内源性蛋白酶对人脑己糖激酶N—末端序列水解作用的研究

HYDROLYSIS OF ENDOGENOUS PROTEASE TO N-TERMINAL SEGMENT OF HUMAN BRAIN HEXOKINASE I

在人脑己糖激酶Ｎ—末端设计和构建两个突变体，Ｔｙｒ－１０→Ｌｅｕ和Ｐｈｅ－１１→Ｌｅｕ，在大肠杆菌中表达，并且分离纯化，通过对这两个突变体Ｎ—末端起始氨基酸的分析结果显示，它们均失去Ｎ—末端序列，酶切位点类似于糜蛋白酶，由此推断来自大肠杆菌表达后的人脑己糖激酶在制备纯化中Ｎ—末端序列是受到类似于来自溶酶体的组织蛋白酶的水解作用。

Human brain hexokinase is imporment modulated enzyme in glyeolysis. Its N-terminal sequence has been determined to be N-Met-Ile-Ala-Ala-Gln-Leu-Leu-Ala-Tyr-Tyr-Phe-Thr-Glu-Leu-Lys-, This hydrophobic N-terminal Sequence is prominently displayed at the enzyme surface. It is responsible for binding to mitochondria, most hexokinase exists as the mitochondria-bound form in human brain, Purification of the enzyme resulted in loss of N-terminal segment and the appearance of nonbindable enzyme, but had no effect on ceaalytic activity. It is known that N-terminal segment of hexokinase is hydrolyzed by cathepsin from lysosome, the cleavage site of hexokinase is the same as chymotrypsin does. Two mutants were designed and constructed, Tyr-10→Leu, Phe-11→Leu. N-terminal amino acids analysis for two mutants after purfication indicated that the cleavage site of hexokinase was the same as chymotrypsin does. It was deduced that some proteinase in E. coli cell cleaves the same site of hexokinase as cathepsin from lysosome does. It is effctive to obtain hexokinase with N-terminal segment.