Chen J T, Mayer R J, Fierke C A, Benkovic S J
J Cell Biochem. 1985;29(2):73-82. doi: 10.1002/jcb.240290203.
Two site-specific mutations of dihydrofolate reductase from Escherichia coli based on the x-ray crystallographic structure were constructed. The first mutation (His-45----Gln) is aimed at assessing the interaction between the imidazole moiety and the pyrophosphate backbone of NADPH. The second (Thr-113----Val) is part of a hydrogen bonding network that contacts the dihydrofolate substrate and may be involved in proton delivery to the N5-C6 imine undergoing reduction. The first mutation was shown to alter both the association and dissociation rate constants for the cofactor so that the dissociation constant was increased 6-40-fold. A corresponding but smaller (fourfold) effect was noted in V/K but not in V compared to the wild-type enzyme. The second was demonstrated to increase the dissociation rate constant for methotrexate 20-30-fold, and presumably dihydrofolate also, with a corresponding 20-30-fold increase in the dissociation constant. In this case an identical effect was noted on V/K but not in V relative to the native enzyme. Thus, in both mutant enzymes the decrease in binding has not been translated into a loss of catalytic efficiency.
基于X射线晶体结构构建了大肠杆菌二氢叶酸还原酶的两个位点特异性突变体。第一个突变（His-45→Gln）旨在评估咪唑部分与NADPH焦磷酸骨架之间的相互作用。第二个突变（Thr-113→Val）是与二氢叶酸底物接触的氢键网络的一部分，可能参与向正在被还原的N5-C6亚胺传递质子。结果表明，第一个突变改变了辅因子的结合和解离速率常数，使得解离常数增加了6至40倍。与野生型酶相比，在V/K中观察到相应但较小的（四倍）影响，但在V中未观察到。第二个突变被证明使甲氨蝶呤的解离速率常数增加了20至30倍，推测二氢叶酸也是如此，解离常数相应增加了20至30倍。在这种情况下，相对于天然酶，在V/K中观察到相同的影响，但在V中未观察到。因此，在两种突变酶中，结合的降低并未转化为催化效率的损失。
