Analysis of the Structural Determinants Underlying Discrimination between Substrate and Solvent in β-Phosphoglucomutase Catalysis

The beta-phosphoglucomutase (beta-PGM) of the haloacid dehalogenase enzyme superfamily (HADSF) catalyzes the conversion of beta-glucose 1-phosphate WG I P) to glucose 6-phosphate (G6P) using Asp8 of the core domain active site to mediate phosphoryl transfer from beta-glucose 1,6-(bis)phosphate (beta G1,6bisP) to beta G1P. Herein, we explore the mechanism by which hydrolysis of the beta-PGM phosphoAsp8 is avoided during the time that the active site must remain open to solvent to allow the exchange of the bound product G6P with the substrate beta G1P. On the basis of structural information, a model of catalysis is proposed in which the general acid/base (Asp10) side chain moves from a position where it forms a hydrogen bond to the Thr16-Ala17 portion of the domain-domain linker to a functional position where it forms a hydrogen bond to the substrate leaving group O and a His20-Lys76 pair of the cap domain. This repositioning of the general acid/base within the core domain active site is coordinated with substrate-induced closure of the cap domain over the core domain. The model predicts that AsplO is required for general acid/base catalysis and for stabilization of the enzyme in the cap-closed conformation. It also predicts that hinge residue Thr16 plays a key role in productive domain-domain association, that hydrogen bond interaction with the Thr16 backbone amide NH group is required to prevent phosphoAsp8 hydrolysis in the cap-open conformation, and that the His20-Lys76 pair plays an important role in substrate-induced cap closure. The model is examined via kinetic analyses of Asp10, Thrl6, His20, and Lys76 site-directed mutants. Replacement of Asp10 with Ala, Ser, Cys, Asn, or Glu resulted in no observable activity. The kinetic consequences of the replacement of linker residue Thrl6 with Pro include a reduced rate of Asp8 phosphorylation by PG1,6bisP, a reduced rate of cycling of the phosphorylated enzyme to convert PGIP to G6P, and an enhanced rate of pho angstrom resolution provides a snapshot of the enzyme in an unnatural cap-open conformation where the Asp10 side chain is located in the core domain active site. The His20 and Lys76 site-directed mutants exhibit reduced activity in catalysis of the Asp8mediated phosphoryl transfer between beta G1,6bisP and PGIP but no reduction in the rate of phosphoAsp8 hydrolysis. Taken together, the results support a substrate induced-fit model of catalysis in which beta G1P binding to the core domain facilitates recruitment of the general acid/base Asp10 to the catalytic site and induces cap closure.