Effects of cavities at the nicotinamide binding site of liver alcohol dehydrogenase on structure, dynamics and catalysis.
Effects of cavities at the nicotinamide binding site of liver alcohol dehydrogenase on structure, dynamics and catalysis.
复制标题
肝醇脱氢酶烟酰胺结合位点空腔对结构、动力学和催化的影响。
DOI：
10.1021/bi401583f
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发表时间：
2014
期刊：
影响因子：
2.9
通讯作者：
Plapp,BryceV
中科院分区：
文献类型：
--
作者：
Yahashiri,Atsushi;Rubach,JonK;Plapp,BryceV
蛋白质动力学在氢转移酶催化中的作用已经得到了大量的科学支持，但蛋白质结构与催化之间的联系仍有待建立。缬氨酸残基203和207位于肝脏酒精脱氢酶中辅酶烟酰胺环的结合位点，被认为可以促进“蛋白质促进振动”（PPV）的催化作用。研究发现，V207A取代对稳态动力学常数和氢转移速率的影响较小；所引入的空腔是空的，对结构的影响最小（与NAD+和2,3,4,5,6-五氟苯甲醇的配合物在1.2 Å下确定）。因此，没有证据支持Val-207在催化动力学中的作用。V203A酶与NAD+和2,3,4,5,6-五氟苯甲醇或2,2,2-三氟乙醇（测定于1.1 Å）络合的蛋白质结构和配体几何形状（包括供体-受体距离）与野生型酶非常相似，除了引入的空腔容纳了一个接触烟酰胺环的新水分子。与先前的研究相比，V203A酶复合物的结构表明，隧道的减少和氢化物转移速率的降低（相对于野生型酶的16倍）不是由于基态配体几何形状的差异。V203A的取代可能会改变PPV和氢转移的重组能，但Michaelis复合物内的蛋白质支架和平衡热运动可能对酶催化作用更重要。
A role for protein dynamics in enzymatic catalysis of hydrogen transfer has received substantial scientific support, but the connections between protein structure and catalysis remain to be established. Valine residues 203 and 207 are at the binding site for the nicotinamide ring of the coenzyme in liver alcohol dehydrogenase and have been suggested to facilitate catalysis with “protein-promoting vibrations” (PPV). We find that the V207A substitution has small effects on steady-state kinetic constants and the rate of hydrogen transfer; the introduced cavity is empty and is tolerated with minimal effects on structure (determined at 1.2 Å for the complex with NAD+and 2,3,4,5,6-pentafluorobenzyl alcohol). Thus, no evidence is found to support a role for Val-207 in the dynamics of catalysis. The protein structures and ligand geometries (including donor–acceptor distances) in the V203A enzyme complexed with NAD+and 2,3,4,5,6-pentafluorobenzyl alcohol or 2,2,2-trifluoroethanol (determined at 1.1 Å) are very similar to those for the wild-type enzyme, except that the introduced cavity accommodates a new water molecule that contacts the nicotinamide ring. The structures of the V203A enzyme complexes suggest, in contrast to previous studies, that the diminished tunneling and decreased rate of hydride transfer (16-fold, relative to that of the wild-type enzyme) are not due to differences in ground-state ligand geometries. The V203A substitution may alter the PPV and the reorganization energy for hydrogen transfer, but the protein scaffold and equilibrium thermal motions within the Michaelis complex may be more significant for enzyme catalysis.