CATALYTIC MECHANISM AND FUNCTION EVOLVEMENT STUDIES OF PHOSPHATASES WITHIN HALOACID DEHALOGENASE SUPERFAMILY (HADSF)

The haloacid dehalogenase superfamily (HADSF) is one of the largest known enzyme superfamilies, having more than 32,000 nonredundant members. Enzymes in this superfamily catalyze a number of different chemical reactions including dehalogenation, phosphoryl transfer, and hydrolysis of phosphate esters and phosphates. The vast majority of HADSF members are phosphatases, which contain a highly conserved core domain that is constructed to catalyze hydrolysis reactions of phosphorylated sugars or nucleotides by using an active site Asp nucleophile and a Mg2+ cofactor. Most phosphatases possess a cap domain, which in association with the core domain assists active site desolvation and binds the region of the substrate that is displaced by water upon Asp nucleophilic attack at the phosphoryl group. In the studies described in this Dissertation, two nucleotidases (AphA and mdN) were chosen to examine how the core Rossmann fold of HADSF members stabilizes the transition state of phosphoryl transfer IV reactions and conserves the stable catalytic scaffold under the pressure of evolutionary changes that expand substrate range. Another phosphatase, YidA, was subjected to studies in order to gain information about how accessory cap domains serve as sophisticated substrate recognition sites. AphA is a nonspecific acid phosphohydrolase (NSAP) from Escherichia coli., which is secreted in the periplasmic space. Although AphA has the highest activity towards hydrolysis of nucleotide phosphates (kcat/Km ~ 105 M-1s-1), it does not yet have an identified biological function. The crystal structures of the apo enzyme as well as complexes of AphA with products of nucleotide monophosphate hydrolysis and with substrate/intermediate analogs have been elucidated. Earlier analysis of these crystal structures shows that a specific hydrophobic substrate binding pocket, formed by Phe56 and Tyr193, exists in AphA. The results of solvent isotope studies carried out in the research effort described i