Department of Chemistry and Biochemistry, Notre Dame University , Notre Dame, Indiana 46556, USA.
Biochemistry. 2012 Oct 9;51(40):7983-95. doi: 10.1021/bi3008593. Epub 2012 Sep 25.
HMG-CoA reductase catalyzes the four-electron reduction of HMG-CoA to mevalonate and is an enzyme of considerable biomedical relevance because of the impact of its statin inhibitors on public health. Although the reaction has been studied extensively using X-ray crystallography, there are surprisingly no computational studies that test the mechanistic hypotheses suggested for this complex reaction. Theozyme and quantum mechanical (QM)/molecular mechanical (MM) calculations up to the B3LYP/6-31g(d,p)//B3LYP/6-311++g(2d,2p) level of theory were employed to generate an atomistic description of the enzymatic reaction process and its energy profile. The models generated here predict that the catalytically important Glu83 is protonated prior to hydride transfer and that it acts as the general acid or base in the reaction. With Glu83 protonated, the activation energies calculated for the sequential hydride transfer reactions, 21.8 and 19.3 kcal/mol, are in qualitative agreement with the experimentally determined rate constant for the entire reaction (1 s(-1) to 1 min(-1)). When Glu83 is not protonated, the first hydride transfer reaction is predicted to be disfavored by >20 kcal/mol, and the activation energy is predicted to be higher by >10 kcal/mol. While not involved in the reaction as an acid or base, Lys267 is critical for stabilization of the transition state in forming an oxyanion hole with the protonated Glu83. Molecular dynamics simulations and MM/Poisson-Boltzmann surface area free energy calculations predict that the enzyme active site stabilizes the hemithioacetal intermediate better than the aldehyde intermediate. This suggests a mechanism in which cofactor exchange occurs before the breakdown of the hemithioacetal. Slowing the conversion to aldehyde would provide the enzyme with a mechanism to protect it from solvent and explain why the free aldehyde is not observed experimentally. Our results support the hypothesis that the pK(a) of an active site acidic group is modulated by the redox state of the cofactor. The oxidized cofactor and deprotonated Glu83 are closer in space after hydride transfer, indicating that indeed the cofactor may influence the pK(a) of Glu83 through an electrostatic interaction. The enzyme is able to catalyze the transfer of a hydride to the structurally and electronically distinct substrates by maintaining the general shape of the active site and adjusting the electrostatic environment through acid-base chemistry. Our results are in good agreement with the well-studied hydride transfer reactions catalyzed by liver alcohol dehydrogenase in calculated energy profile and reaction geometries despite different mechanistic functionalities.
HMG-CoA 还原酶催化 HMG-CoA 的四电子还原为甲羟戊酸，由于其他汀类抑制剂对公众健康的影响，它是一种具有相当重要的生物医学相关性的酶。尽管已经使用 X 射线晶体学对该反应进行了广泛的研究，但令人惊讶的是，没有计算研究测试过该复杂反应的机制假设。Theozyme 和量子力学（QM）/分子力学（MM）计算，最高可达 B3LYP/6-31g(d,p)//B3LYP/6-311++g(2d,2p)理论水平，用于生成酶促反应过程及其能量曲线的原子描述。这里生成的模型预测，催化上重要的 Glu83 在氢化物转移前质子化，并且在反应中充当广义酸或碱。当 Glu83 质子化时，计算出的顺序氢化物转移反应的活化能分别为 21.8 和 19.3 kcal/mol，与整个反应的实验确定的速率常数（1 s(-1) 至 1 min(-1))定性一致。当 Glu83 未质子化时，第一个氢化物转移反应被预测会受到超过 20 kcal/mol 的不利影响，并且活化能预计会高出超过 10 kcal/mol。虽然 Lys267 不作为酸或碱参与反应，但它对于在形成带有质子化 Glu83 的氧阴离子空穴时稳定过渡态至关重要。分子动力学模拟和 MM/泊松-玻尔兹曼表面面积自由能计算预测，酶活性位点比醛中间体更好地稳定半硫缩醛中间体。这表明在半硫缩醛分解之前发生辅助因子交换的机制。减缓向醛的转化将为酶提供一种保护机制，以防止其与溶剂接触，并解释为什么实验中未观察到游离醛。我们的结果支持这样的假设，即活性位点酸性基团的 pK(a) 被辅助因子的氧化还原状态调节。氢化物转移后，氧化的辅助因子和去质子化的 Glu83 空间更接近，这表明实际上辅助因子可能通过静电相互作用影响 Glu83 的 pK(a)。酶通过维持活性位点的一般形状并通过酸碱化学调整静电环境，能够催化氢化物向结构和电子上不同的底物的转移。尽管具有不同的机械功能，但我们的结果与已研究充分的肝醇脱氢酶催化的氢化物转移反应在计算能量曲线和反应几何形状上非常吻合。
