2.8% using CMC-LP.

• 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.

• Journal of the Korean Chemical Society
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• v.14 no.2
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• pp.161-168
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• 1970

In order to obtain the more effective evidence, supporting the hypothesis which have been previously described by former report that pepsin (EC 3.4. 4.1) forms a hydrophobic bond with the nonpolar side chain of its substrate, the inhibitory effect of carboxylic acids(from formic acid to iso-butyric acid) on the activity of pepsin to the synthetic dipeptide, N-Carbobenzoxy-L-glutamyl-L-tyrosine, was discussed. The kinetic study showed that the inhibition by carboxylic acids was competitive. The Kidecreased with increasing size of the inhibitor molecule. The $-{\Delta}F^{\circ}$increased linearly with increasing number of carbon atoms in the hydrocarbon chain of the inhibitor. It was confirmed that the hydrophobic bond between more than one side chain of amino acid residues(phenylalanine) in the binding region of the active center of pepsin and the side chain of amino acid residues in the substrate was formed as the first step of its enzymic mechanism. The inhibitory effect of carboxylic acids was due to the competition of the hydrocarbon group of the carboxylic acids with the side chain of the substrate for the hydrophobic binding site(the side chain of phenylalanine) of the pepsin.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.36 no.5
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• pp.442-448
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• 2003

Melanin concentrating hormone (MCH) regulating color change of fish skin was identified from brain cDNA library of Olive flounder (Paralichthys olivaceus) during the analysis of Expressed Sequence Tags (ESTs). Olive flounder MCH gene consisted of 598 nucleotides encoding 150 amino acids. Olive flounder MCH protein revealed to contain signal peptide of 19 amino acid residues, pro-MCH of 131 amino acids being processed to biologically active and mature form of hormone with 25 amino acid residues at the carboxyl terminus. A comparative structural analysis revealed that Olive flounder MCH precursor had low sequence identity with other fish species and mammalian counterparts, while the amino acid sequences of mature hormone had a relatively high identity and more conserved. RT-PCR analysis revealed that olive flounder MCH precersor gene was expressed spectically only in the brain and not in other tissues.

• Journal of Microbiology
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• v.39 no.4
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• pp.338-342
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• 2001

Pseudomonas fluorescens SM11 is a naphthalene-degrading strain whose dissimilatory genes are cho-mosomally encoded. We have cloned the 2.9 kb Sal I fragment harboring genes for the naphthalene-degrading upper pathway. The nucleotide sequences were determined to be nahQ, napA, and partial regions of nahE genes. The nahQ encods a protein of 188 amino acid residues with a deduced molec-ular wight of 20.8kDa. The high homology with other proteins suggests that NAhQ may be an active and useful protein whtich gives as selective advantage to naphthalene degradatin. Transposase(TnpA)encodes a polypetide chain with a molecular mass of 41.8kDa consisting of 376 amino acid residues. The deduced anino acid sequence of tnpA revealed 96% idenitity with putative transposase of P. stutzeri OX1,. It was assumed that transposase plays an important role in the evloution of the catabloic-path way in the regulation of nah expression.

• Bulletin of the Korean Chemical Society
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• v.29 no.1
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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.

• 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.

• Journal of Photoscience
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• v.9 no.2
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• pp.281-283
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• 2002

S-modulin in frog or its bovine homologue, recoverin, is a 26 kDa EF-hand $Ca^{2+}$-binding protein found in rod photoreceptors. The $Ca^{2+}$ -bound form of S-modulin binds to rhodopsin kinase (Rk) and inhibits its activity. Through this regulation, S-modulin is believed to modulate the light-sensitivity of a rod. In the present study, we tried to identify the interaction site of the $Ca^{2+}$ -bound form of S-modulin to Rk. First, we mapped roughly the interaction regions by using partial peptides of S-modulin. The result suggested that a specific region near the amino terminus is the interaction site of S- modulin. We then identified the essential amino acid residues in this region by using S-modulin mutant proteins: four amino acid residues were suggested to interact with Rk. These residues are located in a small closed pocket in the $Ca^{2+}$-free, inactive form of S-modulin, but exposed to the surface of the molecules in the $Ca^{2+}$ -bound, active form of S-modulin. Two additional amino acid residues were found to be crucial for the $Ca^{2+}$ -dependent conformational changes of S-modulin. The present study firstly identified the functional site of S-modulin, a member of a neuronal calcium sensor protein family.in family..

• BMB Reports
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• v.38 no.4
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• pp.474-480
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• 2005

Curcumin, a major active component of turmeric, has been identified as an inhibitor of the transcriptional activity of activator protein-1 (AP-1). Recently, it was also found that curcumin and synthetic curcumin derivatives can inhibit the binding of Jun-Fos, which are the members of the AP-1 family, to DNA. However, the mechanism of this inhibition by curcumin and its derivatives was not disclosed. Since the binding of Jun-Fos dimer to DNA can be modulated by redox control involving conserved cysteine residues, we studied whether curcumin and its derivatives inhibit Jun-Fos DNA binding activity via these residues. However, the inhibitory mechanism of curcumin and its derivatives, unlike that of other Jun-Fos inhibitors, was found to be independent of these conserved cysteine residues. In addition, we investigated whether curcumin derivatives can inhibit AP-1 transcriptional activity in vivo using a luciferase assay. We found that, among the curcumin derivatives examined, only inhibitors shown to inhibit the binding of Jun-Fos to DNA by Electrophoretic Mobility Shift Assay (EMSA) inhibited AP-1 transcriptional activity in vivo. Moreover, RT-PCR revealed that curcumin derivatives, like curcumin, downregulated c-jun mRNA in JB6 cells. These results suggest that the suppression of the formation of DNA-Jun-Fos complex is the main cause of reduced AP-1 transcriptional activity by curcuminoids, and that EMSA is a suitable tool for identifying inhibitors of transcriptional activation.