Chemical Mechanism of Glycerol 3‑Phosphate Phosphatase: pH-Dependent Changes in the Rate-Limiting Step

The halo-acid dehalogenase (HAD) superfamily comprises a large number of enzymes that share a conserved core domain responsible for a diverse array of chemical transformations (e.g., phosphonatase, dehalogenase, phosphohexomutase, and phosphatase) and a cap domain that controls substrate specificity. Phosphate hydrolysis is thought to proceed via an aspartyl-phosphate intermediate, and X-ray crystallography has shown that protein active site conformational changes are required for catalytic competency. Using a combination of steady-state and pre-steady-state kinetics, pL–rate studies, solvent kinetic isotope effects, 18O molecular isotope exchange, and partition experiments, we provide a detailed description of the chemical mechanism of a glycerol 3-phosphate phosphatase. This phosphatase has been recently recognized as a rate-limiting factor in lipid polar head recycling in Mycobacterium tuberculosis [Larrouy-Maumus, G., et al. (2013) Proc. Natl. Acad. Sci. 110 (28), 11320–11325]. Our results clearly establish the existence of an aspartyl-phosphate intermediate in this newly discovered member of the HAD superfamily. No ionizable groups are rate-limiting from pH 5.5 to 9.5, consistent with the pK values of the catalytic aspartate residues. The formation and decay of this intermediate are partially rate-limiting below pH 7.0, and a conformational change preceding catalysis is rate-limiting above pH 7.0.