Residues putatively involved in binding of ATP and glucose 6-phosphate to a mammalian hexokinase: site-directed mutation at analogous positions in the N- and C-terminal halves of the type I isozyme.
Residues putatively involved in binding of ATP and glucose 6-phosphate to a mammalian hexokinase: site-directed mutation at analogous positions in the N- and C-terminal halves of the type I isozyme.
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推测参与 ATP 和葡萄糖 6-磷酸与哺乳动物己糖激酶结合的残基：I 型同工酶 N 端和 C 端半部类似位置的定点突变。
DOI：
10.1006/abbi.1995.1412
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发表时间：
1995
影响因子：
3.9
通讯作者：
Wilson,JE
中科院分区：
文献类型：
--
作者：
Baijal,M;Wilson,JE
尽管哺乳动物己糖激酶I型同工酶（ATP: d -己糖6-磷酸转移酶；EC 2.7.1.1）的N端和c端有广泛的序列相似性，但它们在功能上是不同的，c端负责催化，N端被认为起调节作用。我们研究了几个位点定向突变对大鼠I型同工酶的动力学和调控特性的影响。c端残基Asp 532突变为Asn， Arg 539突变为Met， Gly 896或Gly 898突变为Val，导致催化活性急剧丧失（<10%的野生型酶），这与之前认为这些残基参与ATP结合的观点一致。酶n端相应残基的突变对酶活性的影响要小得多（约为野生型的50%），但对酶活性的影响却很显著，这可能是由于对酶构象的微妙影响。Lys 899向Met的突变导致其比活性降低约50%，而ATP的km增加约5倍，这与Lys 899通过与磷酸侧链的静电相互作用参与ATP结合的观点一致。与I型己糖激酶的Cys 158和Cys 606相对应的胱氨酸残基存在于其他对葡萄糖6-磷酸（Glc-6-P）抑制表现出明显敏感性的己糖激酶中，但在对Glc-6-P不敏感的己糖激酶中没有发现类似的残基。然而，这种相关性似乎是巧合，因为无论是Cys 158或Cys 606对Ala的突变，还是所检测的任何其他突变，都没有改变I型己糖激酶对Glc-6-P类似物1,5-无氢葡萄糖醇-6- p的抑制敏感性，也没有改变Pi对这种抑制的拮抗作用。
Despite extensive sequence similarity between the N- and C-terminal halves of the Type I isozyme of mammalian hexokinase (ATP:D-hexose 6-phosphotransferase; EC 2.7.1.1), they are functionally distinct, the C-terminal half being responsible for catalysis and the N-terminal half thought to play a regulatory role. We have examined the effects of several site-directed mutations on kinetic and regulatory properties of the rat Type I isozyme. Mutation of the C-terminal residues, Asp 532 to Asn, Arg 539 to Met, and Gly 896 or Gly 898 to Val, resulted in drastic loss of catalytic activity (<10% of wild-type enzyme), consistent with previous suggestions that these residues are involved in binding of ATP. Mutation of the corresponding residues in the N-terminal half of the enzyme caused much less marked (>50% of wild type), but significant, effects on activity which are presumed to result from subtle effects on conformation of the enzyme. Mutation of Lys 899 to Met resulted in an approximately 50% decrease in specific activity and an approximately fivefold increase in the Kmfor ATP, consistent with the view that Lys 899 participates in binding of ATP through electrostatic interactions with the phosphate sidechain. Cys residues corresponding to Cys 158 and Cys 606 of Type I hexokinase are found in other hexokinases that exhibit marked sensitivity to inhibition by the product, glucose 6-phosphate (Glc-6-P), but analogous residues are not found in hexokinases insensitive to Glc-6-P. However, this correlation appears to be coincidental since neither the mutation of Cys 158 or Cys 606 to Ala nor any of the other mutations examined abolished sensitivity of Type I hexokinase to inhibition by the Glc-6-P analog 1,5-anhydroglucitol-6-P or to antagonism of this inhibition by Pi.