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 constructed in which Thr63, a residue which interacts with the 2'-phosphate group of the bound coenzyme, is replaced by alanine. This substitution does not affect kcat, but produces an 800-fold increase in the Km 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 tetrahydrofolate. 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 the structures of the mutant and wild-type enzymes in solution, in their complexes with methotrexate and with methotrexate and NADPH. This showed that only minimal structural changes result from the mutation; a total of 47 residues were monitored from their resolved 1H resonances, and of these nine in the binary complex and six in the ternary differed in chemical shift between mutant and wild-type enzyme. These affected residues are confined to the immediate vicinity of residue 63. There is a substantial difference in the 31P chemical shift of the 2'-phosphate of the bound coenzyme, reflecting the loss of the interaction with the side chain of Thr63. The only changes in nuclear Overhauser effects (NOEs) observed were decreases in the intensity of NOEs between protons of the adenine ring of the bound coenzyme and the nearby residues Leu62 and Ile102, showing that the substitution of Thr63 does cause a change in the position or orientation of the adenine ring in its binding site.
Thomas
J A
JA
Department of Biochemistry and Biological NMR Centre, University of Leicester, UK.
Arnold
J R
JR
Basran
J
J
Andrews
J
J
Roberts
G C
GC
Birdsall
B
B
Feeney
J
J
D000409:Alanine; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008727:Methotrexate / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D012996:Solutions; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D013912:Threonine
D012996:Solutions; D013912:Threonine; D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase; D000409:Alanine; D008727:Methotrexate