| Alkaline Phosphatase (AP) from Escherichia coli is the prototypical two metal ion catalyst, employing two divalent zinc ions to catalyze transfer of a phosphoryl group with great proficiency. The wealth of structural information and prior elucidation of the reaction pathway renders AP a good model system for understanding the potent catalytic power of bimetallic centers. Previous kinetic analyses have been complicated by tight binding of the product, inorganic phosphate. A new assay has been developed that is more sensitive and allows the apparent second-order kinetic parameter, kcat/K m, to be accurately determined. The steep dependence of reactivity on the pKa of the leaving group for a series of primary alkyl phosphates and the smaller magnitude of kcat/Km, relative to aryl phosphates, strongly suggests that the chemical step for phosphorylation of the enzyme is rate-limiting. This new assay has been employed to reinvestigate basic features of the catalytic mechanism, including the pH dependence, the role of the active site arginine, and the nature of the enzymatic transition state.;Recent structural analyses and sequence comparisons have revealed structural homologies between alkaline phosphatases, arylsulfatases, and certain phosphodiesterases, suggesting that these distinct classes of enzymes are evolutionarily related. We have investigated the ability of AP to catalyze hydrolysis of phosphate diesters and sulfate monoesters. These studies strongly suggest that AP does have a low level of phosphodiesterase and sulfatase activity. This activity is much less efficient than the normal reaction, but nonetheless corresponds to substantial rate enhancements. Such low levels of activity toward alternative reactions could facilitate divergence of an enzyme encoded by a duplicated gene by providing a selective advantage, which would then allow optimization via natural selection. Several additional contemporary enzymes are known to catalyze alternative reactions that differ significantly from their normal, biological reactions. In some cases, the alternative reaction is similar to the reaction that is efficiently catalyzed by an evolutionarily related enzyme. The diversity of alternative reactions catalyzed at enzyme active sites suggests that a low level of catalytic promiscuity may be a common characteristic of enzymes. Such catalytic promiscuity may have aided the evolution of new enzymes via divergent evolution, even enzymes that utilize different mechanisms and catalyze different types of reactions. |
