Caldwell J W, Agard D A, Kollman P A
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446.
Proteins. 1991;10(2):140-8. doi: 10.1002/prot.340100207.
We present free energy calculations using molecular dynamics on different substrates of alpha-lytic protease in the gas phase, in solution, while forming a noncovalent Michaelis complex with the enzyme, and in a tetrahedral structure representing a transition state/intermediate for acylation by the enzyme. Various P1 substrates were studied, with P1 = Gly, Ala, Val, and Leu. In qualitative agreement with experiment, the enzyme was calculated to bind and catalyze most effectively substrates with P1 = Ala over those with P1 = Gly, Val or Leu. Also, the calculated relative solvation free energies of Gly----Ala and Ala----Val were in qualitative agreement with experimental values in corresponding model systems. However, the level of quantitative agreement with experiment achieved in our earlier study of relative binding and catalysis of native subtilisin and an Asn-155----Ala mutant was not achieved. We surmise that this is due to the greater difficulty in quantitatively simulating effects that are predominantly van der Waals and hydrophobic compared to those that are hydrogen bonding/electrostatic.
我们利用分子动力学对α-裂解蛋白酶在气相、溶液中、与酶形成非共价米氏复合物时以及代表酶酰化过渡态/中间体的四面体结构中的不同底物进行了自由能计算。研究了各种P1底物，P1分别为甘氨酸、丙氨酸、缬氨酸和亮氨酸。与实验定性一致的是，计算结果表明，与P1为甘氨酸、缬氨酸或亮氨酸的底物相比，该酶与P1为丙氨酸的底物结合和催化最为有效。此外，计算得到的甘氨酸→丙氨酸和丙氨酸→缬氨酸的相对溶剂化自由能与相应模型系统中的实验值定性一致。然而，我们早期对天然枯草杆菌蛋白酶和Asn-155→Ala突变体的相对结合和催化研究中所达到的与实验的定量一致水平并未实现。我们推测，这是由于与氢键/静电作用相比定量模拟主要是范德华力和疏水作用的效应难度更大。
