Mechanistic studies and synthetic applications of phosphite dehydrogenase

Phosphite dehydrogenase (PtxD) is the only enzyme reported to catalyze redox chemistry on an inorganic phosphorus compound by oxidizing phosphite to phosphate with concomitant reduction of NAD+ to NADH. Several possible mechanisms for this unique phosphoryl transfer reaction catalyzed by PtxD, include the highly debated dissociative mechanism with a discrete metaphosphate intermediate, an associative mechanism, a concerted mechanism, and a covalent catalysis mechanism. All mechanisms invoke a hydride transfer from the substrate to the cofactor, NAD+. From stereochemical studies, PtxD was determined to catalyze direct hydride transfer with Re-face stereoselectivity. Primary kinetic isotope effect (KIE), solvent isotope effect, and viscosity assays were also carried out to provide insight into the mechanism of the reaction catalyzed by PtxD. A significant KIE on k_cat indicated that the hydride transfer step is partially rate-determining, which was corroborated with microviscosity assays on k _cat. Additional information such as the suggested stepwise hydride transfer and deprotonation of water and the proposed protonation state for the active form of the enzyrne-substrate complex were obtained from substrate and solvent isotope effects.; Furthermore, phosphite dehydrogenase was found to be an ideal enzyme for regeneration of nicotinamide cofactors for organic synthetic applications. This technology lowers the cost of the enzymatic transformations, simplifies product isolation, and prevents product inhibition of the synthetic enzyme by NADH. The efficiency of PtxD as a regenerative enzyme was determined with the synthetic enzymes malate dehydrogenase, horse liver alcohol dehydrogenase, and D- and L-lactate dehydrogenase yielding high total turnover numbers for NAD+ and PtxD. Moreover, PtxD was employed with the inexpensive deuterated phosphite for the synthesis of an isotopically labeled compound.; Cytochrome c oxidase, an enzyme that catalyzes the reduction of oxygen to water, has a binuclear center as well as a unique post-translational modification in the active site. Specifically, there is a crosslink between the nitrogen (Nϵ₂) of His240 and the carbon (Cϵ ₂) of Tyr244 (numbers correspond to CcO from bovine heart). The significance of the linkage was investigated by comparing the physicochemical properties of a model compound, 2-(imidazolyl-1-yl-4-methylphenol) to p-cresol.