The Molecular Structure of Purple Acid Phosphatases

Purple acid phosphatases are a class of nonspecific acid phosphatases. They are structurally and functionally related to the serine/threonine protein phosphatases which are involved in the regulation of diverse cellular processes. Understanding of the structures and function of purple acid phosphatases will provide valuable insight into the protein phosphatases as well. Of the purple acid phosphatases, the X-ray crystal structure of the kidney bean purple acid phosphatase and the purple acid phosphatases from pig have been reported.^22,23 The pig purple acid phosphatase is a monomeric 35-kD glycoprotein that contains a dinuclear iron center. Biochemical and spectroscopic characterization of other purple acid phosphatases from mammalian sources are consistent with this composition and structure. The red kidney bean purple acid phosphatase is a homodimer with an intersubunit disulfide in which each subunit contains a dinuclear iron-zinc center. Available data for the subunit molecular size, the enzymatic activity, and the electronic absorbance and EPR spectra suggest that the purple acid phosphatases from sweet potato and red kidney beans are highly similar. The proposed research addresses two questions regarding the molecular structure of purple acid phosphatases.

The first concerns the microheterogeneity that is observed in preparations of most purple acid phosphatases. This microheterogeneity is believed to arise from variations in glycosylation of the enzymes and complicates characterization of the metal centers of the phosphatases. HPLC-MS/MS will be used to analyze the carbohydrate content of the purple acid phosphatases from sweet potato and the bacterium, Lysobacter enzymogenes, and determine the effect of the oligosaccharides on the homogeneity of these enzymes.

The second concerns the subunit structure of the various purple acid phosphatases and its effect on the substrate specificity and kinetic properties of the purple acid phosphatases. The proposed work involves covalent modification of a monomeric form of sweet potato purple acid phosphatase and comparison of its properties to those of the native dimeric enzyme from red kidney bean and to those of the mammalian purple acid phosphatases. HPLC-MS/MS will be used to confirm the successful modification of the monomer.