MECHANISM OF FE(III)-ZN(II) PURPLE ACID-PHOSPHATASE BASED ON CRYSTAL-STRUCTURES

Purple acid phosphatase is a widely distributed non-specific phosphomo noesterase. X-ray structures of the dimeric 111-kDa Fe(III)-Zn(II) kid ney bean purple acid phosphatase (kbPAP) complexed with phosphate, the product of the reaction, and with tungstate, a strong inhibitor of th e phosphatase activity, were determined at 2.7 and 3.0 Angstrom resolu tion, respectively. Furthermore the resolution of the unligated enzyme , recently solved at 2.9 Angstrom could be extended to 2.65 Angstrom w ith completely new data. The binding of both oxoanions is not accompan ied by larger conformational changes in the enzyme structure. Small mo vements with a maximal coordinate shift of 1 Angstrom are only observe d for the active site residues His295 and His296. In the inhibitor com plex as well as in the product complex, the oxoanion binds in a bident ate bridging mode to the two metal ions, replacing two of the presumed solvent ligands present in the unligated enzyme form. As also propose d for the unligated structure a bridging hydroxide ion completes the c oordination spheres of both metal ions to octahedral arrangements. All three structures reported herein support a mechanism of phosphate est er hydrolysis involving interaction of the substrate with Zn(II) follo wed by a nucleophilic attack on the phosphorus by an Fe(III)-coordinat ed hydroxide ion. The negative charge evolving at the pentacoordinated transition state is probably stabilized by interactions with the diva lent zinc and the imidazole groups of His202, His295, and His296, the latter protonating the leaving alcohol group. (C) 1996 Academic Press Limited