DELETION OF A HIGHLY MOTIONAL RESIDUE AFFECTS FORMATION OF THE MICHAELIS COMPLEX FOR ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE

Analysis of the dihydrofolate reductase (DHFR)(.) complex with folate by two-dimensional heteronuclear (H-1-N-15) nuclear magnetic relaxatio n revealed that isolated residues exhibit diverse backbone fluctuation s on the nanosecond to picosecond time scale [Epstein, D. M., Benkovic , S. J., and Wright, P. E. (1995) Biochemistry 34, 11037-11048]. These dynamical features may be significant in forming the Michaelis comple x. Of these residues, glycine 121 displays large-amplitude backbone mo tions on the nanosecond time scale. This amino acid, strictly conserve d for prokaryotic DHFRs, is located at the center of the beta F-beta G loop. To investigate the catalytic importance of this residue, we rep ort the effects of Gly121 deletion and glycine insertion into the modi fied beta F-beta G loop. Relative to wild type, deletion of Gly121 dra matically decreases the rate of hydride transfer 550-fold and the stre ngth of cofactor binding 20-fold for NADPH and 7-fold for NADP(+). Fur thermore, Delta G121 DHFR requires conformational changes dependent on the initial binary complex to attain the Michaelis complex poised for hydride transfer. Surprisingly, the insertion mutants displayed a sig nificant decrease in both substrate and cofactor binding. The introduc tion of glycine into the modified beta F-beta G loop, however, general ly eliminated conformational changes required by Delta G121 DHFR to at tain the Michaelis complex. Taken together, these results suggest that the catalytic role for the beta F-beta G loop includes formation of l iganded complexes and proper orientation of substrate and cofactor. Th rough a transient interaction with the Met20 loop, alterations of the beta F-beta G loop can orchestrate proximal and distal effects on bind ing and catalysis that implicate a variety of enzyme conformations par ticipating in the catalytic cycle.