title: Catalysis by dihydrofolate reductase from the psychropiezophile Moritella profunda.
abstract: The influence of temperature and pH on the stability and catalytic activity of dihydrofolate reductase (MpDHFR) from the cold-adapted deep-sea bacterium Moritella profunda was studied. The thermal melting temperature was found to be ∼38 °C and was not affected by pH, while activity measurements demonstrated that its stability was maximal at pH 7 and was reduced dramatically below pH 6 or above pH 8. The steady-state rate constant (k(cat)) was maximal at neutral pH and higher temperatures, while the Michaelis constants (K(M)) for both substrate and cofactor were optimal at lower temperatures and at elevated or reduced pH. For both temperature and pH, any change in k(cat) was therefore offset by a similar change in K(M). Both the activation enthalpy and entropy of the MpDHFR-catalysed reaction were lower than those of DHFR from E. coli leading overall to a very small difference in activation free energy and therefore similar steady-state rate constants at the same temperature. The chemical step of the reaction is not rate limiting at pH 7, but becomes progressively more rate limiting as the pH increases. These results demonstrate adaptation of MpDHFR to its environment and show compromises between enthalpic and entropic contributions to the reaction, and between k(cat) and K(M).
authors: Cardiff University, Cardiff, CF10 3AT, UK.
authors: Rhiannon M
authors: RM
authors: Evans
authors: Enas M
authors: EM
authors: Behiry
authors: Lai-Hock
authors: LH
authors: Tey
authors: Jiannan
authors: J
authors: Guo
authors: E Joel
authors: EJ
authors: Loveridge
authors: Rudolf K
authors: RK
authors: Allemann
mesh_terms: D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D034221:Moritella / Q000201:enzymology*; D013696:Temperature; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D013762:Tetrahydrofolate Dehydrogenase