THE ACTIVITY OF OXIDIZED BOVINE SPLEEN PURPLE ACID-PHOSPHATASE IS DUETO AN FE(III)ZN(II) IMPURITY

Bovine spleen purple acid phosphatase (BSPAP) is a dinuclear iron prot ein with two stable redox states. The Fe3+Fe2+ state is the active sta te, while the fully oxidized protein (BSPAP,,) has been reported to re tain 5-10% activity, corresponding to a k(cat) of ca. 150 s(-1) [Dietr ich, M., Munstermann, D., Suerbaum, H., and Witzel, H. (1991) fur. J. Biochem. 199, 105-113]. Here we show that this activity does not origi nate from Fe3+Fe3+-BSPAP, but rather from an 'impurity' of FeZn-BSPAP. The FeZn form of BSPAP was prepared from apo-BSPAP following a new pr ocedure, and its kinetic properties were carefully determined for comp arison to those of BSPAP(ox). For the hydrolysis of p-NPP at pH 6.00, both k(cat) and KM were affected by the Fe2+-to-Zn2+-substitution [Fe3 +Fe2+-BSPAP, k(cat) = (1.8 +/- 0.1) x 10(3) s(-1) and K-M = 1.2 +/- 0. 2 mM; Fe3+Zn2+-BSPAP; k(cat) = (2.8 +/- 0.2) x 10(3) s(-1) and K-M = 3 .3 +/- 0.4 mM]. The K-M of BSPAP(ox) was the same as that of FeZn-BSPA P. pH profiles of BSPAP(ox) and FeZn-BSPAP were both shifted to lower pH compared to that of BSPAP(red). FeZn-BSPAP, FeZn-BSPAP . PO4, and F eZn-BSPAP . MoO4 all showed characteristic EPR spectra similar to the corresponding complexes of FeZn-Uf. The same species could also be obs erved in concentrated samples of native BSPAP. Spin integration of the se spectra showed a quantitative relation between the spin concentrati on of the FeZn-BSPAP 'impurity' and the residual phosphatase activity after oxidation. Since all activity found after oxidation of BSPAP cou ld be attributed to FeZn-BSPAP, there is no direct evidence that Fe(3)Fe(3+-)BSPAP is catalytically active. These results set an upper limi t to the possible catalytic activity of the Fe3+Fe3+ form of less than or equal to 1% of that of the Fe3+Fe2+ form, a finding that is import ant for understanding the fundamental chemistry by which these dinucle ar enzymes catalyze the hydrolysis of phosphate esters.