Effect of dimerization on dihydrofolate reductase catalysis.

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.