Different reaction mechanisms for mesophilic and thermophilic dihydrofolate reductases.
We report here solvent kinetic isotope effects for two dihydrofolate reductases, namely the monomeric, mesophilic enzyme from E. coli (EcDHFR) and the dimeric, thermophilic enzyme from Thermotoga maritima (TmDHFR). Multiple isotope effects reveal mechanistic differences between the two enzymes. EcDHFR follows a stepwise mechanism in which proton transfer precedes hydride transfer, whereas the two steps are concerted in TmDHFR. At elevated pH, EcDHFR also follows a concerted reaction pathway. TmDHFR at pH 7 behaves more like EcDHFR at elevated pH suggesting that the restricted motions of TmDHFR resulting from dimerization preclude it from modulating the pK(a) of its substrate as efficiently as EcDHFR. The reduced reaction rates of TmDHFR therefore appear to be a consequence of its quaternary structure, which is required for increased thermostability but which also prevents active modulation of the reactivity of the active site bound substrate observed in EcDHFR.
School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff, CF10 3AT, UK.
E Joel
EJ
Loveridge
Enas M
EM
Behiry
Richard S
RS
Swanwick
Rudolf K
RK
Allemann
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D020124:Thermotoga maritima / Q000201:enzymology*
D001426:Bacterial Proteins; D013762:Tetrahydrofolate Dehydrogenase