title: Probing the origins of catalytic discrimination between phosphate and sulfate monoester hydrolysis: comparative analysis of alkaline phosphatase and protein tyrosine phosphatases.
abstract: Catalytic promiscuity, the ability of enzymes to catalyze multiple reactions, provides an opportunity to gain a deeper understanding of the origins of catalysis and substrate specificity. Alkaline phosphatase (AP) catalyzes both phosphate and sulfate monoester hydrolysis reactions with a ∼10(10)-fold preference for phosphate monoester hydrolysis, despite the similarity between these reactions. The preponderance of formal positive charge in the AP active site, particularly from three divalent metal ions, was proposed to be responsible for this preference by providing stronger electrostatic interactions with the more negatively charged phosphoryl group versus the sulfuryl group. To test whether positively charged metal ions are required to achieve a high preference for the phosphate monoester hydrolysis reaction, the catalytic preference of three protein tyrosine phosphatases (PTPs), which do not contain metal ions, were measured. Their preferences ranged from 5 × 10(6) to 7 × 10(7), lower than that for AP but still substantial, indicating that metal ions and a high preponderance of formal positive charge within the active site are not required to achieve a strong catalytic preference for phosphate monoester over sulfate monoester hydrolysis. The observed ionic strength dependences of kcat/KM values for phosphate and sulfate monoester hydrolysis are steeper for the more highly charged phosphate ester with both AP and the PTP Stp1, following the dependence expected based on the charge difference of these two substrates. However, the dependences for AP were not greater than those of Stp1 and were rather shallow for both enzymes. These results suggest that overall electrostatics from formal positive charge within the active site is not the major driving force in distinguishing between these reactions and that substantial discrimination can be attained without metal ions. Thus, local properties of the active site, presumably including multiple positioned dipolar hydrogen bond donors within the active site, dominate in defining this reaction specificity.
authors.affiliation: Department of Chemical and Systems Biology, ‡Department of Chemistry, and §Department of Biochemistry, Stanford University , Stanford, California 94305-5307, United States.
authors.forename: Logan D
authors.initials: LD
authors.lastname: Andrews
authors.forename: Jesse G
authors.initials: JG
authors.lastname: Zalatan
authors.forename: Daniel
authors.initials: D
authors.lastname: Herschlag
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D010755:Organophosphates / Q000737:chemistry*; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry*; D055672:Static Electricity; D013379:Substrate Specificity; D013463:Sulfuric Acid Esters / Q000737:chemistry*
chemical_list: D010755:Organophosphates; D013463:Sulfuric Acid Esters; D000469:Alkaline Phosphatase; D017027:Protein Tyrosine Phosphatases