Department of Chemistry, Digital Technology Center, and Supercomputing Institute, University of Minnesota , 207 Pleasant Street Southeast, Minneapolis, Minnesota 55455, United States.
Biochemistry. 2013 Mar 26;52(12):2036-49. doi: 10.1021/bi301559q. Epub 2013 Jan 16.
Combined quantum mechanics/molecular mechanics molecular dynamics simulations reveal that the M20 loop conformational dynamics of dihydrofolate reductase (DHFR) is severely restricted at the transition state of the hydride transfer as a result of the M42W/G121V double mutation. Consequently, the double-mutant enzyme has a reduced entropy of activation, i.e., increased entropic barrier, and altered temperature dependence of kinetic isotope effects in comparison with those of wild-type DHFR. Interestingly, in both wild-type DHFR and the double mutant, the average donor-acceptor distances are essentially the same in the Michaelis complex state (~3.5 Å) and the transition state (2.7 Å). It was found that an additional hydrogen bond is formed to stabilize the M20 loop in the closed conformation in the M42W/G121V double mutant. The computational results reflect a similar aim designed to knock out precisely the dynamic flexibility of the M20 loop in a different double mutant, N23PP/S148A.
结合量子力学/分子力学分子动力学模拟揭示，二氢叶酸还原酶（DHFR）的 M20 环构象动力学在氢化物转移的过渡态受到严重限制，这是由于 M42W/G121V 双突变的结果。因此，与野生型 DHFR 相比，双突变酶的活化熵减小，即活化熵垒增加，并且动力学同位素效应的温度依赖性发生改变。有趣的是，在野生型 DHFR 和双突变体中，Michaelis 复合物状态（~3.5 Å）和过渡态（2.7 Å）下的平均供体-受体距离基本相同。发现双突变体中形成了额外的氢键以稳定 M20 环的封闭构象。计算结果反映了一种相似的目的，旨在精确消除不同的双突变体 N23PP/S148A 中 M20 环的动态灵活性。
