• BMB Reports
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• v.37 no.5
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• pp.533-537
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• 2004

Previously published kinetic data on the interactions of seventeen different enzymes with their physiological substrates are re-examined in order to understand the connection between ground state binding energy and transition state stabilization of the enzyme-catalyzed reactions. When the substrate ground state binding energies are normalized by the substrate molar volumes, binding of the substrate to the enzyme active site may be thought of as an energy concentration interaction; that is, binding of the substrate ground state brings in a certain concentration of energy. When kinetic data of the enzyme/substrate interactions are analyzed from this point of view, the following relationships are discovered: 1) smaller substrates possess more binding energy concentrations than do larger substrates with the effect dropping off exponentially, 2) larger enzymes (relative to substrate size) bind both the ground and transition states more tightly than smaller enzymes, and 3) high substrate ground state binding energy concentration is associated with greater reaction transition state stabilization. It is proposed that these observations are inconsistent with the conventional (Haldane) view of enzyme catalysis and are better reconciled with the shifting specificity model for enzyme catalysis.

• Journal of Microbiology and Biotechnology
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• v.28 no.8
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• pp.1339-1345
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• 2018

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, which catalyzes aldol cleavage and condensation reactions, has two distinct substrate-binding sites. The substrate-binding mode at the catalytic site and Schiff-base formation have been well studied. However, structural information on the phosphate-binding loop (P-loop) is limited. Zymomonas mobilis KDPG aldolase is one of the aldolases with a wide substrate spectrum. Its structure in complex with the substrate-mimicking 3-phosphoglycerate (3PG) shows that the phosphate moiety of 3PG interacts with the P-loop and a nearby conserved serine residue. 3PG-binding to the P-loop replaces water molecules aligned from the P-loop to the catalytic site, as observed in the apostructure. The extra electron density near the P-loop and comparison with other aldolases suggest the diversity and flexibility of the serine-containing loop among KDPG aldolases. These structural data may help to understand the substrate-binding mode and the broad substrate specificity of the Zymomonas KDPG aldolase.

• Reproductive and Developmental Biology
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• v.33 no.3
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• pp.119-123
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• 2009

The two dimensional quantitative structure-activity relationships (2D-QSARs) models concerning the binding affinity constants ($p[Od.]_{50}$) between 2-cyclohexyltetrahydropyrane and 2-cyclohexyltetrahydrofurane analogues as substrates, and bovine odorant binding protein (bOBP) as receptor were derived by multiple regression analyses method and discussed. The statistical quality of the optimized 2D-QSAR model (5) was good (r=0.907). From the model, the binding affinity constants ($p[Od.]_{50}$) were dependent upon the optimal value ($(TL)_{opt.}$=2.737) of total lipole (TL) of substrate molecules. Based on these findings, the high active compounds predicted by optimized 2D-QSAR model (5) were 2-(dimethylcyclohexyl)tetrahydropyrane molecule and their isomer molecules. The binding affinity constants regarding bOBP of the tetrahydrofuryl-2-yl family compounds were dependent upon the hydrophobicity (logP) of whole substrate molecules. In any case of porcine odorant-binding proteins (pOBP), the constants were dependent upon the hydrophobicity (${\pi}x={\log}P_X-{\log}P_H$) of substituents (R) in substrate molecules. Also, from the optimal values of hydrophobic constant, the hydrophobicity for bOBP influenced ca. twice time bigger (bOBP>pOBP) than that for pOBP.

• BMB Reports
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• v.42 no.3
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• pp.166-171
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• 2009

Hsp70s assist in the process of protein folding through nucleotide-controlled cycles of substrate binding and release by alternating from an ATP-bound state in which the affinity for substrate is low to an ADP-bound state in which the affinity for substrate is high. It has been long recognized that the two-domain structure of Hsp70 is critical for these regulated interactions. Therefore, it is important to obtain information about conformational changes in the relative positions of Hsp70 domains caused by nucleotide binding. In this study, analytical ultracentrifugation and dynamic light scattering were used to evaluate the effect of ADP and ATP binding on the conformation of the human stress-induced Hsp70.1 protein. The results of these experiments showed that ATP had a larger effect on the conformation of Hsp70 than ADP. In agreement with previous biochemical experiments, our results suggest that conformational changes caused by nucleotide binding are a consequence of the movement in position of both nucleotide- and substrate-binding domains.

• Journal of Microbiology and Biotechnology
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• v.16 no.12
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• pp.1897-1903
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• 2006

In order to investigate the functions of the C-terminal region of chitinase ChiCW of Bacillus cereus 28-9, a C-terminal truncated enzyme, ChiCW$\Delta$FC, was expressed in Escherichia coli and purified to homogeneity for biochemical characterization. Compared with ChiCW, ChiCW$\Delta$FC exhibited higher chitinase activity at high temperature and pH, but expressed lower hydrolytic and binding activities toward insoluble substrates. In addition, kinetic properties indicated that ChiCW$\Delta$MC hydrolyzed oligomeric and polymeric substrates less efficiently than ChiCW. These results suggest that the C-terminal region of ChiCW plays important roles in substrate binding and hydrolysis of chitin. In addition, the biological meaning of C-terminal proteolytic modification of ChiCW is discussed.

