PMID: 2496755Feb 7, 1989Paper

NMR studies of differences in the conformations and dynamics of ligand complexes formed with mutant dihydrofolate reductases

Biochemistry
B BirdsallH T Cheung

Abstract

Two mutants of Lactobacillus casei dihydrofolate reductase, Trp 21----Leu and Asp 26----Glu, have been prepared by using site-directed mutagenesis methods, and their ligand binding and structural properties have been compared with those of the wild-type enzyme. 1H, 13C, and 31P NMR studies have been carried out to characterize the structural changes in the complexes of the mutant and wild-type enzymes. Replacement of the conserved Trp 21 by a Leu residue causes a decrease in activity of the enzyme and reduces the NADPH binding constant by a factor of 400. The binding of substrates and substrate analogues is only slightly affected. 1H NMR studies of the Trp 21----Leu enzyme complexes have confirmed the original resonance assignments for Trp 21. In complexes formed with methotrexate and the mutant enzyme, the results indicate some small changes in conformation occurring as much as 14 A away from the site of substitution. For the enzyme-NADPH complexes, the chemical shifts of nuclei in the bound coenzyme indicate that the nicotinamide ring binds differently in complexes with the mutant and the wild-type enzyme. There are complexes where the wild-type enzyme has been shown to exist in solution as a mixture of conformations, and stu...Continue Reading

References

Mar 18, 1977·Proceedings of the Royal Society of London. Series B, Containing Papers of a Biological Character·B BirdsallA Burgen
May 30, 1978·Biochemistry·B BirdsallG C Roberts
Jun 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·M S SearleA J Geddes
Dec 21, 1984·Nucleic Acids Research·W KramerH J Fritz
Nov 18, 1983·Science·J E VillafrancaJ Kraut
Jul 15, 1983·Analytical Biochemistry·B BirdsallG C Roberts
May 15, 1981·Biochemical and Biophysical Research Communications·L CoccoR L Blakley

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Citations

Oct 14, 2005·Journal of Biomolecular NMR·Nadezhda V KovalevskayaJames Feeney
Jul 1, 1990·Biochemical Pharmacology·J Feeney
Jan 1, 1990·Annals of the New York Academy of Sciences·H UedairaS Ohashi
Jun 5, 2007·The FEBS Journal·Ronnie A BöckWilliam W Barrow

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