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结果1.将极端嗜热菌Thermus caldophilus GK24的LDH基因克隆到大肠杆菌中，建立了该基因的表达系统。乳酸脱氢酶效应器结合部位的分析。通过对白头翁和脊椎动物LDH的化学修饰和结构比较分析，发现LDH的效应结合部位对应于脊椎动物LDH的阴离子结合部位。定点突变获得的突变酶的分析。将His-188、Arg-216、Arg-256、Arg-259、Gly-267、Try-269突变为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)。用核磁共振技术分析蛋白质结构与调控机制的关系。利用转移核Overhauser效应(TRNOE)技术，分析了LDH结合辅酶的构象变化。结果表明，这种变化是由于酶蛋白的结构变化引起的，即效应物与酶的效应物结合部位。

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.