Kiema T R, Engel C K, Schmitz W, Filppula S A, Wierenga R K, Hiltunen J K
Biocenter Oulu, Department of Biochemistry, University of Oulu, Finland.
Biochemistry. 1999 Mar 9;38(10):2991-9. doi: 10.1021/bi981646v.
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.
结构和酶学研究表明，Glu144和Glu164对2-烯酰辅酶A水合酶-1（巴豆酸酶）的催化作用至关重要。在此，我们报告大鼠线粒体2-烯酰辅酶A水合酶-1的Glu144Ala和Glu164Ala变体的酶学性质。尺寸排阻色谱法和圆二色光谱表明，野生型蛋白和突变体具有相似的寡聚化状态和折叠结构。活性位点突变体Glu144Ala和Glu164Ala的kcat值降低了约2000倍，但Km值未变。为了研究线粒体2-烯酰辅酶A水合酶-1（mECH-1）潜在的内在Δ3-Δ2-烯酰辅酶A异构酶活性，引入了一种以2-烯酰辅酶A水合酶-2和（R）-3-羟酰基辅酶A脱氢酶作为辅助酶的新检测方法。结果表明，大鼠野生型mECH-1也能够催化异构化反应，其活性比（异构化/水合作用）为1/5000。Glu144Ala和Glu164Ala中异构化反应的kcat值分别降低了10倍和1000倍。这些数据与以下观点一致：Glu164作为质子氨基酸残基参与水合作用和异构化反应。通过研究Gln162Ala、Gln162Met和Gln162Leu变体的酶学性质，探讨了有利于水合酶反应而非异构酶反应的结构因素。Gln162侧链与Glu164侧链形成氢键；然而，这些突变体的酶学性质与野生型相似，这表明Glu164侧链在水合酶和异构酶反应中的催化功能不依赖于与Gln162侧链的相互作用。
