Hydride Transfer Mechanism of Enzymatic Sugar Nucleotide C2 Epimerization Probed with a Loose-Fit CDP-Glucose Substrate

Transient oxidation-reduction through hydride transfer with tightly bound NAD coenzyme is used by a large class of sugar nucleotide epimerases to promote configurational inversion of carbon stereocenters in carbohydrate substrates. A requirement for the epimerases to coordinate hydride abstraction and re-addition with substrate rotation in the binding pocket poses a challenge for dynamical protein conformational selection linked to enzyme catalysis. Here, we studied the thermophilic C2 epimerase from Thermodesulfatator atlanticus (TaCPa2E) in combination with a slow CDP-glucose substrate (kcat ≈ 1.0 min-1 60 °C) to explore the sensitivity of the enzymatic hydride transfer toward environmental fluctuations affected by temperature (20-80 °C). We determined noncompetitive primary kinetic isotope effects (KIE) due to 2H at the glucose C2 and showed that a normal KIE on the kcat (Dkcat) reflects isotope sensitivity of the hydrogen abstraction to enzyme-NAD+ in a rate-limiting transient oxidation. The Dkcat peaked at 40 °C was 6.1 and decreased to 2.1 at low (20 °C) and 3.3 at high temperature (80 °C). The temperature profiles for kcat with the 1H and 2H substrate showed a decrease in the rate below a dynamically important breakpoint (40 °C), suggesting an equilibrium shift to an impaired conformational landscape relevant for catalysis in the low-temperature region. Full Marcus-like model fits of the rate and KIE profiles provided evidence for a high-temperature reaction via low-frequency conformational sampling associated with a broad distribution of hydride donor-acceptor distances (long-distance population centered at 3.31 ± 0.02 Å), only poorly suitable for quantum mechanical tunneling. Collectively, dynamical characteristics of TaCPa2E-catalyzed hydride transfer during transient oxidation of CDP-glucose reveal important analogies to mechanistically simpler enzymes such as alcohol dehydrogenase and dihydrofolate reductase. A loose-fit substrate (in TaCPa2E) resembles structural variants of these enzymes by extensive dynamical sampling to balance conformational flexibility and catalytic efficiency.