ATP-binding site of human brain hexokinase as studied by molecular modeling and site-directed mutagenesis.
ATP-binding site of human brain hexokinase as studied by molecular modeling and site-directed mutagenesis.
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通过分子建模和定点诱变研究人脑己糖激酶的 ATP 结合位点。
DOI：
10.1021/bi960750e
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Fromm,HJ
中科院分区：
文献类型：
--
作者：
Zeng,C;Aleshin,AE;Hardie,JB;Harrison,RW;Fromm,HJ
ATP与己糖激酶活性位点的相互作用是未知的，因为己糖激酶-ATP复合物的晶体结构是不可用的。发现脑己糖激酶的ATP结合位点与肌动蛋白、热休克蛋白hsc 70和甘油激酶的ATP结合位点同源。基于这些相似性，ATP分子被定位在人脑己糖激酶的催化结构域中，该催化结构域是根据酵母己糖激酶的X射线结构建模的。进行定点诱变以测试推测参与与ATP的三聚磷酰基部分相互作用的残基的功能。Asp 532被认为参与结合MgATP 2-复合物的Mg 2+离子，其被突变为Lys和Glu。两个突变体的kcat值分别降低了1000倍和200倍。另一个残基Thr 680被突变为瓦尔和Ser，与ATP的γ-磷酰基通过氢键相互作用，Thr 680瓦尔突变体的kcat值下降了2000倍，而Thr 680 Ser突变体的kcat值仅下降了2.5倍，表明羟基的重要性。所有上述突变体的ATP或葡萄糖的Kmand解离常数值相对于野生型酶几乎没有或没有变化。葡萄糖6-磷酸类似物1，5-脱水葡萄糖醇6-磷酸的Ki值与野生型酶的Ki值相同，无机磷酸盐（Pi）对所有四种突变体的抑制作用均被逆转。突变体的圆二色性光谱与野生型酶相同。定点突变的结果表明，推测的相互作用的研究残基与ATP的过渡态的稳定是重要的。
The interaction of ATP with the active site of hexokinase is unknown since the crystal structure of the hexokinase−ATP complex is unavailable. It was found that the ATP binding site of brain hexokinase is homologous to that of actin, heat shock protein hsc70, and glycerol kinase. On the basis of these similarities, the ATP molecule was positioned in the catalytic domain of human brain hexokinase, which was modeled from the X-ray structure of yeast hexokinase. Site-directed mutagenesis was performed to test the function of residues presumably involved in interaction with the tripolyphosphoryl moiety of ATP. Asp532, which is thought to be involved in binding the Mg2+ion of the MgATP2-complex, was mutated to Lys and Glu. Thekcatvalues decreased 1000- and 200-fold, respectively, for the two mutants. Another residue, Thr680 was proposed to interact with the γ-phosphoryl group of ATP through hydrogen bonds and was mutated to Val and Ser. Thekcatvalue of the Thr680Val mutant decreased 2000-fold, whereas thekcatvalue of the Thr680Ser decreased only 2.5-fold, implying the importance of the hydroxyl group. TheKmand dissociation constant values for either ATP or glucose of all the above mutants showed little or no change relative to the wild-type enzyme. TheKivalues for the glucose 6-phosphate analogue 1,5-anhydroglucitol 6-phosphate, were the same as that of the wild-type enzyme, and the inhibition was reversed by inorganic phosphate (Pi) for all four mutants. The circular dichroism spectra of the mutants were the same as that of the wild-type enzyme. The results from the site-directed mutagenesis demonstrate that the presumed interactions of investigated residues with ATP are important for the stabilization of the transition state.