• Bulletin of the Korean Chemical Society
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• v.24 no.5
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• pp.627-632
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• 2003

The pH dependence of the kinetic parameters of recombinant acetohydroxy acid synthase catalyzed reaction was determined in order to obtain information about the chemical mechanism, particularly acid-base chemistry. The maximum velocity and V/K for pyruvate were bell-shaped with estimated pK values of 6.5-6.7 and 8.6-8.9, respectively. The maximum velocity and V/K for 2-ketobutyrate were also bell-shaped with estimated pK values of 6.6-7.0 and 8.4-8.6. The pH dependence of 1/Ki for 3-bromopyruvate, a competitive inhibitor of pyruvate, was also bell-shaped, giving pK values almost identical with those obtained for pyruvate. Since the same pK values were observed in the $pK_{i 3-bromopyruvate}$, V/K pH profiles and $V_{max}$ profiles, both enzyme groups must be in their optimum protonation state for efficient binding of reactants. These results reflect that two enzyme groups are necessary for binding of substrate and/or catalysis.

• Bulletin of the Korean Chemical Society
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• v.25 no.1
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• pp.115-118
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• 2004

We have examined the feasibility of using the specific interaction between mistletoe lectin I (ML I) and ${\beta}$-Dgalactose instead of the anti-ML I antibody in developing a homogeneous type competitive binding assay for ML I. We also have examined the feasibility of adapting the biotin/avidin mediated homogeneous assay for this system. Alkaline phosphatase (AKP) was employed as a single substrate enzyme label. The dose-response curve shows a detection range of 1-25 ${\mu}$g/mL and a linear response with a correlation coefficient of 0.99. To demonstrate the analytical utility of this method, 10 ${\mu}$g/mL of ML I was spiked into distilled water. The results show that the mean recovery was 10.03 ${\mu}$g/mL with an SD of 0.18. The difference between the spiked value and the mean recovery was 0.03 ${\mu}$g/mL, with a relative error of 0.3 and 1.6 % of RSD.

• Journal of Sensor Science and Technology
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• v.22 no.5
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• pp.346-351
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• 2013

We developed a one-step method for fabrication of addressable suspended SWNT films and demonstrate excellent detection performance of paraoxon based on OPH-immobilized SWNT films for environmental monitoring. For dispersed SWNT suspension, COOH-SWNT was prepared by the oxidation of carbon nanotubes using acid treatment and sonication. Suspended SWNT-film was fabricated between cantilever electrodes by dielectrophoretic force and surface tension of the water meniscus. After that, OPH were immobilized on suspended SWNT-films by nonspecific binding for enzymatic hydrolysis of paraoxon. The electrical properties of the SWNT films were measured in real time at room temperature. Structurally suspended SWNT films from substrate surface made possible rapid and highly sensitive detection of target molecules with increased convectional and diffusional fluxes of the molecules and with a large binding surface area. SWNT film FET resulted in a real-time, label-free, and electrical detection of paraoxon to the concentration of ca. $10{\mu}m$ with a step-wise rapid response time of several seconds.

• Biomolecules & Therapeutics
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• v.22 no.6
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• pp.497-502
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• 2014

In the present study, we found that the natural compound arctigenin inhibited hydrogen peroxide-induced reactive oxygen species (ROS) production in rat primary astrocytes. Since hemeoxygenase-1 (HO-1) plays a critical role as an antioxidant defense factor in the brain, we examined the effect of arctigenin on HO-1 expression in rat primary astrocytes. We found that arctigenin increased HO-1 mRNA and protein levels. Arctigenin also increases the nuclear translocation and DNA binding of Nrf2/c-Jun to the antioxidant response element (ARE) on HO-1 promoter. In addition, arctigenin increased ARE-mediated transcriptional activities in rat primary astrocytes. Further mechanistic studies revealed that arctigenin increased the phosphorylation of AKT, a downstream substrate of phosphatidylinositol 3-kinase (PI3K). Treatment of cells with a PI3K-specific inhibitor, LY294002, suppressed the HO-1 expression, Nrf2 DNA binding and ARE-mediated transcriptional activities in arctigenin-treated astrocyte cells. The results collectively suggest that PI3K/AKT signaling pathway is at least partly involved in HO-1 expression by arctigenin via modulation of Nrf2/ARE axis in rat primary astrocytes.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.74-74
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• 1995

GABA transaminase is inactivated by preincubation with p$^1$, p$^2$-bis(5'-pyridoxal) diphosphate at pH 7.0. The inactivation under pseudo-first order conditions proceeds at a slow rate (K$\_$obs/=0.035 min$\^$-1/). The degree of labeling of the enzyme by p$^1$, p$^2$-bis(5'-pyridoxal) diphosphate was determined by absorption spectroscopy, The blocking of 2 lysyl residues/dimer is needed for inactivation of the transaminase. The time course of the reaction is significantly affected by the substrate ${\alpha}$-ketoglutarate, which afforded complete protection against the loss of the catalytic activity. Whereas cofator pyridoxal phosphate failed to prevent the inactivation of the enzyme. Therefore, it is postulated that binding of ${\alpha}$-ketoglutarate tn lysyl residues is the major factor contributing to stabilization of the catalytic site and bifuctional reagent p$^1$, p$^2$bis(5'-pyridoxal) diphosphate blocks lysyl residues other than those involved in the binding of the cofactor.

