Chen L H, Borders C L, Vásquez J R, Kenyon G L
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143, USA.
Biochemistry. 1996 Jun 18;35(24):7895-902. doi: 10.1021/bi952798i.
Creatine kinase (CK; EC 2.7.3.2) catalyzes the reversible conversion of creatine and MgATP to phosphocreatine and MgADP. In the absence of an X-ray crystal structure, we have used the sequence homology of creatine kinases and other guanidino kinases from a variety of sources to identify the conserved histidine residues in rabbit muscle CK, as well as to try to pinpoint a reactive histidine that has been implicated in the active site. This residue has been proposed to act as a general acid/base catalyst assisting in the phosphoryl transfer mechanism [Cook et al. (1981) Biochemistry 20, 1204-1210]. There are 17 histidine residues in rabbit muscle CK, and of these, only five have been conserved in all guanidino kinase sequences published to date [Mühlebach et al. (1994) Mol. Cell. Biochem. 133, 245-62]. In rabbit muscle CK, these residues are H96, H105, H190, H233, and H295. We have carried out site-specific mutagenesis of these five histidine residues, replacing each with an asparagine. Each of these mutants exhibited enzymatic activity but to varying degrees. The H105N, H190N, and H233N mutants displayed specific activities similar to that of the wild-type enzyme. H96N has high activity, but appears to be quite unstable, losing catalytic activity upon cell lysis by sonication and/or chromatographic steps involved in purification. H295N shows a significantly reduced catalytic activity relative to the native enzyme, due to marked decreases in kcat and the affinities for both substrates. Each of the five mutants is inactivated by diethyl pyrocarbonate (DEP), and inactivation is reversible upon incubation with hydroxylamine. However, only H295N shows a dramatically reduced rate of inactivation relative to native CK, consistent with H295 being the residue modified by DEP in the native enzyme. These intriguing results indicate that four of the conserved histidines (H96, H105, H295, and H233) are not essential for activity, and while H295 may be at the active site of CK, it is unlikely to play the role of a general acid/base catalyst.
肌酸激酶（CK；EC 2.7.3.2）催化肌酸和MgATP可逆转化为磷酸肌酸和MgADP。由于缺乏X射线晶体结构，我们利用来自多种来源的肌酸激酶和其他胍基激酶的序列同源性，来确定兔肌肉CK中保守的组氨酸残基，并试图找出一个与活性位点有关的反应性组氨酸。有人提出这个残基作为一般酸碱催化剂协助磷酸转移机制[库克等人（1981年）《生物化学》20，1204 - 1210]。兔肌肉CK中有17个组氨酸残基，其中只有5个在迄今公布的所有胍基激酶序列中保守[米尔巴赫等人（1994年）《分子与细胞生物化学》133，245 - 62]。在兔肌肉CK中，这些残基是H96、H105、H190、H233和H295。我们对这5个组氨酸残基进行了定点诱变，将每个残基都替换为天冬酰胺。这些突变体均表现出酶活性，但程度不同。H105N、H190N和H233N突变体的比活性与野生型酶相似。H96N具有高活性，但似乎相当不稳定，在通过超声破碎细胞裂解和/或纯化过程中的色谱步骤后失去催化活性。H295N相对于天然酶显示出显著降低的催化活性，这是由于催化常数（kcat）以及对两种底物的亲和力都明显降低。这5个突变体均被焦碳酸二乙酯（DEP）灭活，并且与羟胺孵育后灭活是可逆的。然而，相对于天然CK，只有H295N显示出显著降低的失活速率，这与H295是天然酶中被DEP修饰的残基一致。这些有趣的结果表明，4个保守的组氨酸（H96、H105、H295和H233）对活性并非必不可少，虽然H295可能位于CK的活性位点，但它不太可能起到一般酸碱催化剂的作用。
