Active site modifications in a double mutant of liver alcohol dehydrogenase: Structural studies of two enzyme-ligand complexes
Active site modifications in a double mutant of liver alcohol dehydrogenase: Structural studies of two enzyme-ligand complexes
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DOI：
10.1021/bi973184b
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发表时间：
1998-06-30
期刊：
影响因子：
2.9
通讯作者：
Goldstein, BM
中科院分区：
文献类型：
--
作者：
Colby, TD;Bahnson, BJ;Goldstein, BM
马肝酒精脱氢酶（LADH）将酒精氧化为醛，需要将一个氢化物离子从酒精底物转移到辅助因子烟酰胺腺嘌呤二核苷酸（NAD）上。量子力学隧穿对氢化物转移步骤的贡献已经在许多LADH突变体中得到证明，这些突变体旨在增强或减弱这种效应[Bahnson， b.j.等人，（1997）Proc. Natl.]。学会科学。[j].美国地质学报，1999,19(4):12797-12802。活性位点双突变体Phe(93) -> Trp/Val(203) -> Ala的催化效率比天然酶降低了75倍，与任何单一突变体相比，隧道效应也减少了。我们得到了双突变体的两种晶体结构：与NAD和底物类似物三氟乙醇的2.0埃配合物和与NAD类似物CPAD和乙醇的2.6埃配合物。在这两种复合物中观察到的活性部位的变化与活性降低和隧道化一致。nad1 -三氟乙醇配合物以活性酶的封闭构象结晶。然而，NAD烟酰胺环旋转远离底物，朝向Val（203）被较小的丙氨酸取代而空出的空间。用更大的色氨酸取代苯丙氨酸（93）也会与烟酰胺羧基产生不利的空间接触，潜在地破坏维持封闭构象所需的氢键。这些接触在第二个配合物中通过CPAD吡啶环旋转成不寻常的同步取向而减轻。羧酸酰胺氢键的损失产生了脱酶特征的开放构象。
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.