title: Effect of dimerization on dihydrofolate reductase catalysis.
abstract: Dihydrofolate reductase (DHFR) from the hyperthermophile Thermotoga maritima (TmDHFR) forms a very stable homodimer, while DHFRs from other organisms are monomers. We investigated the effect of dimerization on DHFR catalysis by preparing a dimeric variant, Xet-3, of DHFR from Escherichia coli (EcDHFR). Introducing residues located at the TmDHFR dimer interface into EcDHFR increases the melting temperature to ∼60 °C, approximately 9 °C higher than that measured for EcDHFR. The steady-state and pre-steady-state rate constants measured for Xet-3 were similar to those of dimeric TmDHFR but significantly lower than those of the parent EcDHFR. This reduction in the degree of catalytic competence is likely a consequence of the loss of flexibility of catalytically important loop regions of EcDHFR on dimerization and a compromise of the electrostatic environment of the active site. In contrast, the reduced catalytic ability of TmDHFR relative to that of EcDHFR is not simply a consequence of reduced loop flexibility in the dimeric enzyme. Our studies demonstrate that EcDHFR is not a good model for understanding the properties of other DHFRs, including TmDHFR.
authors.affiliation: School of Chemistry and Cardiff Catalysis Institute, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, United Kingdom.
authors.forename: Jiannan
authors.initials: J
authors.lastname: Guo
authors.forename: E Joel
authors.initials: EJ
authors.lastname: Loveridge
authors.forename: Louis Y P
authors.initials: LY
authors.lastname: Luk
authors.forename: Rudolf K
authors.initials: RK
authors.lastname: Allemann
mesh_terms: D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008958:Models, Molecular; D017510:Protein Folding; D055503:Protein Multimerization; D013696:Temperature; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D020124:Thermotoga maritima / Q000201:enzymology*
chemical_list: D013762:Tetrahydrofolate Dehydrogenase