He X Y, Yang S Y
Department of Pharmacology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA.
Biochemistry. 1997 Sep 9;36(36):11044-9. doi: 10.1021/bi970901t.
Glu139 of the large alpha-subunit of the multienzyme complex of fatty acid oxidation from Escherichia coli was identified as the catalytic residue of enoyl-CoA hydratase [Yang, S.-Y., He, X.-Y., & Schulz, H. (1995) Biochemistry 34, 6441-6447]. To determine whether any of the other conserved protic residues is directly involved in the hydratase catalysis, the multienzyme complexes with either an alpha/Asp69 --> Asn or an alpha/Glu119 --> Gln mutation were overproduced and characterized. The catalytic properties of 3-ketoacyl-CoA thiolase and l-3-hydroxyacyl-CoA dehydrogenase of the mutant complexes were almost unaffected. The amidation of Asp69 and Glu119 caused a 7.6- and 88-fold decrease, respectively, in the kcat of enoyl-CoA hydratase without a significant change in the Km value of the hydratase as well as a 5.9- and 62-fold increase, respectively, in the Km of Delta3-cis-Delta2-trans-enoyl-CoA isomerase with a very small decrease in the kcat of the latter enzyme. The data suggest that the carboxyl group of Glu119 is particularly important to the catalytic activity of enoyl-CoA hydratase. Furthermore, the wild-type hydratase shows a bell-shaped pH dependence of the kcat/Km with pKa values of 5.9 and 9.2, whereas the Glu119 --> Gln mutant hydratase has only a single pKa of 9.5. A simple explanation for these observations is that a deprotonated Glu119 and a protonated Glu139 are required for the high kcat of the enoyl-CoA hydratase. The results of site-directed mutagenesis studies, together with the structural information about the spatial arrangement of two conserved glutamate residues of rat liver enoyl-CoA hydratase [Engel, C. K., Mathieu, M., Zeelen, J. P., Hiltunen, J. K., and Wierenga, R. K. (1996) EMBO J. 15, 5135-5145] to which Glu119 and Glu139 of the large alpha-subunit correspond, lead to the conclusion that the gamma-carboxyl group of Glu119 serves as the second general acid-base functional group in catalyzing the hydration of 2-trans-enoyl-CoA.
来自大肠杆菌的脂肪酸氧化多酶复合体大亚基中的Glu139被鉴定为烯酰辅酶A水合酶的催化残基[杨，S.-Y.，何，X.-Y.，& 舒尔茨，H.（1995年）《生物化学》34卷，6441 - 6447页]。为了确定其他保守的质子供体残基是否直接参与水合酶催化，对具有α/Asp69→Asn或α/Glu119→Gln突变的多酶复合体进行了过量表达和特性分析。突变复合体中3 - 酮酰辅酶A硫解酶和l - 3 - 羟酰辅酶A脱氢酶的催化特性几乎未受影响。Asp69和Glu119的酰胺化分别导致烯酰辅酶A水合酶的kcat下降7.6倍和88倍，而水合酶的Km值无显著变化，同时分别使Δ3 - 顺式 - Δ2 - 反式 - 烯酰辅酶A异构酶的Km增加5.9倍和62倍，而后一种酶的kcat仅有非常小的下降。数据表明Glu119的羧基对烯酰辅酶A水合酶的催化活性尤为重要。此外，野生型水合酶的kcat/Km呈现钟形pH依赖性，pKa值分别为5.9和9.2，而Glu119→Gln突变型水合酶仅有一个pKa值为9.5。对这些观察结果的一个简单解释是，烯酰辅酶A水合酶的高kcat需要一个去质子化的Glu119和一个质子化的Glu139。定点诱变研究的结果，连同大鼠肝脏烯酰辅酶A水合酶两个保守谷氨酸残基空间排列的结构信息[恩格尔，C. K.，马蒂厄，M.，泽伦，J. P.，希尔图宁，J. K.，和维伦加，R. K.（1996年）《欧洲分子生物学组织杂志》15卷，5135 - 5145页]（大亚基中的Glu119和Glu139与之对应），得出结论：Glu119的γ - 羧基在催化2 - 反式 - 烯酰辅酶A水合反应中作为第二个一般酸碱功能基团。
