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首页> 外文期刊>Biochemistry >Active site modifications in a double mutant of liver alcohol dehydrogenase: Structural studies of two enzyme-ligand complexes
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Active site modifications in a double mutant of liver alcohol dehydrogenase: Structural studies of two enzyme-ligand complexes

机译：肝酒精脱氢酶的双突变体中的活性位点修饰：两种酶-配体复合物的结构研究

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The oxidation of alcohol to aldehyde by horse liver alcohol dehydrogenase (LADH) requires the transfer of a hydride ion from the alcohol substrate to the cofactor nicotinamide adenine dinucleotide (NAD). A quantum mechanical tunneling contribution to this hydride transfer step has been demonstrated in a number of LADH mutants designed to enhance or diminish this effect [Bahnson, B. J., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 12797-12802]. The active site double mutant Phe(93) --> Trp/Val(203) --> Ala shows a 75-fold reduction in catalytic efficiency relative to that of the native enzyme, and reduced tunneling relative to that of either single mutant. We present hen two crystal structures of the double mutant: a 2.0 Angstrom complex with NAD and the substrate analogue trifluoroethanol and a 2.6 Angstrom complex with the isosteric NAD analogue CPAD and ethanol. Changes at the active site observed in both complexes are consistent with reduced activity and tunneling. The NAD-trifluoroethanol complex crystallizes in the closed conformation characteristic of the active enzyme. However, the NAD nicotinamide ring rotates away from the substrate, toward the space vacated by replacement of Val(203) with the smaller alanine. Replacement of Phe(93) with the larger tryptophan also produces unfavorable steric contacts with the nicotinamide carboxamide group, potentially destabilizing hydrogen bonds required to maintain the closed conformation. These contacts are relieved in the second complex by rotation of the CPAD pyridine ring into an unusual syn orientation. The resulting loss of the carboxamide hydrogen bonds produces an open conformation characteristic of the apoenzyme. [References: 44]
机译：通过马肝醇脱氢酶（LADH）将醇氧化为醛，需要将氢离子从醇底物转移至辅因子烟酰胺腺嘌呤二核苷酸（NAD）。在许多设计用来增强或减弱这种作用的LADH突变体中，已经证明了量子机械隧穿对这一氢化物转移步骤的贡献[Bahnson，B.J。等人，J.Biol.Chem.Soc。，1993，8，1897]。 （1997年）过程。 Natl。学院科学U.S.A. 94，12797-12802]。活性位点双突变体Phe（93）-> Trp / Val（203）->丙氨酸相对于天然酶显示出75倍的催化效率降低，相对于任一单个突变体而言，其隧穿降低。我们介绍了双突变体的两个晶体结构：一个2.0埃配合物与NAD和底物类似物三氟乙醇和一个2.6埃配合物与等距NAD类似物CPAD和乙醇。在两种复合物中观察到的活性位点的变化与活性降低和隧穿一致。 NAD-三氟乙醇复合物以活性酶的闭合构象特征结晶。但是，NAD烟酰胺环从底物旋转，朝着用较小的丙氨酸替换Val（203）所腾空的空间旋转。用较大的色氨酸替换Phe（93）也会与烟酰胺羧酰胺基团产生不利的空间接触，可能会破坏维持闭合构象所需的氢键。通过将CPAD吡啶环旋转到异常的syn方向，可以缓解第二个络合物中的这些接触。所产生的羧酰胺氢键的丧失产生了脱辅酶的开放构象特征。 [参考：44]

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