Inhibition and pH dependence of phosphite dehydrogenase

Heather A. Relyea, Jennifer M. Vrtis, Ryan Woodyer, Stacey A. Rimkus, Wilfred A. Van Der Donk

Research output: Contribution to journalArticlepeer-review


Phosphite dehydrogenase (PTDH) catalyzes the NAD-dependent oxidation of phosphite to phosphate, a reaction that is 15 kcal/mol exergonic. The enzyme belongs to the family of D-hydroxy acid dehydrogenases. Five other family members that were analyzed do not catalyze the oxidation of phosphite, ruling out the possibility that this is a ubiquitous activity of these proteins. PTDH does not accept any alternative substrates such as thiophosphite, hydrated aldehydes, and methylphosphinate, and potential small nucleophiles such as hydroxylamine, fluoride, methanol, and trifluoromethanol do not compete with water in the displacement of the hydride from phosphite. The pH dependence of kcat/Km,phosphite is bell-shaped with a pKa of 6.8 for the acidic limb and a pKa of 7.8 for the basic limb. The pKa of 6.8 is assigned to the second deprotonation of phosphite. However, whether the dianionic form of phosphite is the true substrate is not clear since a reverse protonation mechanism is also consistent with the available data. Unlike kcat/Km,phosphite, kcat and kcat/Km,NAD are pH-independent. Sulfite is a strong inhibitor of PTDH that is competitive with respect to phosphite and uncompetitive with respect to NAD+. Incubation of the enzyme with NAD+ and low concentrations of sulfite results in a covalent adduct between NAD+ and sulfite in the active site of the enzyme that binds very tightly. Fluorescent titration studies provided the apparent dissociation constants for NAD+, NADH, sulfite, and the sulfite-NAD+ adduct. Substrate isotope effect studies with deuterium-labeled phosphite resulted in small normal isotope effects (1.4-2.1) on both kcat and kcat/Km,phosphite at pH 7.25 and 8.0. Solvent isotope effects (SIEs) on kcat are similar in size; however, the SIE of kcat/Km,phosphite at pH 7.25 is significantly larger (4.4), whereas at pH 8.0, it is the inverse (0.6). The pH-rate profile of k cat/Km,phosphite, which predicts that the observed SIEs will have a significant thermodynamic origin, can account for these effects.

Original languageEnglish (US)
Pages (from-to)6640-6649
Number of pages10
Issue number17
StatePublished - May 3 2005

ASJC Scopus subject areas

  • Biochemistry


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