{'fraction_non_alphanumeric': 0.0322180916976456, 'fraction_numerical': 0.0161090458488228, 'mean_word_length': 4.9375, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Temperature independent kinetic isotope effects as evidence for a Marcus-like model of hydride tunneling in phosphite dehydrogenase.

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 °C 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 (AH/AD) and the activation energy (ΔEa) were 1.6 ± 0.1 and 0.21 ± 0.05, respectively, suggesting that 17X-PTDH facilitates extensive tunneling of both isotopes via a Marcus-like model. Site-directed mutagenesis was used to evaluate the role of an active-site threonine (Thr104) found on the back face of the nicotinamide in promoting the close packing of the substrates. In mutants with reduced steric bulk at this position, values of AH/AD and ΔEa fall within the range describing semi-classical "over the barrier" reactivity, suggesting that Thr104 acts as a steric backstop to promote tunneling in 17X-PTDH. Whereas hydrogen tunneling is now a widely appreciated feature of C-H activating enzymes, these observations with a P-H activating system are consistent with the proposal that that tunneling is likely to be a common feature on all enzymes that catalyze hydrogen transfers.