Residues M42 and G121 of Escherichia coli dihydrofolate reductase (ecDHFR) are on opposite sides of the catalytic centre (15 and 19 Å away from it, respectively). Theoretical studies have suggested that these distal residues might be part of a dynamics network coupled to the reaction catalysed at the active site. The ecDHFR mutant G121V has been extensively studied and appeared to have a significant effect on rate, but only a mild effect on the nature of H-transfer. The present work examines the effect of M42W on the physical nature of the catalysed hydride transfer step. Intrinsic kinetic isotope effects (KIEs), their temperature dependence and activation parameters were studied. The findings presented here are in accordance with the environmentally coupled hydrogen tunnelling. In contrast to the wild-type (WT), fluctuations of the donor–acceptor distance were required, leading to a significant temperature dependence of KIEs and deflated intercepts. A comparison of M42W and G121V to the WT enzyme revealed that the reduced rates, the inflated primary KIEs and their temperature dependences resulted from an imperfect potential surface pre-arrangement relative to the WT enzyme. Apparently, the coupling of the enzyme's dynamics to the reaction coordinate was altered by the mutation, supporting the models in which dynamics of the whole protein is coupled to its catalysed chemistry.
↵1 Equation (2.3) cannot be solved analytically. Northrop (1991) offers a table (its Appendix 1) that has numerically calculated values for a range of observed KIEs. More recently, we posted on our web site a free of charge JAVA script-based program that can numerically solve equation (2.1) for any experiment of interest (URL: cricket.chem. uiowa.edu/kohen/tools.html).
2 For the purpose of this work hydrogen-bonds are not considered covalent bonds.
- © 2006 The Royal Society