Djordjevic S, Dong Y, Paschke R, Frerman F E, Strauss A W, Kim J J
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Biochemistry. 1994 Apr 12;33(14):4258-64. doi: 10.1021/bi00180a021.
We have used molecular modeling and site-directed mutagenesis to identify the catalytic residues of human long chain acyl-CoA dehydrogenase. Among the acyl-CoA dehydrogenases, a family of flavoenzymes involved in beta-oxidation of fatty acids, only the three-dimensional structure of the medium chain fatty acid specific enzyme from pig liver has been determined (Kim, J.-J.P., Wang, M., & Paschke, R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7523-7527). Despite the overall sequence homology, the catalytic residue (E376) of medium chain acyl-CoA dehydrogenase is not conserved in isovaleryl- and long chain acyl-CoA dehydrogenases. A molecular model of human long chain acyl-CoA dehydrogenase was derived using atomic coordinates determined by X-ray diffraction studies of the pig medium chain specific enzyme, interactive graphics, and molecular mechanics calculations. The model suggests that E261 functions as the catalytic base in the long-chain dehydrogenase. An altered dehydrogenase in which E261 was replaced by a glutamine was constructed, expressed, purified, and characterized. The mutant enzyme exhibited less than 0.02% of the wild-type activity. These data strongly suggest that E261 is the base that abstracts the alpha-proton of the acyl-CoA substrate in the catalytic pathway of this dehydrogenase.
我们利用分子建模和定点诱变来确定人类长链酰基辅酶A脱氢酶的催化残基。在酰基辅酶A脱氢酶中，这是一族参与脂肪酸β-氧化的黄素酶，目前仅确定了猪肝中链脂肪酸特异性酶的三维结构（Kim, J.-J.P., Wang, M., & Paschke, R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7523 - 7527）。尽管存在整体序列同源性，但中链酰基辅酶A脱氢酶的催化残基（E376）在异戊酰基辅酶A脱氢酶和长链酰基辅酶A脱氢酶中并不保守。人类长链酰基辅酶A脱氢酶的分子模型是利用通过对猪中链特异性酶进行X射线衍射研究确定的原子坐标、交互式图形和分子力学计算推导出来的。该模型表明E261在长链脱氢酶中起催化碱基的作用。构建了一个将E261替换为谷氨酰胺的变异脱氢酶，对其进行表达、纯化和特性鉴定。该突变酶的活性不到野生型活性的0.02%。这些数据有力地表明E261是该脱氢酶催化途径中夺取酰基辅酶A底物α-质子的碱基。
