Multiple approaches for the establishment of the basis of Protein Engineering.

为了建立“蛋白质工程”的基础，我们已经完成了下面所述的各种研究。1、通过替换氨基酸的方法：(1)T4内切酶的Glu23残基被证明是糖基酶活性的重要因素。(2)H~+-ATPase的Thr156位残基对激活活性有重要作用。(3)研究NADH细胞色素b_5的结构与功能的关系，以了解遗传性高铁血红蛋白血症的原因。(4)Trp108在溶菌酶中对诱导活性的作用变得明显。2、构建目的蛋白表达系统的途径：(1)凝血因子VII中牛组织因子的结合部位位于Gal和EGF结构域之间。(2)枯草杆菌中性蛋白酶的活性部位及其催化机理。Amylosaccharticus的结构特征。(3)分析了L赖氨酸和三磷酸腺苷与嗜热脂肪芽孢杆菌赖氨酰tRNA合成酶的结合方式。3、COM…方法蛋白质一级结构和三级结构的进一步细化：(1)赖氨酰内肽酶的HIS210残基在诱导活性的高选择性中起重要作用。(2)分析了哈布蛇磷脂酶A_2的结构与功能的关系。(3)通过制备不同的人醛缩酶嵌合蛋白，分析了其催化机理。(4)通过对磷酸丙糖异构酶和核糖核酸酶的模块结构分析，探讨了它们的结构、功能和遗传性。4、化学修饰方法：(1)引入新型聚乙二醇类和直链淀粉衍生物，使核糖核酸酶T1和胰酶稳定。5、核磁共振和X射线结晶学方法：(1)对长双歧杆菌L乳酸脱氢酶的变构现象进行了分析。(2)用核磁共振和距离几何算法确定了新卡西宁的三级结构。(3)通过对Bowman-Birk酶抑制剂与胰酶络合物的X-射线结晶分析，验证了该酶抑制剂的功能。较少

We have accomplished various Investigations described below for the establishment of the basis of 'Protein Engineering'.1, The approach by replacing amino acid : (1) Glu23 residue of T4 endonuclease was proved to be Important in glycosylase activity. (2) Thrl56 residue of H^+ ATPase was found to be important to evoke activity. (3) The structure-function relationship of NADHcytochrome b_5 was examined to understand the cause of hereditary methemoglobinemia. (4) The role of Trp108 in lysozyme on evoking activity became apparent.2, The approach by constructing expression system of aimed protein : (1) The binding site of bovine tissue factor was present in the region between Gal and EGF domain in blood coagulation factor VII. (2) The active sites and the catalytic mechanism of neutral protease from Bacillus subtilis var. amylosacchariticus were elucidated. (3) The binding mode of L-lysine and ATP with lysyl tRNA synthase from Bacillus stearothermophilus were analyzed.3, The approach by com … More paring primarx and tertiary structure of proteins : (1) HIs210 residue of lysylendopeptidase was proved to be important in high-selectivity on evoking activity. (2) The relationship between structure and function in phospholipase A_2 from Habu snake was analyzed. (3) The catalytic mechanism of human aldolase was analyzed by preparing its various chimeric proteins. (4) The structure, function and heredity of both triosephosphate isomerase and ribonuclease were examined discussed by the analysis of their module structures.4, The approach by means of chemical modification : (1) RNase T1 and trypsin became stable by introducing novel polyethyleneglycol and amylose derivative.5, The approach by use of NMR and X-ray crystallography : (1) The allosteric phenomena of L-lactic acid dehydrogenase from Bifidobacterium longum was analyzed. (2) The tertiary structure of neocarzinostatin was determined by use of NMR and distance geometry algorism. (3) The function of Bowman-Birk protease inhibitor was examined from the X-ray crystallographic analysis of its complex with trypsin. Less