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 relaxation revealed that isolated residues exhibit diverse backbone fluctuations 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 complex. Of these residues, glycine 121 displays large-amplitude backbone motions on the nanosecond time scale. This amino acid, strictly conserved for prokaryotic DHFRs, is located at the center of the beta F-beta G loop. To investigate the catalytic importance of this residue, we report the effects of Gly121 deletion and glycine insertion into the modified beta F-beta G loop. Relative to wild type, deletion of Gly121 dramatically decreases the rate of hydride transfer 550-fold and the strength of cofactor binding 20-fold for NADPH and 7-fold for NADP(+). Furthermore, 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 significant decrease in both substrate and cofactor binding. The introduction of glycine into the modified beta F-beta G loop, however, generally eliminated conformational changes required by Delta G121 DHFR to attain the Michaelis complex. Taken together, these results suggest that the catalytic role for the beta F-beta G loop includes formation of liganded complexes and proper orientation of substrate and cofactor. Through a transient interaction with the Met20 loop, alterations of the beta F-beta G loop can orchestrate proximal and distal effects on binding and catalysis that implicate a variety of enzyme conformations participating in the catalytic cycle.