Functional Role of a Mobile Loop of Escherichia coli Dihydrofolate Reductase in Transition-State Stabilization

The function of a highly mobile loop in Escherichia coli dihydrofolate reductase was studied by constructing a mutant (DL1) using cassette mutagenesis that had four residues deleted in the middle section of the loop (Metl6-Alal9) and a glycine inserted to seal the gap. This part of the loop involves residues 16-20 and is disordered in the X-ray crystal structures of the apoprotein and the NADP⁺binary complex but forms a hairpin turn that folds over the nicotinamide moiety of NADP⁺and the pteridine moiety of folate in the ternary complex [Bystroff, C., & Kraut, J. (1991) Biochemistry 30, 2227-2239]. The steady-state and pre-steady-state kinetics and two-dimensional ‘H NMR spectra were analyzed and compared to the wild-type protein. The kinetics on the DL1 mutant enzyme show that the K_M value for NADPH (5.3 µM), the K_M for dihydrofolate (2 µM), the rate constant for the release of the product tetrahydrofolate (10.3 s^-1). and the intrinsic pK_a value (6.2) are similar to those exhibited by the wild-type enzyme. However, the hydride-transfer rate declines markedly from the wild-type value of 950 s^-1to 1.7 s^-1for the DL1 mutant and when taken with data for substrate binding indicates that the loop contributes to substrate flux by a factor of 3.5 X 10⁴. Thus, the mobility of loop I may provide a mechanism of recruiting hydrophobic residues which can properly align the nicotinamide and pteridine rings for the hydride-transfer process (a form of transition-state stabilization). Two-dimensional NMR spectra show a slow (on the NMR time scale) exchange process at 298 K in the wild-type protein which cannot be detected in the deletion mutant. This slow process in the wild-type protein can be partially attributed to a two-site exchange of some of the residues in loop I. Therefore, the deletion of the hairpin-forming residues in loop I also affects the observed dynamics of the secondary structure in the wild-type protein and thus ties the changes in the kinetic behavior of the DL1 protein to the restricted movement of its loop I relative to the wild-type enzyme.