title: Hydrophobic interactions via mutants of Escherichia coli dihydrofolate reductase: separation of binding and catalysis.
abstract: The strictly conserved residue leucine-54 of Escherichia coli dihydrofolate reductase forms part of the hydrophobic wall which binds the p-aminobenzoyl side chain of dihydrofolate. In addition to the previously reported glycine-54 mutant, isoleucine-54 and asparagine-54 substitutions have been constructed and characterized with regard to their effects on binding and catalysis. NADP+ and NADPH binding is virtually unaffected with the exception of a 15-fold decrease in NADPH dissociation from the Gly-54 mutant. The synergistic effect of NADPH on tetrahydrofolate dissociation seen in the wild-type enzyme is lost in the isoleucine-54 mutant: little acceleration is seen in tetrahydrofolate dissociation when cofactor is bound, and there is no discrimination between reduced and oxidized cofactor. The dissociation constants for dihydrofolate and methotrexate increase in the order Leu less than Ile less than Asn less than Gly, varying by a maximum factor of 1700 for dihydrofolate and 6300 for methotrexate. Despite these large changes in binding affinity, the hydride transfer rate of 950 s-1 in the wild-type enzyme is decreased by a constant factor of ca. 30 (2 kcal/mol) regardless of the mutant. Thus, the contributions of residue 54 to binding and catalysis appear to have been separated.
authors.affiliation: Department of Chemistry, Pennsylvania State University, University Park 16802.
authors.forename: D J
authors.initials: DJ
authors.lastname: Murphy
authors.forename: S J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D009154:Mutation; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D013762:Tetrahydrofolate Dehydrogenase