The role of enzyme dynamics and tunnelling in catalysing hydride transfer: studies of distal mutants of dihydrofolate reductase

Abstract

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 prearrangement 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.

Allosteric Communication in Dihydrofolate Reductase: Signaling Network and Pathways for Closed to Occluded Transition and Back

“…Benkovic and coworkers showed that mutations of residues (M42 and G121) that are far from the active site affect the hydride transfer rates [18,46]. Based on equilibrium covariance matrix fluctuations of the C α atoms obtained from all atom MD simulations, Rod et al showed that interactions of M42 with other residues (H45, D28, S49) would also be involved in the CS→OS conformational transition [13].…”

Coordinated effects of distal mutations on environmentally coupled tunneling in dihydrofolate reductase

“…Competitive KIE experiments were conducted with G121V-M42W by using a method as described (37,40). The findings were compared with those for the WT enzyme (35) and the two single mutants, G121V (40) and M42W (41). Fig.…”

Quantum Effects in Enzyme Kinetics

“…Three distal mutants were also studied (G121 V, M42W and G121V-M42W). [49][50][51] The mutants were found to exhibit different tunnelling patterns from the wild type (WT) and from each other: while the WT enzyme required no modification of the donor-acceptor distance to facilitate tunnelling, the single mutants did require some rearrangement to arrive at the appropriate tunnelling distance. The double mutant, on the other hand, required significant rearrangement before tunnelling could occur, thus showing a stronger temperature dependence than either of the single mutants.…”