Ja. Thomas et al., EFFECTS OF SUBSTITUTION OF THR63 BY ALANINE ON THE STRUCTURE AND FUNCTION OF LACTOBACILLUS-CASEI DIHYDROFOLATE-REDUCTASE, Protein engineering, 7(6), 1994, pp. 783-792
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.