Previous studies of E. coli dihydrofolate reductase (ecDHFR) have demonstrated that residue G121, which is 19Å away from the catalytic center, is involved in catalysis and long distance dynamical motions were implied. Specifically, the ecDHFR mutant G121V has been extensively studied by various experimental and theoretical tools, and the mutation's effect on kinetic, structural, and dynamical features of the enzyme explored. The current work examined the effect of this mutation on the physical nature of the catalyzed hydride transfer step by means of intrinsic kinetic isotope effects (KIEs), their temperature dependence and activation parameters as described before for the wild type ecDHFR (Sikorski et al. 2004, J. Am. Chem. Soc., 126, 4778-4779). The temperature dependence of initial velocities was used to estimate activation parameters. Isotope effects on the preexponential Arrhenius factors, and the activation energy could be rationalized by an environmentally coupled hydrogen tunneling model, similar to the one used for the wild type enzyme. Yet, in contrast to the wild type, fluctuations of the donor-acceptor distance were now required. Secondary (2°) KIEs were also measured for both H and D transfer and, as in the case of the wild type enzyme, no coupled motion was detected. Despite these similarities, the reduced rates, the slightly inflated 1° KIEs and their temperature dependence, together with relatively deflated 2° KIEs, indicate that the potential surface prearrangement was not as ideal as for the wild type enzyme. These findings support theoretical studies suggesting that the G121V mutation lead to a different conformational ensemble of reactive states and less effective rearrangement of the potential surface, but has only small effect on H-tunneling.Dihydrofolate reductase from Escherichia coli (ecDHFR) is a small monomeric enzyme (18 kDa) that consists of eight-stranded β-sheet and four α-helices connected with several loop regions. It catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7, with the concomitant oxidation of NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) to NADP + , in which a hydride is stereospecifically transferred from the pro-R C4 position of the nicotinamide ring to the si face of the C6 position of pterin. This enzyme helps maintain intracellular pools of THF used in the biosynthesis of purine nucleotides and some amino acids. Furthermore the essential role of DHFR in DNA synthesis and in a variety of anabolic pathways makes it a common target for antiproliferative therapeutics. Due to its biological and pharmacological importance, and it being a small monomeric enzyme, DHFR has been the subject of intensive structural and kinetic investigation over many years, serving as a paradigm of enzymatic systems in many experimental and theoretical studies (1)(2)(3)(4)(5)(6)(7)(8). † This work was supported by NIH R01 GM65368-01 and NSF CHE-0133117 to A.K. The complete kinetic scheme for wild type ecDHFR was derived from equilibrium binding, steady-sta...