Increased Dynamic Effects in a Catalytically Compromised Variant of Escherichia coli Dihydrofolate Reductase

Isotopic substitution (15N, 13C, 2H) of a catalytically compromised variant of Escherichia coli dihydrofolate reductase, EcDHFR-N23PP/S148A, has been used to investigate the effect of these mutations on catalysis. The reduction of the rate constant of the chemical step in the EcDHFR-N23PP/S148A catalyzed reaction is essentially a consequence of an increase of the quasi-classical free energy barrier and to a minor extent of an increased number of recrossing trajectories on the transition state dividing surface. Since the variant enzyme is less well set up to catalyze the reaction, a higher degree of active site reorganization is needed to reach the TS. Although millisecond active site motions are lost in the variant, there is greater flexibility on the femtosecond time scale. The “dynamic knockout” EcDHFR-N23PP/S148A is therefore a “dynamic knock-in” at the level of the chemical step, and the increased dynamic coupling to the chemical coordinate is in fact detrimental to catalysis. This finding is most likely applicable not just to hydrogen transfer in EcDHFR but also to other enzymatic systems.This work was supported by grant BB/J005266/1 (R.K.A.) from the UK Biotechnology and Biological Sciences Research Council (BBSRC), by the Vice Chancellor Fund of Cardiff University, by the Spanish Ministerio de Economiá y Competitividad (project CTQ2012-36253-C03), by MICINN (project CTQ2009-14541-C02), by Generalitat Valenciana (projects ACOMP/2012/119, ACOMP/2012/243, GV/ 2012/044, and Prometeo/2009/053), and by Universitat Jaume I-Bancaixa (projects P1·1A2010-08 and P1·1B2011- 23). J.J.R.-P. thanks the Spanish Ministerio de Ciencia e Innovacioń for a “Juan de la Cierva” contract. R.G.-M. acknowledges a FPU fellowship of the Ministerio de Economiá y Competitividad. We acknowledge Prof. D. G. Truhlar for helpful comments on the original manuscript. I.T. acknowl- edges many helpful discussions with Prof. J. T. Hynes and D. Laage during his sabbatical stay at the Ecole Normale Supeŕ ieure (Paris).We acknowledge the computational facilities of Universitat Jaume I, Universitat de Valeǹcia (Tirant Supercomputer), and the Spanish Superconducting Network (Picasso Supercomputer)