Thymidylate synthase catalyzed H-transfers: two chapters in one tale

Abstract

Examination of the nature of different bond activations along the same catalytic path is of general interest in chemistry and biology. In this report, we compare the physical nature of two sequential H-transfers in the same enzymatic reaction. Thymidylate synthase (TSase) catalyzes a complex reaction that involves many chemical transformations including two different C-H bond cleavages, a rate-limiting C-H-C hydride transfer and a non-rate-limiting C-H-O proton transfer. Although the large kinetic complexity imposes difficulties in studying the proton transfer catalyzed by TSase, we are able to experimentally extract the intrinsic kinetic isotope effects (KIEs) on both steps. In contrast with the hydride transfer, the intrinsic KIEs of the proton transfer are temperature dependent. The results are interpreted within the framework of the Marcus-like model. This interpretation suggests that TSase optimizes the donor-acceptor geometries for the slower and overall rate-limiting hydride transfer but not for the faster proton transfer.

Figure 1

An illustration of the Marcus-like model (see text). The blue and green curves correspond to the reactant and product states, respectively. The red curve represents the hydrogen wave-function overlap at tunneling ready state (TRS, see text).

Figure 2

Arrhenius plots of observed (empty structures) and intrinsic (filled structures) primary KIEs on the proton transfer (A) and the hydride transfer (B) catalyzed by E. coli TSase. The KIEs are shown as average values and standard deviations, while the lines are the non-linear fittings of all calculated intrinsic KIEs (see text) to the Arrhenius equation.

Figure 3

The forward commitment (C_f) of the proton transfer step plotted on the logarithmic scale vs. the reciprocal of the absolute temperature. The data are presented as average values and standard deviations (red: H/T; blue: D/T). The lines are the exponential fittings of all calculated C_f values (see text) to the Arrhenius equation.

Figure 4

The observed H/T (filled structures) and D/T (empty structures) KIEs plotted vs. fraction conversion as measured at 25 °C with 3 μM CH₂H₄folate. The different colors and shapes represent different independent experiments.

Scheme 1

Proposed chemical mechanisms of TSase-catalyzed reaction (revised from Ref. 30). R=2'-deoxyribose-5'-phosphate; R'=(p-aminobenzoyl)glutamate.

Scheme 2

A simplified kinetic scheme for of wild-type E. coli TSase (revised from Ref 34). A represents dUMP and B CH₂H₄folate. The only isotopically sensitive step here is represented by the rate constant k₅.