Deletion of a Highly Motional Residue Affects Formation of the Michaelis Complex for Escherichia coli Dihydrofolate Reductase

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Abstract
Analysis of the dihydrofolate reductase (DHFR) complex with folate by two-dimensional heteronuclear (1H−15N) 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 βF−βG loop. To investigate the catalytic importance of this residue, we report the effects of Gly121 deletion and glycine insertion into the modified βF−βG 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, ΔG121 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 βF−βG loop, however, generally eliminated conformational changes required by ΔG121 DHFR to attain the Michaelis complex. Taken together, these results suggest that the catalytic role for the βF−βG loop includes formation of liganded complexes and proper orientation of substrate and cofactor. Through a transient interaction with the Met20 loop, alterations of the βF−βG loop can orchestrate proximal and distal effects on binding and catalysis that implicate a variety of enzyme conformations participating in the catalytic cycle.