Mizushima N, Spellmeyer D, Hirono S, Pearlman D, Kollman P
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
J Biol Chem. 1991 Jun 25;266(18):11801-9.
We present the results of free energy perturbation calculations on binding and catalysis of a tetrapeptide substrate, acetyl-Phe-Ala-Ala-Phe-NMe, by native subtilisin BPN' and a subtilisin BPN' mutant (Thr220----Ala220). The calculated difference in the free energy of binding was 0.70 +/- 0.72 kcal/mol. The calculated difference in the free energy of catalysis was 1.48 +/- 0.89 kcal/mol. These calculated values compare well with the experimental values in which another substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, was used. These findings suggest that Thr220 is more important for catalysis than substrate binding.
我们展示了关于天然枯草杆菌蛋白酶BPN'和一种枯草杆菌蛋白酶BPN'突变体（苏氨酸220突变为丙氨酸220）对四肽底物乙酰 - 苯丙氨酸 - 丙氨酸 - 苯丙氨酸 - N - 甲基酯的结合和催化作用的自由能扰动计算结果。计算得到的结合自由能差异为0.70±0.72千卡/摩尔。计算得到的催化自由能差异为1.48±0.89千卡/摩尔。这些计算值与使用另一种底物琥珀酰 - 丙氨酸 - 丙氨酸 - 脯氨酸 - 苯丙氨酸 - 对硝基苯胺的实验值相当。这些发现表明，苏氨酸220对催化作用比对底物结合更为重要。
