Probing the Role of the Trivalent Metal in Phosphate Ester Hydrolysis: Preparation and Characterization of Purple Acid Phosphatases Containing AlIIIZnII and InIIIZnII Active Sites, Including the First Example of an Active Aluminum Enzyme

Abstract

Purple acid phosphatases contain a dinuclear Fe3+M2+ center in their active site (M = Fe2+ or Zn2+). To resolve the specific role of the ferric ion in catalysis, a series of metal-substituted forms of bovine spleen purple acid phosphatase (BSPAP) of general formula MIIIZnII-BSPAP has been prepared, in which the trivalent metal ion was systematically varied (MIII = Al, Fe, Ga, and In). The activity of the AlZn-BSPAP form was only slightly lower (kcat ≈ 2000 s-1) than that of the previously reported GaZn and FeZn forms (kcat ≈ 3000 s-1). The InZn form was inactive. The kinetics parameters and pH profile of AlZn-BSPAP were remarkably similar to those of FeZn-BSPAP and GaZn-BSPAP, but AlZn-BSPAP was readily distinguished from the GaZn and FeZn forms by its 50−70-fold lower inhibition constant for fluoride. These results are not, at first sight, consistent with intrinsic properties of the trivalent metal ions as they are known from coordination chemistry. In particular, aluminum has generally been believed to be of little use as a Lewis acid in the active site of an enzyme because of the slow ligand exchange rates typically observed for aluminum complexes. The present results are thus in conflict with this general wisdom. The conflict can be resolved either by assuming that the protein modulates the properties of the aluminum ion such that ligand exchange rates are substantially enhanced and thus not rate-limiting, or by assuming a catalytic mechanism in which ligand exchange does not take place at the trivalent metal ion.