title: Probing the functional role of phenylalanine-31 of Escherichia coli dihydrofolate reductase by site-directed mutagenesis.
abstract: The role of Phe-31 of Escherichia coli dihydrofolate reductase in binding and catalysis was probed by amino acid substitution. Phe-31, a strictly conserved residue located in a hydrophobic pocket and interacting with the pteroyl moiety of dihydrofolate (H2F), was replaced by Tyr and Val. The kinetic behavior of the mutant enzymes in general is similar to that of the wild type. The rate-limiting step for both mutant enzymes is the release of tetrahydrofolate (H4F) from the E X NADPH X H4F ternary complex as determined for the wild type. The 2-fold increase in V for the two mutant enzymes arises from faster dissociation of H4F from the enzyme-product complex. The quantitative effect of these mutations is to decrease the rate of hydride transfer, although not to the extent that this step becomes partially rate limiting, but to accelerate the dissociation rates of tetrahydrofolate from product complexes so that the opposing effects are nearly compensating.
authors.affiliation: Department of Chemistry, Pennsylvania State University, University Park 16802.
authors.forename: J T
authors.initials: JT
authors.lastname: Chen
authors.forename: K
authors.initials: K
authors.lastname: Taira
authors.forename: C P
authors.initials: CP
authors.lastname: Tu
authors.forename: S J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: D000596:Amino Acids / Q000494:pharmacology; D001665:Binding Sites; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology*; D005492:Folic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D009249:NADP / Q000378:metabolism; D010649:Phenylalanine / Q000502:physiology*; D010957:Plasmids; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D014443:Tyrosine / Q000494:pharmacology; D014633:Valine / Q000494:pharmacology
chemical_list: D000596:Amino Acids; C010920:dihydrofolate; D014443:Tyrosine; D010649:Phenylalanine; D009249:NADP; D005492:Folic Acid; D003903:Deuterium; D013762:Tetrahydrofolate Dehydrogenase; D014633:Valine