title: Evidence that a 'dynamic knockout' in Escherichia coli dihydrofolate reductase does not affect the chemical step of catalysis.
abstract: The question of whether protein motions play a role in the chemical step of enzymatic catalysis has generated much controversy in recent years. Debate has recently reignited over possible dynamic contributions to catalysis in dihydrofolate reductase, following conflicting conclusions from studies of the N23PP/S148A variant of the Escherichia coli enzyme. By investigating the temperature dependence of kinetic isotope effects, we present evidence that the reduction in the hydride transfer rate constants in this variant is not a direct result of impairment of conformational fluctuations. Instead, the conformational state of the enzyme immediately before hydride transfer, which determines the electrostatic environment of the active site, affects the rate constant for the reaction. Although protein motions are clearly important for binding and release of substrates and products, there appears to be no detectable dynamic coupling of protein motions to the hydride transfer step itself.
authors.affiliation: School of Chemistry, Cardiff University, Park Place, Cardiff, CF10 3AT, UK.
authors.forename: E Joel
authors.initials: EJ
authors.lastname: Loveridge
authors.forename: Enas M
authors.initials: EM
authors.lastname: Behiry
authors.forename: Jiannan
authors.initials: J
authors.lastname: Guo
authors.forename: Rudolf K
authors.initials: RK
authors.lastname: Allemann
mesh_terms: D055162:Biocatalysis; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D013762:Tetrahydrofolate Dehydrogenase