• Bulletin of the Korean Chemical Society
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• v.27 no.4
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• pp.529-534
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• 2006

Chemical modification of the S. cerevisiae farnesyl protein transferase (FPT) with CMC, phenylglyoxal and DEPC resulted in enzyme inactivation, depending upon the reagent concentration. The peptide substrate GST-PEP-I, a GST-fused undecapeptide mimicking the C-terminus of $p21^{Ki-ras}$, protected the enzyme against inactivation by CMC which is specific to either aspartate or glutamate, while the other substrate farnesyl pyrophosphate (FPP) showed protection against phenylglyoxal which is the specific modifier of arginine residues, dependent on the substrate concentrations. Neither of the two substrates protected the enzyme against histidine inactivation by DEPC. It is suggested that there is at least one aspartate or glutamate residue at the peptide substrate binding site, and that at least one arginine residue is located at the binding site of FPP. There also seems to be at least one histidine residue which is critical for enzymic activity and is exposed toward the bulk solution, excluded from the substrate binding sites.

• Journal of Applied Biological Chemistry
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• v.48 no.3
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• pp.113-119
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• 2005

O-methylation mediated by O-methyltransferases (OMTs) is a common modification in natural product biosynthesis and contributes to diversity of secondary metabolites. OMTs use phenylpropanoids, flavonoids, other phenolics and alkaloids as substrates, and share common domains for S-adenosyl-L-methionine (AdoMet) and substrate binding. We searched Arabiposis genome and found 17 OMTs genes (AtOMTs). AdoMet- and substrate-binding sites were predicted. AdoMet binding domain of AtOMTs is highly conserved, while substrate-binding domain is diverse, indicating use of different substrates. In addition, expressions of six AtOMT genes in response to UV and in different tissues were investigated using real-time quantitative reverse transcriptase-polymerase chain reaction. All the AtOMTs investigated were expressed under normal growth condition and most, except AtOMT10, were induced after UV illumination. AtOMT1 and AtOMT8 were expressed in all the tissues, whereas AtOMT10 showed flower-specific expression. Analysis of these AtOMT gene expressions could provide some clues on AtOMT involvement in the cellular processes.

• Bulletin of the Korean Chemical Society
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• v.28 no.12
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• pp.2248-2252
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• 2007

SOMT-9 is an O-methyltransferase that utilizes quercetin to produce 3'-methoxy quercetin. In order to determine which amino acids of SOMT-9 are important for this reaction and its regioselectivity, molecular docking experiments followed by site directed mutagenesis were performed. Molecular modeling and molecular docking experiments identified several amino acid residues involved in metal binding, AdoMet binding, and substrate binding. Site-directed mutagenesis showed that Asp188 is critical for metal binding and that Lys165 assists other metal binding residues in maintaining quercetin in the proper position during the reaction. In addition, Tyr207 was shown to play an important role in the determination of the regioselectivity and Met60 was shown to be involved in formation of the hydrophobic pocket necessary for substrate binding. The molecular modeling and docking experiments discussed in this study could be applicable to future research including prediction of substrate binding and regioselectivity of an enzyme.

• Journal of Microbiology and Biotechnology
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• v.12 no.1
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• pp.149-152
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• 2002

The effect of pressure on the catalytic properties of glutamate racemase from Aquifex pyrophilus, an extremophilic bacterium, was investigated. The activation volume for the overall reaction $({\Delta}V^{\neq})$ and catalysis $({{Delta}V_{cat}}^{\neq})$ was -96.97 ml/mol and 4.97 ml/mol, respectively, while the reaction volume for the substrate binding (${\Delta}V_{K_m^-1}$) was -101.94 ml/mol. The large negative ${\Delta}V^{\neq}$ for the overall reaction indicated that the pressurization of glutamate racemase resulted in enhanced catalytic efficiencies. In addition, this value was also due to the large negative ${Delta}V_{K_m^-1}$ for the substrate binding. The negative value of ${Delta}V_{K_m^-1}$ implied that the conformational changes in the enzyme molecule occurred during the substrate binding process, thereby increasing the degree of hydration. The small value of ${{Delta}V_{cat}}^{\neq}$suggested that the pressure did not affect the glutamate racemase catalysis after the substrate binding.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.7-13
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• 2010

The binding affinity constants ($p(Od)_{50}$) and molecular docking scores (OS) between porcine odorant binding proteins pOBP (1HQP) and pPBP (1GM6) as receptor and a series of tetrahydrofuran-2-yl (A & B) analogues as substrate, and their interactions were discussed quantitatively using three-dimensional quantitative structure-activity relationship (30-QSAR) models. The statistical qualities of the optimized CoMF A models for pOBP were better than those of the CoMSIA models. The binding affinity constants and OS between substrate and receptor molecules were dependent upon steric and hydrophobic interaction. The DS constants of the substrates into the binding site of OBP (1HQP) were bigger than those of PBP (1GM6). The resulting contour maps produced by the optimized CoMFA model were used to identify the structural features relevant to the binding affinity in binding site of pOBP.