摘要
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.
出处
《生物技术》
CAS
CSCD
1999年第2期7-10,共4页
Biotechnology
关键词
己糖激酶
糖酵解
内源性
蛋白酶
水解
hexokinase,
glycolysis, endogenous protease, cathepsin