Temperature-Independent Kinetic Isotope Effects as Evidence for a Marcus-like Model of Hydride Tunneling in Phosphite Dehydrogenase.

ABSTRACT: Phosphite dehydrogenase catalyzes the transfer of a hydride from phosphite to NAD⁺, producing phosphate and NADH. We have evaluated the role of hydride tunneling in a thermostable variant of this enzyme (17X-PTDH) by measuring the temperature dependence of the primary ²H kinetic isotope effects (KIEs) between 5 and 45 °C. Pre-steady-state kinetic measurements were used to demonstrate that the hydride transfer is rate-determining across this temperature range and that the observed KIEs are equal to the intrinsic isotope effect on the chemical step. The KIEs on the pre-exponential factor (A_H/A_D) and the activation energy (ΔE_a) were 1.6 ± 0.1 and 0.21 ± 0.05 kcal/mol, respectively, suggesting that 17X-PTDH facilitates

SUBMITTER: Howe GW 

PROVIDER: S-EPMC6852621 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature