Investigating mechanistic and evolutionary relationships between members of the alkaline phosphatase superfamily

Abstract

Phosphatases can accelerate the rate of phosphate ester hydrolysis by greater than 1020 fold. To achieve this enormous rate enhancement, enzymes must preferentially stabilize the transition state relative to the ground state. Alkaline phosphatase preferentially catalyzes phosphate monoester hydrolysis with a rate acceleration of >1017. AP also catalyzes phosphate diester hydrolysis, albeit to a lesser extent (1011 fold rate enhancement). Conversely, nucleotide pyrophosphates/phosphodiesterase (NPP), a member of the same superfamily, preferentially catalyzes phosphate diester hydrolysis. Homology models indicate that AP and NPP have similar active sites, raising the question of how these enzymes are able to discriminate between monoesters and diesters to preferentially catalyze one reaction over the other. In AP there is a large positive correlation between the amount of negative charge on the non-bridging oxygen atom of the substrate and the rate acceleration of the reaction. To understand this electrostatic discrimination, we are using site-directed mutagenesis to systematically perturb the ligand field charge of the metallocluster in AP and evaluating the effects of these mutations on substrate specificity and catalytic proficiency. Results indicate that mutations made at the bimetallocluster in AP alter the relative reactivity towards phosphate monoester and phosphate diester substrates. Understanding the catalytic features of phosphoryl transfer enzymes that gives rise to their differential substrate specificities will provide insight into the evolutionary changes that occurred in this superfamily leading to the different enzymatic functions. Funding Source: NIH