Analysis of enzymatic regulation by protein engineering.

1.嗜热菌L-乳酸脱氢酶基因的克隆与表达。将极端嗜热菌Thermus caldophilus GK 24的LDH基因克隆到大肠杆菌中，并建立了该基因的表达系统. LDH的效应物结合位点的分析。通过对T. caldopilus和脊椎动物LDH。通过定点诱变获得的突变酶的分析。突变的酶，其中His-188、Arg-216、Arg-256、Arg-259、Gly-267、Try-269被替换为Gln-173（173 Q）、Lys-173（173 K）、Glu-173（173 E）、Phe-188（188 F）、Glu-216（216 E）、Asp-256（256 D）、Gln-256（256 Q）、Gln-259（259 Q）、Arg-267（267 R），His 269（269 H），并分析其酶促调控特性.核磁共振分析蛋白质结构与调控机制的关系。用转移核欧沃豪泽效应（TRNOE）技术分析了辅酶与LDH结合后的构象变化。结果表明，这种变化是由酶蛋白的结构变化引起的，效应物与酶的效应物结合位点结合。

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.