Pre-steady-state studies of phosphite dehydrogenase demonstrate that hydride transfer is fully rate limiting

Emily J. Fogle, Wilfred A. Van Der Donk

Research output: Contribution to journalArticlepeer-review

Abstract

Phosphite dehydrogenase (PTDH) is a unique NAD-dependent enzyme that catalyzes the oxidation of inorganic phosphite to phosphate. The enzyme has great potential for cofactor regeneration, and mechanistic studies have provided some insight into the residues that are important for catalysis. In this investigation, pre-steady-state studies were performed on the His 6-tagged wild-type (WT) enzyme, several active site mutants, a thermostable mutant (12X-PTDH), and a thermostable mutant with dual cofactor specificity (NADP-12X-PTDH). Stopped-flow kinetic experiments indicate that slow steps after hydride transfer do not significantly limit the rate of reaction for the WT enzyme, the active site mutants, or the thermostable mutant. Pre-steady-state kinetic isotope effects (KIEs) and single-turnover experiments further confirm that slow steps after the chemical step do not significantly limit the rate of reaction for any of these proteins. Collectively, these results suggest that the hydride transfer step is fully rate determining in PTDH and that the observed KIE on kcat is the intrinsic effect in WT PTDH and the mutants examined. In contrast, a slow step after catalysis may partially limit the rate of phosphite oxidation by NADP-12X-PTDH with NADP as the cofactor. Finally, site-directed mutagenesis of Asp79 indicates that this residue is important in orienting Arg237 for proper interaction with phosphite.

Original languageEnglish (US)
Pages (from-to)13101-13108
Number of pages8
JournalBiochemistry
Volume46
Issue number45
DOIs
StatePublished - Nov 13 2007

ASJC Scopus subject areas

  • Biochemistry

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