摘要
哺乳动物己糖激酶Ⅰ的分子量是１００ｋＤ．目前已经认为是由分子量５０ｋＤ酵母型己糖激酶通过基因复制和融合进化来的．己糖激酶Ⅰ的Ｃ端半分子包含了底物葡萄糖的结合位点即催化位点．Ｘ射线衍射结构的结果已经推测在酵母型的己糖激酶分子中Ｓｅｒ－１５８、Ａｓｐ－２１１是和葡萄糖的结合及催化活性有关，这些氨基酸残基相当于人脑己糖激酶Ⅰ分子中的Ｓｅｒ－６０３、Ａｓｐ－６５７，它们正好位于该酶分子的Ｃ端半分子中．定位突变这两个氨基酸残基得到４个该酶的Ｃ端半分子酶（ｍｉｎｉ－ＨＫⅠ）的突变体，它们是Ｓｅｒ－６０３→Ｃｙｓ，Ｓｅｒ－６０３→Ｔｈｒ，Ａｓｐ－６７５→Ｇｌｕ，Ａｓｐ－６７５→Ｖａｌ．实验结果指出４个突变体酶的Ｋｍ值变化不大，但酶活性只保留野生型酶的０．２８％～１１％，园二色谱分析４个突变体的ＣＤ谱与野生型酶基本一致，因此说明二级结构没有变化．这些研究结果和Ｘ射线衍射结构的推断是一致的，显示了Ｓｅｒ－６０３和Ａｓｐ－６５７氨基酸残基在该酶结合底物葡萄糖或催化作用上起了重要的作用．
Mammlian hexokinase type Ⅰ is a 100 kD enzyme.It has been considered to be evolved from an ancestral 50 kD yeast type hexokinase by gene duplication and fusion.The catalytic site is associated with C terminal half of the enzyme.the X ray structure of yeast hexokinase is known and putative glucose binding domain has been suggested.Ser 158 and Asp 211 are considered to interact directly with glucose in the yeast enzyme,The correspon ding residues in human brain hexokinase Ⅰ are Ser 603 and Asp 657 which are in the C terminal domain.Site directed mutagenesis was performed to obtain 4 mutants of mini hexokinase Ⅰ (C terminal half of human brain hexokinase Ⅰ).Ser 603→Cys,Ser 603→Thr,Asp 657→Glu and Asp 657→Val mutants retained as much as 0 28%-11% of the wild type enzyme activity.circular dichroism spectrometry analysis showed that four mutants had almost identical CD spectrum to the wild type enzyme,so the mutants did not change their secondry structure.These results are idetical to the view that all two residues predicted to interact with glucose from X ray studies may play a role in binding glucose or catalysis.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
1998年第6期742-745,共4页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金
