Isoleucine 69 and valine 325 form a specificity pocket in human muscle creatine kinase.
Isoleucine 69 and valine 325 form a specificity pocket in human muscle creatine kinase.
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DOI：
10.1021/bi049060y
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发表时间：
2004-10
期刊：
影响因子：
2.9
通讯作者：
W. R. Novak;Pan‐Fen Wang;M. McLeish;G. L. Kenyon;P. Babbitt
中科院分区：
文献类型：
--
作者：
W. R. Novak;Pan‐Fen Wang;M. McLeish;G. L. Kenyon;P. Babbitt
肌酸激酶(CK)催化三磷酸腺苷对肌酸的可逆磷酸化。从结构的角度来看，该酶利用两个灵活的环区来隔离和定位催化底物。关于柔性环在底物特异性和催化CK和其他相关的磷酸原激酶中的具体作用一直存在争议。在CK中，两个疏水环残基I69和V325与肌酸的N-甲基接触。在本研究中，我们报道了通过V325的突变改变CK的底物专一性。V325到谷氨酸的突变导致对糖胞胺的偏好超过100倍，而V325到丙氨酸的突变导致对环肌酸(1-羧甲基-2-亚氨基咪唑烷)的轻微偏好。这项研究加深了我们对磷酸原激酶活性部位是如何进化到识别它们各自的底物并催化它们的反应的理解。
Creatine kinase (CK) catalyzes the reversible phosphorylation of creatine by ATP. From a structural perspective, the enzyme utilizes two flexible loop regions to sequester and position the substrates for catalysis. There has been debate over the specific roles of the flexible loops in substrate specificity and catalysis in CK and other related phosphagen kinases. In CK, two hydrophobic loop residues, I69 and V325, make contacts with the N-methyl group of creatine. In this study, we report the alteration of the substrate specificity of CK through the mutagenesis of V325. The V325 to glutamate mutation results in a more than 100-fold preference for glycocyamine, while mutation of V325 to alanine results in a slight preference of the enzyme for cyclocreatine (1-carboxymethyl-2-iminoimidazolidine). This study enhances our understanding of how the active sites of phosphagen kinases have evolved to recognize their respective substrates and catalyze their reactions.