MECHANISM OF FE(III)-ZN(II) PURPLE ACID PHOSPHATASE BASED ON CRYSTAL STRUCTURES. T. Klabundea, N. Strätera, H. Witzelb, B. Krebsa, aAnorganisch-Chemisches Institut, Westfälische Wilhelms-Universität, Wilhelm-Klemm-Strasse 8, D48149 Münster, Germany, bInstitut für Biochemie, Westfälische, Wilhelms-Universität, Wilhelm-Klemm-Strasse 2, D48149 Münster, Germany
Purple acid phosphatases (PAPs) containing a dinuclear Fe(III) -Me(II) center (where Me can be Fe or Zn) in their active sites catalyse the hydrolysis of activated phosphoric acid esters and anhydrides, like ATP, at a pH range from 4 to 7. All mammalian PAPs characterized so far are monomeric proteins with a molecular mass of approximately 35 kDa containing an Fe(III)-Fe(II) center in the active site. In contrast, the most intensively studied plant enzyme from kidney bean (kbPAP) is a homodimeric Fe(III)-Zn(II) metalloprotein of molecular mass 111 kDa.
X-ray structures of kbPAP complexed with phosphate, the product of the reaction, and with tungstate, a strong inhibitor of the phosphatase activity, were determined at 2.7 and 3.0 Å resolution, respectively. Furthermore the resolution of the unliganded enzyme, recently solved at 2.9 Å [1] could be extended to 2.65 Å with completely new data. In the inhibitor complex as well as in the product complex, the oxoanion binds in a bidentate bridging mode to the two metal ions, replacing two of the presumed solvent ligands present in the unliganded enzyme form. All three structures support a mechanism of phosphate ester hydrolysis involving interaction of the substrate with Zn(II) followed by a nucleophilic attack on the phosphorus by an Fe(III)-coordinated hydroxide ion. The negative charge evolving at the pentacoordinated transition state is probably stabilized by interactions with the divalent zinc and the imidazole groups of His-202, His-295 and His-296, the later protonating the leaving alcohol group.
[1] N. Sträter, T. Klabunde, P. Tucker, H. Witzel & B. Krebs, Science 268,1489