• Journal of Microbiology and Biotechnology
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• v.33 no.10
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• pp.1361-1369
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• 2023

Corynebacterium glutamicum (C. glutamicum) has been considered a very important and meaningful industrial microorganism for the production of amino acids worldwide. To produce amino acids, cells require nicotinamide adenine dinucleotide phosphate (NADPH), which is a biological reducing agent. The pentose phosphate pathway (PPP) can supply NADPH in cells via the 6-phosphogluconate dehydrogenase (6PGD) enzyme, which is an oxidoreductase that converts 6-phosphogluconate (6PG) to ribulose 5-phosphate (Ru5P), to produce NADPH. In this study, we identified the crystal structure of 6PGD_apo and 6PGD_NADP from C. glutamicum ATCC 13032 (Cg6PGD) and reported our biological research based on this structure. We identified the substrate binding site and co-factor binding site of Cg6PGD, which are crucial for understanding this enzyme. Based on the findings of our research, Cg6PGD is expected to be used as a NADPH resource in the food industry and as a drug target in the pharmaceutical industry.

• BMB Reports
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• v.38 no.1
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• pp.58-64
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• 2005

Myo-inositol monophosphate phosphatase (IMPP) is a key enzyme in the phosphoinositide cell-signaling system. This study found that incubating the IMPP from a porcine brain with pyridoxal-5'-phosphate (PLP) resulted in a time-dependent enzymatic inactivation. Spectral evidence showed that the inactivation proceeds via the formation of a Schiff's base with the amino groups of the enzyme. After the sodium borohydride reduction of the inactivated enzyme, it was observed that 1.8 mol phosphopyridoxyl residues per mole of the enzyme dimer were incorporated. The substrate, myo-inositol-1-phosphate, protected the enzyme against inactivation by PLP. After tryptic digestion of the enzyme modified with PLP, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. Amino acid sequencing of the peptide identified a portion of the PLP-binding site as being the region containing the sequence L-Q-V-S-Q-Q-E-D-I-T-X, where X indicates that phenylthiohydantoin amino acid could not be assigned. However, the result of amino acid composition of the peptide indicated that the missing residue could be designated as a phosphopyridoxyl lysine. This suggests that the catalytic function of IMPP is modulated by the binding of PLP to a specific lysyl residue at or near its substrate-binding site of the protein.

• The Korean Journal of Pesticide Science
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• v.14 no.3
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• pp.183-190
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• 2010

To search the new potent herbicidal agents by receptor-based approach, the interactions between receptor and substrate molecules from molecular docking to acetyl-CoA carboxylase(PDB code: 3K8X) of 2-(4-(6-chloro-2-benzoxazolyl)oxy)phenoxy-N-phenylpropionamide analogues (1-38) as substrate molecules were performed and discussed quantitatively. The most of the substrate molecules were formated 2 H-bonds between carbonyl oxygen atom of the substrate molecules and the amino acid residues (Ala1627 and Ile1735) in binding site of acetyl-CoA carboxylase (ACCase). But, the substrate molecules such as $R_l$=Acetyl substituents (6 & P9) were formated 3 H-bonds between H-bond acceptors in the substrate molecules and the H-bond donors in three amino acid residues including the rest residue (Gly 1998). Therefore, the inhibitory activity factors of the substrate molecules against ACCase are due to the H-bonding characters that will be able to apply to the optimization of herbicidal agents.

• Molecules and Cells
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• v.27 no.1
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• pp.99-103
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• 2009

Ribose-5-phosphate isomerase A (RpiA) plays an important role in interconverting between ribose-5-phosphate (R5P) and ribulose-5-phosphate in the pentose phosphate pathway and the Calvin cycle. We have determined the crystal structures of the open form RpiA from Vibrio vulnificus YJ106 (VvRpiA) in complex with the R5P and the closed form with arabinose-5-phosphate (A5P) in parallel with the apo VvRpiA at $2.0{\AA}$ resolution. VvRpiA is highly similar to Escherichia coli RpiA, and the VvRpiA-R5P complex strongly resembles the E. coli RpiA-A5P complex. Interestingly, unlike the E. coli RpiA-A5P complex, the position of A5P in the VvRpiA-A5P complex reveals a different position than the R5P binding mode. VvRpiA-A5P has a sugar ring inside the binding pocket and a phosphate group outside the binding pocket: By contrast, the sugar ring of A5P interacts with the Asp4, Lys7, Ser30, Asp118, and Lys121 residues; the phosphate group of A5P interacts with two water molecules, W51 and W82.

• Molecules and Cells
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• v.19 no.1
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• pp.97-103
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• 2005

The role of residue C323 in catalysis by human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was examined by substituting Arg, Gly, Leu, Met, or Tyr at C323 by cassette mutagenesis using synthetic human GDH isozyme genes. As a result, the $K_m$ of the enzyme for NADH and ${\alpha}-ketoglutarate$ increased up to 1.6-fold and 1.1-fold, respectively. It seems likely that C323 is not responsible for substrate-binding or coenzyme-binding. The efficiency ($k_{cat}/K_m$) of the mutant enzymes was only 11-14% of that of the wild-type isozymes, mainly due to a decrease in $k_{cat}$ values. There was a linear relationship between incorporation of [$^{14}C$]p-chloromercuribenzoic acid and loss of enzyme activity that extrapolated to a stoichiometry of one mol of [$^{14}C$] incorporated per mol of monomer for wild type hGDHs. No incorporation of [$^{14}C$]p-chloromercuribenzoic acid was observed with the C323 mutants. ADP and GTP had no effect on the binding of p-chloromercuribenzoic acid, suggesting that C323 is not directly involved in allosteric regulation. There were no differences between the two hGDH isozymes in sensitivities to mutagenesis at C323. Our results suggest that C323 plays an important role in catalysis by human GDH isozymes.