Appleman J R, Howell E E, Kraut J, Blakley R L
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennesse 38101.
J Biol Chem. 1990 Apr 5;265(10):5579-84.
The apoenzyme of wild-type (WT) dihydrofolate reductase (DHRF) from Escherichia coli exists in two conformational states, Et and Ew, which differ in affinity for NADPH and in kinetic competence. Dissociation constants for the binary complex of NADPH with the two conformers differ by over 100-fold (KDt = 0.17 microM, KDw = 22 microM). Rate constants governing the interconversion of conformers are small (t1/2 for Ew----Et = 71 s), and since Ew is not catalytically competent, this conversion is accompanied by an increase in catalytic velocity. The equilibrium proportion of Et in the absence of ligands is 63%, but binding of NADPH greatly increases this proportion, and t1/2 for conversion of Ew.NADPH to Et.NADPH is 30 s. This conformational equilibrium has also been examined in mutant enzyme in which aspartate 27 is replaced by asparagine (D27N E. coli DHFR). Although ASp27 is an active site residue, it does not interact directly with bound NADPH, and in the mutant the rate constant for NADPH binding to Et is unchanged as are the dissociation constants for NADPH complexes with Et or Ew. However, for mutant apoenzyme, the proportion of Et is decreased to 18% in the absence of ligands so that the overall KD for NADPH is increased (0.15 microM for WT E. coli DHFR, 0.68 microM for D27N E. coli DHFR). The lower proportion of Et is due to a decreased rate for Ew----Et (t1/2 = 221 s) and an increased rate for Et----Ew (t1/2 = 50 s versus 120 s for WT E. coli DHFR).
来自大肠杆菌的野生型（WT）二氢叶酸还原酶（DHRF）的脱辅酶存在两种构象状态，即Et和Ew，它们对NADPH的亲和力以及动力学活性不同。NADPH与这两种构象体形成的二元复合物的解离常数相差超过100倍（KDt = 0.17微摩尔，KDw = 22微摩尔）。控制构象体相互转化的速率常数很小（Ew→Et的半衰期为71秒），由于Ew没有催化活性，这种转化伴随着催化速度的增加。在没有配体的情况下，Et的平衡比例为63%，但NADPH的结合大大增加了这一比例，Ew·NADPH转化为Et·NADPH的半衰期为30秒。还对天冬氨酸27被天冬酰胺取代的突变酶（D27N大肠杆菌DHFR）中的这种构象平衡进行了研究。尽管Asp27是一个活性位点残基，但它不与结合的NADPH直接相互作用，在突变体中，NADPH与Et结合的速率常数不变，NADPH与Et或Ew形成的复合物的解离常数也不变。然而，对于突变体脱辅酶，在没有配体的情况下，Et的比例降至18%，因此NADPH的总体KD增加（野生型大肠杆菌DHFR为0.15微摩尔，D27N大肠杆菌DHFR为0.68微摩尔）。Et比例较低是由于Ew→Et的速率降低（半衰期为221秒）以及Et→Ew的速率增加（野生型大肠杆菌DHFR的半衰期为120秒，而此处为50秒）。
