Biochemical characterization and crystal structure determination of human heart short chain L-3-Hydroxyacyl-CoA dehydrogenase provide insights into catalytic mechanism
Biochemical characterization and crystal structure determination of human heart short chain L-3-Hydroxyacyl-CoA dehydrogenase provide insights into catalytic mechanism
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DOI：
10.1021/bi9829027
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发表时间：
1999-05-04
期刊：
影响因子：
2.9
通讯作者：
Banaszak, LJ
中科院分区：
文献类型：
--
作者：
Barycki, JJ;O'Brien, LK;Banaszak, LJ
人心脏短链L-3-羟酰基-CoA脱氢酶（SCHAD）催化L-3-羟酰基-CoA的羟基氧化为酮基，同时NAD（+）还原为NADH，作为β-氧化途径的一部分。同源二聚体酶已在大肠杆菌中过表达，纯化至均一，并使用生物化学和晶体学技术进行研究。NAD（+）和NADH的解离常数已在较宽的pH范围内测定，表明SCHAD优先结合还原型辅因子。表观催化常数的检查表明，SCHAD显示最佳的酶活性接近中性pH值，与催化效率迅速下降对pH极端。用多波长反常衍射技术和硒代蛋氨酸取代的酶类似物解决了与NAD（+）络合的SCHAD的晶体结构。亚基结构由两个结构域组成。第一个结构域类似于其他α/β二核苷酸折叠，但包括一个不寻常的螺旋-转角-螺旋基序，其从中心β-折叠延伸。第二，或C-末端，结构域主要是α-螺旋和介导亚基二聚化，并推测，L-3-羟酰辅酶A结合。将L-3-羟基丁酰-CoA对接到酶-NAD（+）复合物中的分子建模研究表明，His 158作为一般碱基，从底物的3-OH基团中提取质子。此外，His 158执行这种功能的能力可以通过与Glu 170的静电相互作用来增强，这与先前的生化观察一致。这些研究提供了与L-3-羟酰辅酶A脱氢酶缺乏相关的几种遗传性代谢疾病状态的分子基础的进一步理解。
Human heart short chain L-3-hydroxyacyl-CoA dehydrogenase(SCHAD) catalyzes the oxidation of the hydroxyl group of L-3-hydroxyacyl-CoA to a keto group, concomitant with the reduction of NAD(+) to NADH, as part of the beta-oxidation pathway. The homodimeric enzyme has been overexpressed in Escherichia coli, purified to homogeneity, and studied using biochemical and crystallographic techniques. The dissociation constants of NAD(+) and NADH have been determined over a broad pH range and indicate that SCHAD binds reduced cofactor preferentially. Examination of apparent catalytic constants reveals that SCHAD displays optimal enzymatic activity near neutral pH, with catalytic efficiency diminishing rapidly toward pH extremes. The crystal structure of SCHAD complexed with NAD(+) has been solved using multiwavelength anomalous diffraction techniques and a selenomethionine-substituted analogue of the enzyme. The subunit structure is comprised of two domains. The first domain is similar to other alpha/beta dinucleotide folds but includes an unusual helix-turn-helix motif which extends from the central beta-sheet. The second, or C-terminal, domain is primarily alpha-helical and mediates subunit dimerization and, presumably, L-3-hydroxyacyl-CoA binding. Molecular modeling studies in which L-3-hydroxybutyryl-CoA was docked into the enzyme-NAD(+) complex suggest that His 158 serves as a general base, abstracting a proton from the 3-OH group of the substrate. Furthermore, the ability of His 158 to perform such a function may be enhanced by an electrostatic interaction with Glu 170, consistent with previous biochemical observations. These studies provide further understanding of the molecular basis of several inherited metabolic disease states correlated with L-3-hydroxyacyl-CoA dehydrogenase deficiencies.