| Research Abstract |
1. Cloning and expression of gene of L-lactate dehydrogenase (LDH) from thermophilic bacterium. LDH gene of an extremely-thermophilic bacterium, Thermus caldophilus GK24, was cloned into Escherichia coli, and the expression system of the gene was established.
2. Analysis of the effector-binding site of the LDH. The effector-binding site of the LDH has been found to be corresponded to an anion-binding site of vertebrate LDH, by the analysis of the chemical modification and structural comparison between T. caldopilus and vertebrate LDHs.
3. Analysis of mutated enzymes obtained by site-directed Mutagenesis. mutated enzymes, in which His-188,Arg-216,Arg-256,Arg-259,Gly-267,Try-269 were replaced into Gln-173(173Q),Lys-173(173K),Glu-173(173E),Phe-188(188F),Glu-216(216E),Asp-256(256D),Gln-256(256Q),Gln-259(259Q),Arg-267(267R),His269(269H), were obtained, and their characteristics in the ehzymatic regulation were analyzed.
4. Analysis of relationship between protein structure and regulation mechanism by NMR. By the technique of transferred nuclear Overhauser effect (TRNOE),conformational change of a coenzyme bound to LDH has been analyzed. The results indicated that this change was induced by the structural change of the enzyme protein with the binding of the effector to the effector-binding site of the enzyme.
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