• BMB Reports
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• v.31 no.1
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• pp.25-30
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• 1998

Kinetic studies of L-Alanine dehydrogenase from Bacillus subtilis-catalyzed reactions in the presence of $Zn^{2+}$ were carried out. The substrate (L-alanine) saturation curve is hyperbolic in the absence of the metal ion but it becomes sigmoidal when $Zn^{2+}$ is added to the reaction mixture indicating the positive cooperative binding of the substrate in the presence of zinc ion. The cooperativity of substrate binding depends on the xinc ion concentration: the Hill coefficients ($n_H$) varied from 1.0 to 1.95 when the zinc ion concentration varied from 0 to $60\;{\mu}m$. The inhibition of AlaDH by $Zn^{2+}$ is reversible and noncompetitive with respect to $NAD^+$ ($K_i\;=\;5.28{\times}10^{-5}\;M$). $Zn^{2+}$ itself binds to AlaDH with positive cooperativity and the cooperativity is independent of substrate concentration. The Hill coefficients of substrate biding in the presence of $Zn^{2+}$ are not affected by the enzyme concentration indicating that $Zn^{2+}$ binding does not change the polymerization-depolymerization equilibria of the enzyme. Among other metal ions, $Zn^{2+}$ appears to be a specific reversible inhibitor inducing conformational change through the intersubunit interaction. These results indicate that $Zn^{2+}$ is an allosteric competitive inhibitor and substrate being a non-cooperative per se, excludes the $Zn^{2+}$ from its binding site and thus exhibits positive cooperativity. The allosteric mechanism of AlaDh from Bacillus subtilis is consistent with both MWC and Koshland's allosteric model.

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.226-232
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• 2016

Dihydrodipicolinate reductase is an enzyme that converts dihydrodipicolinate to tetrahydrodipicolinate using an NAD(P)H cofactor in L-lysine biosynthesis. To increase the understanding of the molecular mechanisms of lysine biosynthesis, we determined the crystal structure of dihydrodipicolinate reductase from Corynebacterium glutamicum (CgDapB). CgDapB functions as a tetramer, and each protomer is composed of two domains, an Nterminal domain and a C-terminal domain. The N-terminal domain mainly contributes to nucleotide binding, whereas the C-terminal domain is involved in substrate binding. We elucidated the mode of cofactor binding to CgDapB by determining the crystal structure of the enzyme in complex with NADP⁺ and found that CgDapB utilizes both NADH and NADPH as cofactors. Moreover, we determined the substrate binding mode of the enzyme based on the coordination mode of two sulfate ions in our structure. Compared with Mycobacterium tuberculosis DapB in complex with its cofactor and inhibitor, we propose that the domain movement for active site constitution occurs when both cofactor and substrate bind to the enzyme.

• Bulletin of the Korean Chemical Society
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• v.33 no.1
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• pp.248-254
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• 2012

During the monolayer assembly of zeolite microcrystals using sonication with stacking (SS) method, the factors that govern the degree of close packing (DCP) between the microcrystals, the rate of attachment (rA) of microcrystals onto the substrate, the degree of coverage (DOC), and the binding strength (BS) between each crystal and the substrate were investigated for each mode of binding linkage (MBL). The tested MBLs were covalent linkage (CL), ionic linkage (IL), and polyelectrolyte-mediated ionic linkage (p-IL). Unlike the monolayers of zeolite crystals assembled on glass with a covalent linkage, the strong BS, very high DOC, and very high DCP do not decrease during monolayer assembly on glass through ionic linkages. This results indicate that the surface migration of crystals undergo linkage-nondestructively when crystals were attached to the substrates through ionic linkages.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.70-70
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• 2003

