Differences in the roles of conserved glutamic acid residues in the active site of human class 3 and class 2 aldehyde dehydrogenases
Differences in the roles of conserved glutamic acid residues in the active site of human class 3 and class 2 aldehyde dehydrogenases
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DOI：
10.1110/ps.8.10.1922
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发表时间：
1999-10-01
期刊：
影响因子：
8
通讯作者：
Weiner, H
中科院分区：
文献类型：
--
作者：
Mann, CJ;Weiner, H
虽然二聚体3类大鼠乙醛脱氢酶的三维结构最近已经发表(Liu ZJ et al.，1997，Natural Struct Biol 4：317-326)，但对该同工酶的机理研究很少。我们对重组人胃醛脱氢酶的酶学性质进行了鉴定，该酶在氨基酸序列上与大鼠胃醛脱氢酶非常相似。我们已经确定人类3类同工酶的限速步骤是氢化物转移，而不是像观察到的人类肝脏2类线粒体酶那样的去酰化。NADH在与3类酶结合时没有观察到荧光增强，而先前在与2类同工酶结合时观察到NADH的荧光增强。还观察到NAD辅因子的结合抑制了3类酶的酯酶活性，而激活了2类酶的酯酶活性。两个保守的谷氨酸残基(209和333)对谷氨酰胺残基的定点突变表明，与第二类酶不同，Glu333在催化反应中作为一般碱基，而E209Q对酶活性只有轻微的影响，从而证实了所提出的机制(Hempel J等，1999，Adv Exp Med Bioi 436：53-59)。综上所述，这些数据表明，尽管同工酶的亚基结构和活性部位残基相似，但除了低聚状态(二聚体与四聚体)和底物专一性外，这些酶还有非常不同的性质。
Although the three-dimensional structure of the dimeric class 3 rat aldehyde dehydrogenase has recently been published (Liu ZJ et al., 1997, Nature Struct Biol 4:317-326), few mechanistic studies have been conducted on this isoenzyme. We have characterized the enzymatic properties of recombinant class 3 human stomach aldehyde dehydrogenase, which is very similar in amino acid sequence to the class 3 rat aldehyde dehydrogenase. We have determined that the rate-limiting step for the human class 3 isozyme is hydride transfer rather than deacylation as observed for the human liver class 2 mitochondrial enzyme. No enhancement of NADH fluorescence was observed upon binding to the class 3 enzyme, while fluorescence enhancement of NADH has been previously observed upon binding to the class 2 isoenzyme. It was also observed that binding of the NAD cofactor inhibited the esterase activity of the class 3 enzyme while activating the esterase activity of the class 2 enzyme. Site-directed mutagenesis of two conserved glutamic acid residues (209 and 333) to glutamine residues indicated that, unlike in the class 2 enzyme, Glu333 served as the general base in the catalytic reaction and E209Q had only marginal effects on enzyme activity, thus confirming the proposed mechanism (Hempel J et al., 1999, Adv Exp Med Bioi 436:53-59). Together, these data suggest that even though the subunit structures and active site residues of the isozymes are similar, the enzymes have very distinct properties besides their oligomeric state (dimer vs. tetramer) and substrate specificity.