Kinetic isotope effects as a probe of hydrogen transfers to and from common enzymatic cofactors

Abstract

Enzymes use a number of common cofactors as sources of hydrogen to drive biological processes, but the physics of the hydrogen transfers to and from these cofactors is not fully understood. Researchers study the mechanistically important contributions from quantum tunneling and enzyme dynamics and connect those processes to the catalytic power of enzymes that use these cofactors. Here we describe some progress that has been made in studying these reactions, particularly through the use of kinetic isotope effects (KIEs). We first discuss the general theoretical framework necessary to interpret experimental KIEs, and then describe practical uses for KIEs in the context of two case studies. The first example is alcohol dehydrogenase, which uses a nicotinamide cofactor to catalyze a hydride transfer, and the second example is thymidylate synthase, which uses a folate cofactor to catalyze both a hydride and a proton transfer.

Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii

“…2,12,54–59 The inflated KIEs in the recombinant CbFDH reflect a longer average DAD than the average DAD in the commercial mixture. 60 Importantly, both systems have temperature independent intrinsic KIEs, indicating that the DAD is narrowly distributed in both the recombinant enzyme and the isozyme mixture as isolated from yeast – validating the use of the recombinant enzyme and its prospective mutants in biophysical studies similar to those of other enzymes (e.g., dihydrofolate reductase; 12,59,61 soybean lipoxygenase; 13,62,63 alcohol dehydrogenase; 14,64,65 and others).…”

Decarboxylation involving a ferryl, propionate, and a tyrosyl group in a radical relay yields heme b

“…(8) The rate constants in each case had identical though modest (~3-fold) pH dependencies, with a pKa = 7.4 and a kD/kH kinetic isotope effect (KIE) of approximately 2 across the entire pH range. We conclude that the C-H bond is cleaved during the decarboxylation reaction; because the theoretically expected value for a primary H/D KIE is 7 (21,22), this step likely only partially limits the rate of coproheme/heme b conversion. The D-coproheme/H2O2 reaction was subsequently monitored over time by freezequench EPR (Fig.…”

Theory of single-molecule controlled rotation experiments, predictions, tests, and comparison with stalling experiments in F 1 -ATPase

“…In our previous study (16) the binding and release of nucleotides were treated in F 1 -ATPase based on a formalism originally proposed for electron transfers (17) and adapted to other transfers (18), including proton (19) and methyl cation (20) transfers. In the theory a thermodynamic driving force that determines the rate and equilibrium constants in the experiments for any reaction step, including nucleotide binding, is the change in the relevant Gibbs free energy of reaction for that step.…”