Mechanistic studies of purple acid phosphatases: Spectroscopic investigations of oxoanions and fluoride inhibition

Purple acid phosphatases (PAPs) belong to a family of metallohydrolases containing bimetallic active sites that catalyze the hydrolysis of phosphomonoesters. They are similar to Ser/Thr protein phosphatases (PPs) with respect to protein phosphatase activity, sequence homology at the catalytic regions and active site structure. Thus, PAPs by analogy may play a similar role in regulating some cellular process as PPs.; Uteroferrin, the PAP from porcine uterus, is one of the best characterized enzymes of the family. It contains a native Fe(III)Fe(II) active center and the Fe(II) site can be substituted by Zn with full activity. However, so far no crystal structure is available for uteroferrin. X-ray absorption spectroscopy is a powerful technique to examine the structure of the metal centers in the absence of a crystal structure. The FeZn derivative of uteroferrin (FeZnUf) and its oxoanion complexes are characterized by X-ray absorption spectroscopy at both the iron and zinc K-edges to gain insight into the nature of the FeZn active site as well as the oxoanion binding mode (Chapter 2). The results show that the oxoanions bridge the FeZn active site. Chapter 3 focuses on mechanistic studies using fluoride as a probe for water or hydroxide binding. Fluoride is an uncompetitive inhibitor, which implies that fluoride inhibits by acting on the enzyme-substrate (ES) complex. A ternary ES-fluoride complex is generated using FeZnUf as demonstrated by UV-vis and EPR spectroscopy; such a ternary complex models the activated ES complex which is the key to the catalytic mechanism. The ternary enzyme-phosphate-fluoride complex is further characterized by X-ray absorption and resonance Raman spectroscopy. Phosphate most likely forms a bidentate bridge between the two metal ions, and fluoride replaces the hydroxide bridge. A mechanism is proposed based on these observations which involves the attack of the bridging hydroxide on the substrate which binds to the dinuclear site in a bidentate bridging mode.