Study of Regulatory Mechanism of Catalysis by Cobalamin-dependent Enzymes

1.甘油脱水酶的X射线分析：对肺炎克雷伯菌重组甘油脱水酶进行纯化和鉴定。在2.1A分辨率下测定了甘油脱水酶与氰基钴胺和丙烷-1，2-二醇的络合物的晶体结构。所阐明的结构特征与二醇脱水酶非常相似。虽然外消旋丙烷-1，2-二醇用于结晶，但与甘油脱水酶结合的底物被指定为(R)-异构体。这与二醇脱水酶形成了鲜明的对比，解释了两种酶对甘油自杀性失活的敏感性差异。2.二醇脱水酶活性部位残基的突变分析。X射线结构显示，二醇脱水酶的7个氨基酸残基直接与底物的活性部位K和/或羟基相互作用。为了研究这些残基的作用，构建了这些残基的丙氨酸突变体并进行了分析。The Results…进一步说明了H143和E170残基在催化过程中的重要性。通过构建H143E、H143K、H143Q和H143L突变体，进一步研究了残基H143的作用。结果表明组氨酸与丙二醇2-羟基之间的氢键对催化反应具有重要意义。用定点突变方法研究了两个钴胺结合残基的作用。结果表明，Sα224对于激活ADOCbl的Co-C键，防止催化剂失活具有重要意义。3.构建大鼠甲硫氨酸合成酶突变体H773G。为构建甲钴胺碱基结合突变的甲硫氨酸合成酶突变体，采用定点突变的方法将His773~Gly突变引入蛋氨酸合成酶基因。利用杆状病毒表达系统在昆虫细胞中高效表达大鼠蛋氨酸合成酶的方法，试图获得突变的蛋氨酸合成酶，但未能获得突变蛋白的表达。较少

1.X-ray analysis of glycerol dehydratase.Recombinant glycerol dehydratase of Klebsiella pneumoniae was purified to homogeneity and characterized. The crystal structure of glycerol dehydratase in complex with cyanocobalamin and propane-1,2-diol was determined at 2.1 A resolution. The structural features elucidated are quite similar to those of diol dehydratase. Although racemic propane-1,2-diol was used for crystallization, the substrate bound to glycerol dehydratase was assigned to the (R)-isomer. This is in clear contrast to diol dehydratase and accounts for the difference between the two enzymes in the susceptibility of suicide inactivation by glycerol.2.Mutational analysis of active-site residues of diol dehydratase.X-ray structure of diol dehydratase showed the seven amino acid residues interacting directly with active site K^+ and/or hydroxy group of substrate. To investigate the roles of these residues, alanine mutants of these residues were constructed and analyzed. The results … More indicated the importance of H143 and E170 residues in the catalytic process. Role of the residue, H143, was examined further by constructing H143E,H143K,H143Q, and H 143L mutants. The results indicated the importance of hydrogen bond between His and 2-hydroxy group of propanediol for the catalytic reaction.Role of two cobalamin-binding residues was investigated with site-directed mutagenesis. The results indicated that Sα224 is important for the activation of the Co-C bond of AdoCbl and for the prevention from inactivation during catalysis. The importance of Kβ135 for the binding of AdoCbl was also suggested.3.Construction of H773G mutant of rat methionine synthase.To construct mutant methionine synthase to which methylcobalamin was bound in base-on mode, His773 to Gly mutation was introduced into methionine synthase gene by site-directed mutagenesis. The method of high-level expression of rat methionine synthase in insect cells using a baculovirus expression system was tried to get the mutant methionine synthase, however, expression of mutant protein failed. Less