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 A 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.

Figure 1

Structure of E. coli DHFR with bound NADP⁺ (orange) and folic acid (red; PDB 1r×2). Amino acid side chains of M42 and G121 are shown as green and blue spheres, respectively.

Figure 2

Arrhenius plot of observed (open squares) and intrinsic (filled squares) KIEs for the M42W-ecDHFR (from tables 1 and 2 in the electronic supplementary material). H/T KIEs in dark grey, H/D KIEs in light grey and D/T KIEs in black. The lines represent the nonlinear regression to equation (2.4).

Figure 3

Comparison of the Arrhenius plots of intrinsic H/T KIEs of the wild-type (dark grey; Sikorski et al. 2004), G121V (light grey; Wang et al. 2006) and M42W (black; table 2 in the electronic supplementary material) DHFRs.

Figure 4

Comparison of the Arrhenius plots of the commitment to catalysis (C_f) on k_cat/K_M for the wild-type (dark grey; Sikorski et al. 2004), G121V (light grey; Wang et al. 2006) and M42W (black; tables 1 and 2 in the electronic supplementary material and equation (3.1)) DHFR.

Figure 5

Illustration of ‘Marcus-like’ models: energy surface of environmentally coupled hydrogen tunnelling. Two orthogonal coordinates are presented: p, the environmental energy parabolas for the reactant state (R in grey) and the product state (P in black); and q, the H-transfer potential surface at each p configuration. The grey shapes represent the populated states (e.g. the location of the particle). The original Marcus expression would have a fixed q distance between donor and acceptor. Thermal fluctuations of that distance (denoted ‘gating’ by Knapp & Klinman 2002) lead to the temperature dependency of the KIE.

Scheme 1

The DHFR-catalysed reaction. R≡adenine dinucleotide 2′-P, R′≡(p-aminobenzoyl) glutamate.