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首页> 外文期刊>Biochemistry >Mutagenic and enzymological studies of the hydratase and isomerase activities of 2-enoyl-CoA hydratase-1.
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Mutagenic and enzymological studies of the hydratase and isomerase activities of 2-enoyl-CoA hydratase-1.

机译：2-enoyl-CoA水合酶-1的水合酶和异构酶活性的诱变和酶学研究。

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摘要

Structural and enzymological studies have shown the importance of Glu144 and Glu164 for the catalysis by 2-enoyl-CoA hydratase-1 (crotonase). Here we report about the enzymological properties of the Glu144Ala and Glu164Ala variants of rat mitochondrial 2-enoyl-CoA hydratase-1. Size-exclusion chromatography and CD spectroscopy showed that the wild-type protein and mutants have similar oligomerization states and folding. The kcat values of the active site mutants Glu144Ala and Glu164Ala were decreased about 2000-fold, but the Km values were unchanged. For study of the potential intrinsic Delta3-Delta2-enoyl-CoA isomerase activity of mECH-1, a new assay using 2-enoyl-CoA hydratase-2 and (R)-3-hydroxyacyl-CoA dehydrogenase as auxiliary enzymes was introduced. It was demonstrated that rat wild-type mECH-1 is also capable of catalyzing isomerization with the activity ratio (isomerization/hydration) of 1/5000. The kcat values of isomerization in Glu144Ala and Glu164Ala were decreased 10-fold and 1000-fold, respectively. The data are in line with the proposal that Glu164 acts as a protic amino acid residue for both the hydration and the isomerization reaction. The structural factors favoring the hydratase over the isomerase reaction have been addressed by investigating the enzymological properties of the Gln162Ala, Gln162Met, and Gln162Leu variants. The Gln162 side chain is hydrogen bonded to the Glu164 side chain; nevertheless, these mutants have enzymatic properties similar to that of the wild type, indicating that catalytic function of the Glu164 side chain in the hydratase and isomerase reaction does not depend on the interactions with the Gln162 side chain.
机译：结构和酶学研究表明，Glu144和Glu164对于2-烯酰基-CoA水合酶-1（巴豆酶）催化的重要性。在这里，我们报告有关大鼠线粒体2-enoyl-CoA水合酶-1 Glu144Ala和Glu164Ala变体的酶学性质。尺寸排阻色谱法和CD光谱法表明，野生型蛋白质和突变体具有相似的低聚状态和折叠。活性位点突变体Glu144Ala和Glu164Ala的kcat值降低了约2000倍，但Km值没有变化。为了研究mECH-1的潜在固有固有Delta3-Delta2-enoyl-CoA异构酶活性，引入了一种新的测定方法，该方法使用2-enoyl-CoA水合酶-2和（R）-3-羟酰基-CoA脱氢酶作为辅助酶。已经证明，大鼠野生型mECH-1还能够以1/5000的活性比（异构化/水合）催化异构化。 Glu144Ala和Glu164Ala中异构化的kcat值分别降低了10倍和1000倍。数据符合以下建议：Glu164既可以作为水合反应又可以作为异构化反应的质子氨基酸残基。通过研究Gln162Ala，Gln162Met和Gln162Leu变体的酶学性质，已经解决了比异构酶反应更倾向于水合酶的结构因素。 Gln162侧链通过氢键连接到Glu164侧链;然而，这些突变体具有与野生型相似的酶促性质，表明在水合酶和异构酶反应中Glu164侧链的催化功能不取决于与Gln162侧链的相互作用。

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