EVIDENCE FOR A FUNCTIONAL-ROLE OF THE DYNAMICS OF GLYCINE-121 OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE OBTAINED FROM KINETIC-ANALYSIS OFA SITE-DIRECTED MUTANT

Two-dimensional heteronuclear (H-1-N-15) nuclear magnetic relaxation s tudies of dihydrofolate reductase (DHFR) from Escherichia coli have de monstrated that glycine-121 which is 19 Angstrom from the catalytic ce nter of the enzyme has large-amplitude backbone motions on the nanosec ond time scale [Epstein, D. M., Benkovic, S. J., and Wright, P. E. (19 95) Biochemistry 34, 11037-11048]. in order to probe the dynamic-funct ion relationships of this residue, we constructed a mutant enzyme in w hich this glycine was changed to valine. Equilibrium binding studies i ndicated that the Val-121 mutant retained wild-type binding properties with respect to dihydrofolate and tetrahydrofolate; however, binding to NADPH and NADP(+) was decreased by 40-fold and 2-fold, respectively , relative to wild-type DHFR. Single-turnover experiments indicated th at hydride transfer was reduced by 200-fold to a rate of 1.3 s(-1) and was the rate-limiting step in the steady state. Interestingly, pre-st eady-state kinetic analysis of the Val-121 mutant revealed a conformat ional change which preceded chemistry that occurred at a rate of 3.5 s (-1). If this step exists in the kinetic mechanism of the wild-type en zyme, then it would be predicted to occur at a rate of approximately 2 000 s(-1). Glycine-121 was also changed to alanine, serine, leucine, a nd proline. While the Ala-121 and Ser-121 mutants behaved similar to w ild-type DHFR, the Leu-121 and Pro-121 mutants behaved like Val-121 DH FR in that hydride transfer was the rate-limiting step in the steady s tate and a conformational change preceding chemistry was observed. Fin ally, insertion of a glycine or valine between amino acids 121 and 122 produced mutant enzymes with properties similar to wild-type or Val-1 21 DHFRs, respectively. Taken together, these results provide compelli ng evidence for dynamic coupling of a remote residue to kinetic events at the active site of DHFR.