Probing the Origins of Catalytic Discrimination betweenPhosphate and Sulfate Monoester Hydrolysis: Comparative Analysis ofAlkaline Phosphatase and Protein Tyrosine Phosphatases

摘要

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 ∼10¹⁰-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 × 10⁶ to 7 × 10⁷, lower than that for AP but still substantial, indicating that metal ionsand a high preponderance of formal positive charge within the activesite are not required to achieve a strong catalytic preference forphosphate monoester over sulfate monoester hydrolysis. The observedionic strength dependences of kcat/KM values for phosphate and sulfate monoesterhydrolysis are steeper for the more highly charged phosphate esterwith both AP and the PTP Stp1, following the dependence expected basedon the charge difference of these two substrates. However, the dependencesfor AP were not greater than those of Stp1 and were rather shallowfor both enzymes. These results suggest that overall electrostaticsfrom formal positive charge within the active site is not the majordriving force in distinguishing between these reactions and that substantialdiscrimination can be attained without metal ions. Thus, local propertiesof the active site, presumably including multiple positioned dipolarhydrogen bond donors within the active site, dominate in definingthis reaction specificity.