title: Chemical mechanism of glycerol 3-phosphate phosphatase: pH-dependent changes in the rate-limiting step.
abstract: 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, (18)O 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.
authors.affiliation: Mycobacterial Research Division, MRC, National Institute for Medical Research , and ‡MRC Biomolecular NMR Centre, The Ridgeway, Mill Hill, London NW7 1AA, U.K.
authors.forename: Gérald
authors.initials: G
authors.lastname: Larrouy-Maumus
authors.forename: Geoff
authors.initials: G
authors.lastname: Kelly
authors.forename: Luiz Pedro Sório
authors.initials: LP
authors.lastname: de Carvalho
mesh_terms: D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D005994:Glycerophosphates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D012997:Solvents / Q000494:pharmacology; D013379:Substrate Specificity; D014783:Viscosity
chemical_list: D005994:Glycerophosphates; D012997:Solvents; C032197:beta-aspartyl phosphate; D001224:Aspartic Acid; C029620:alpha-glycerophosphoric acid; C583817:glycerol 3-phosphate phosphatase, Mycobacterium tuberculosis; D010744:Phosphoric Monoester Hydrolases