INVESTIGATION OF THE ACTIVE-SITE CYSTEINE RESIDUE OF RAT-LIVER MITOCHONDRIAL ALDEHYDE DEHYDROGENASE BY SITE-DIRECTED MUTAGENESIS
INVESTIGATION OF THE ACTIVE-SITE CYSTEINE RESIDUE OF RAT-LIVER MITOCHONDRIAL ALDEHYDE DEHYDROGENASE BY SITE-DIRECTED MUTAGENESIS
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DOI：
10.1021/bi00008a025
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发表时间：
1995-02-28
期刊：
影响因子：
2.9
通讯作者：
WEINER, H
中科院分区：
文献类型：
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作者：
FARRES, J;WANG, TTY;WEINER, H
为了确定醛脱氢酶的活性位点半胱氨酸残基，我们突变重组表达的大鼠肝线粒体（2类）醛脱氢酶的氨基酸残基49，162和302。C49 A和C162 A突变体是完全活性的四聚体酶，尽管C162 A突变体被发现是高度不稳定的。C302 A突变体也是一个四聚体和结合辅酶，但缺乏脱氢酶和酯酶活性。为了测试半胱氨酸302作为亲核试剂的作用，将残基突变为丝氨酸，一种弱亲核试剂。该C302 S突变体具有活性，但催化剂差得多，k（cat）/K-m值比重组天然酶低7 × 10（5）倍。与天然酶不同，脱酰作用是限速的，丝氨酸半缩醛中间体的形成似乎是限速步骤。半胱氨酸302是醛脱氢酶超家族的所有可用序列中唯一严格保守的半胱氨酸残基，支持该残基作为醛脱氢酶的活性位点亲核体的作用。
To determine the active site cysteine residue in aldehyde dehydrogenase, we mutated amino acid residues 49, 162, and 302 of recombinantly expressed rat liver mitochondrial (class 2) aldehyde dehydrogenase. The C49A and C162A mutants were fully active tetrameric enzymes, although the C162A mutant was found to be highly unstable. The C302A mutant was also a tetramer and bound coenzyme, but lacked both dehydrogenase and esterase activities. To test for the role of cysteine 302 as a nucleophile, the residue was mutated to a serine, a poor nucleophile. This C302S mutant was active but was a much poorer catalyst, with a k(cat)/K-m value 7 x 10(5) times lower than that of the recombinant native enzyme. Unlike with native enzyme where deacylation is rate limiting, formation of the serine hemiacetal intermediate appeared to be the rate-limiting step. Cysteine 302 is the only strictly conserved cysteine residue among all the available sequences of the aldehyde dehydrogenase superfamily, supporting the role of this residue as the active site nucleophile of aldehyde dehydrogenase.