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首页> 外文期刊>Biochemistry >A histidine residue in the catalytic mechanism distinguishes Vibrio harveyi aldehyde dehydrogenase from other members of the aldehyde dehydrogenase superfamily.
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A histidine residue in the catalytic mechanism distinguishes Vibrio harveyi aldehyde dehydrogenase from other members of the aldehyde dehydrogenase superfamily.

机译：催化机理中的组氨酸残基将哈维弧菌醛脱氢酶与醛脱氢酶超家族的其他成员区分开。

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摘要

Aldehyde dehydrogenases (ALDHs) catalyze the transfer to NAD(P) of a hydride ion from a thiohemiacetal derivative of the aldehyde coupled with a cysteine residue in the active site. In Vibrio harveyi aldehyde dehydrogenase (Vh-ALDH), a histidine residue (H450) is in proximity (3.8 A) to the cysteine nucleophile (C289) and is thus capable of increasing its reactivity in sharp contrast to other ALDHs in which more distantly located glutamic acid residues are proposed to act as the general base. Mutation of H450 in Vh-ALDH to Gln and Asn resulted in loss of dehydrogenase, (thio)esterase, and acyl-CoA reductase activities; the residual activity of H450Q was higher than that of the H450N mutant in agreement with the capability of Gln but not Asn to partially replace the epsilon-imino group of H450. Coupled with a change in the rate-limiting step, these results indicate that H450 increases the reactivity of C289. Moreover, for the first time, the acylated enzyme intermediate could be directly monitored after reaction with [(3)H]tetradecanoyl-CoA showing that the H450Q mutant was acylated more rapidly than the H450N mutant. Inactivation of the wild-type enzyme with N-ethylmaleimide was much more rapid than the H450Q mutant which in turn was faster than the H450N mutant, demonstrating directly that the nucleophilicity of C289 was affected by H450. As the glutamic acid residue implicated as the general base in promoting cysteine nucleophilicity in other ALDHs is conserved in Vh-ALDH, elucidation of why a histidine residue has evolved to assist in this function in Vh-ALDH will be important to understand the mechanism of ALDHs in general, as well as help delineate the specific roles of the active site glutamic acid residues.
机译：醛脱氢酶（ALDHs）催化醛的硫半缩醛衍生物与活性位点中的半胱氨酸残基偶联的氢化物离子转移至NAD（P）。在哈维弧菌醛脱氢酶（Vh-ALDH）中，组氨酸残基（H450）与半胱氨酸亲核试剂（C289）接近（3.8 A），因此能够与其他距离更远的其他ALDH形成鲜明对比，从而提高其反应活性谷氨酸残基被提议作为一般碱。 Vh-ALDH中的H450突变为Gln和Asn导致脱氢酶，（硫）酯酶和酰基辅酶A还原酶活性的丧失; H450Q的残余活性高于H450N突变体，这与Gln的能力一致，但Asn不能部分取代H450的ε-亚氨基。结合限速步骤的变化，这些结果表明H450增加了C289的反应性。此外，首次与[（3）H]十四烷酰-辅酶A反应后，可以直接监测酰化酶中间体，表明H450Q突变体比H450N突变体被更快地酰化。用N-乙基马来酰亚胺灭活野生型酶比H450Q突变体快得多，而H450Q突变体又比H450N突变体快，这直接证明C289的亲核性受H450影响。由于在Vh-ALDH中保守了谷氨酸残基，它被认为是促进其他ALDHs中半胱氨酸亲核性的通用碱基，因此阐明为什么组氨酸残基已经进化为在Vh-ALDH中有助于此功能的原因对于理解ALDHs的机制非常重要。通常，以及有助于描述活性位点谷氨酸残基的特定作用。

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