Active sites and substrate bindings of 1-aminoxyclopropane-1-carboxylate oxidase (MD-ACO1) catalyzing the oxidative conversion of ACC to ethylene have been determined based on site-directed mutagenesis and comparative modeling methods. Molecular modeling based on the crystal structure of Isopenicillin N synthase (IPNS) provided MD-ACO1 structure. MD-ACO1 protein folds into a compact jelly roll shape, consisting of 9 ${\alpha}$-helices, 10 ${\beta}$-strands and several long loops. The MD-ACO1/ACC/Fe(II)/Ascorbate complex conformation was determined from automated docking program, AUTODOCK. The MD-ACO1/Fell complex model was consistent with well known binding motif information (HIS177-ASP179-HIS234). The cosubstrate, ascorbate is placed between iron binding pocket and Arg244 of MD-ACO1 enzyme, supporting the critical role of Arg244 for generating reaction product. These findings are strongly supported by previous biochemical data as well as site-directed mutagenesis data. The structure of enzyme/substrate suggests the structural mechanism for the biochemical role as well as substrate specificity of MD-ACO1 enzyme.

• Bulletin of the Korean Chemical Society
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• v.23 no.8
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• pp.1157-1159
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• 2002

• Journal of Life Science
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• v.4 no.3
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• pp.92-101
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• 1994

With the rapid development of bioorganic chemistry recently, a field of artificial enzymes has a great concern from the industrial point of view. A number of possibilities now exist ofr the construction of artificial enzymes. They must posses two structural entities, a substrate-binding site and a catalytically effective site. It has been found that producing the facility for substrate binding is relatively straightforward but catalytic sites are somewhat more difficult. Therefore, synthetic catalysts do not yet match all the properties of an enzyme, however, the design of catalysts has lead to very powerful effects. This article reviews the existing literature on the modeling of artificial enzymes using cyclodextrin, modified cyclodextrin and crown compounds.

• Journal of Microbiology and Biotechnology
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• v.30 no.1
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• pp.146-154
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• 2020

With the advantages of biocatalytic method, enzymes have been excavated for the synthesis of chiral amino acids by the reductive amination of ketones, offering a promising way of producing pharmaceutical intermediates. In this work, a robust phenylalanine dehydrogenase (PheDH) with wide substrate spectrum and high catalytic efficiency was constructed through rational design and active-site-targeted, site-specific mutagenesis by using the parent enzyme from Bacillus halodurans. Active sites with bonding substrate and amino acid residues surrounding the substrate binding pocket, 49L-50G-51G, 74M,77K, 122G-123T-124D-125M, 275N, 305L and 308V of the PheDH, were identified. Noticeably, the new mutant PheDH (E113D-N276L) showed approximately 6.06-fold increment of k_cat/Km in the oxidative deamination and more than 1.58-fold in the reductive amination compared to that of the wide type. Meanwhile, the PheDHs exhibit high capacity of accepting benzylic and aliphatic ketone substrates. The broad specificity, high catalytic efficiency and selectivity, along with excellent thermal stability, render these broad-spectrum enzymes ideal targets for further development with potential diagnostic reagent and pharmaceutical compounds applications.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.13-19
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• 2006

To search of new porcine pheromonal odorants for biostimulation control system technologies to improve reproductive efficiency in livestock species, the comparative molecular field analysis (CoMFA) for binding affinity constant $(p(Od)_{50})$ between porcine odorant binding protein (pOBP) and ligands of odorant 2-(cyclohexyloxy) tetrahydrofurane derivatives as substrate molecule was conducted and discussed. In the optimized CoMFA model AIV with chirality $(C_1'(R),\;C_2(S))$ in substrate molecule and atom based fit alignment (A) of odorants, the statistical results showed the best predictability of the binding affinities $(p(Od)_{50})$ based on the LOO cross-validated value $r^2_{cv}.\;(q^2=0.886)$ and non-cross-validated conventional coefficient $(r^2_{ncv}.=0.984)$. the binding affinity constants exhibited a good correlation with steric (40.8%), electrostatic (14.6%) and hydrophobic (44.6%) factors of the substrate molecules. from the analytical results of the contour maps, which may give us some valuable informations to the modification of odorants for effective binding affinity.

• 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.

• Toxicological Research
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• v.15 no.1
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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.