title: The role of large-scale motions in catalysis by dihydrofolate reductase.
abstract: Dihydrofolate reductase has long been used as a model system to study the coupling of protein motions to enzymatic hydride transfer. By studying environmental effects on hydride transfer in dihydrofolate reductase (DHFR) from the cold-adapted bacterium Moritella profunda (MpDHFR) and comparing the flexibility of this enzyme to that of DHFR from Escherichia coli (EcDHFR), we demonstrate that factors that affect large-scale (i.e., long-range, but not necessarily large amplitude) protein motions have no effect on the kinetic isotope effect on hydride transfer or its temperature dependence, although the rates of the catalyzed reaction are affected. Hydrogen/deuterium exchange studies by NMR-spectroscopy show that MpDHFR is a more flexible enzyme than EcDHFR. NMR experiments with EcDHFR in the presence of cosolvents suggest differences in the conformational ensemble of the enzyme. The fact that enzymes from different environmental niches and with different flexibilities display the same behavior of the kinetic isotope effect on hydride transfer strongly suggests that, while protein motions are important to generate the reaction ready conformation, an optimal conformation with the correct electrostatics and geometry for the reaction to occur, they do not influence the nature of the chemical step itself; large-scale motions do not couple directly to hydride transfer proper in DHFR.
authors: School of Chemistry, Cardiff University, Park Place, Cardiff CF10 3AT, United Kingdom.
authors: E Joel
authors: EJ
authors: Loveridge
authors: Lai-Hock
authors: LH
authors: Tey
authors: Enas M
authors: EM
authors: Behiry
authors: William M
authors: WM
authors: Dawson
authors: Rhiannon M
authors: RM
authors: Evans
authors: Sara B-M
authors: SB
authors: Whittaker
authors: Ulrich L
authors: UL
authors: Günther
authors: Christopher
authors: C
authors: Williams
authors: Matthew P
authors: MP
authors: Crump
authors: Rudolf K
authors: RK
authors: Allemann
mesh_terms: D055162:Biocatalysis; D002942:Circular Dichroism; D007700:Kinetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D013762:Tetrahydrofolate Dehydrogenase