EFFECTS OF SUBSTITUTION OF THR63 BY ALANINE ON THE STRUCTURE AND FUNCTION OF LACTOBACILLUS-CASEI DIHYDROFOLATE-REDUCTASE

A mutant of Lactobacillus casei dihydrofolate reductase has been const ructed in which Thr63, a residue which interacts with the 2'-phosphate group of the bound coenzyme, is replaced by alanine. This substitutio n does not affect k(cat), but produces an 800-fold increase in the K, for NADPH, which reflects dissociation of NADPH from the enzyme-NADPH- tetrahydrofolate complex, and a 625-fold increase (corresponding to 3. 8 kcal/mol) in the dissociation constant for the enzyme-NADPH complex. The difference in magnitude of these effects indicates a small effect of the substitution on the negative cooperativity between NADPH and t etrahydrofolate. Stopped-flow studies of the kinetics of NADPH binding show that the weaker binding arises predominantly from a decrease in the association rate constant. NMR spectroscopy was used to compare th e structures of the mutant and wild-type enzymes in solution, in their complexes with methotrexate and with methotrexate and NADPH. This sho wed that only minimal structural changes result from the mutation; a t otal of 47 residues were monitored from their resolved H-1 resonances, and of these nine in the binary complex and six in the ternary differ ed in chemical shift between mutant and wild-type enzyme. These affect ed residues are confined to the immediate vicinity of residue 63. Ther e is a substantial difference in the P-31 chemical shift of the 2'-pho sphate of the bound coenzyme, reflecting the loss of the interaction w ith the side chain of Thr63. The only changes in nuclear Overhauser ef fects (NOEs) observed were decreases in the intensity of NOEs between protons of the adenine ring of the bound coenzyme and the nearby resid ues Leu62 and Ile102, showing that the substitution of Thr63 does caus e a change in the position or orientation of the adenine ring in its b inding site.