The activity of oxidized bovine spleen purple acid phosphatase is due to an Fe(III)Zn(II) 'impurity'.
Bovine spleen purple acid phosphatase (BSPAP) is a dinuclear iron protein with two stable redox states. The Fe3+Fe2+ state is the active state, while the fully oxidized protein (BSPAPox) has been reported to retain 5-10% activity, corresponding to a kcat of ca. 150 s-1 [Dietrich, M., Münstermann, D., Suerbaum, H., and Witzel, H. (1991) Eur. J. Biochem. 199, 105-113]. Here we show that this activity does not originate 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 procedure, and its kinetic properties were carefully determined for comparison to those of BSPAPox. For the hydrolysis of p-NPP at pH 6.00, both kcat and KM were affected by the Fe2+-to-Zn2+-substitution [Fe3+Fe2+-BSPAP, kcat = (1.8 +/- 0.1) x 10(3) s-1 and KM = 1.2 +/- 0.2 mM; Fe3+Zn2+-BSPAP; kcat = (2.8 +/- 0.2) x 10(3) s-1 and KM = 3.3 +/- 0.4 mM]. The KM of BSPAPox was the same as that of FeZn-BSPAP. pH profiles of BSPAPox and FeZn-BSPAP were both shifted to lower pH compared to that of BSPAPred. FeZn-BSPAP, FeZn-BSPAP.PO4, and FeZn-BSPAP.MoO4 all showed characteristic EPR spectra similar to the corresponding complexes of FeZn-Uf. The same species could also be observed in concentrated samples of native BSPAP. Spin integration of these spectra showed a quantitative relation between the spin concentration of the FeZn-BSPAP 'impurity' and the residual phosphatase activity after oxidation. Since all activity found after oxidation of BSPAP could be attributed to FeZn-BSPAP, there is no direct evidence that Fe3+Fe3+-BSPAP is catalytically active. These results set an upper limit to the possible catalytic activity of the Fe3+Fe3+ form of </=1% of that of the Fe3+Fe2+ form, a finding that is important for understanding the fundamental chemistry by which these dinuclear enzymes catalyze the hydrolysis of phosphate esters.
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E.C. Slater Institute, Biocentrum Amsterdam, University of Amsterdam, The Netherlands.
Averill
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BA
D000135:Acid Phosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D001149:Arsenates / Q000494:pharmacology; D001667:Binding, Competitive; D002384:Catalysis; D002417:Cattle; D004492:Edetic Acid; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000187:drug effects; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism*; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008982:Molybdenum / Q000494:pharmacology; D010084:Oxidation-Reduction; D010710:Phosphates / Q000494:pharmacology; D013154:Spleen / Q000201:enzymology; D017973:Tungsten Compounds / Q000494:pharmacology; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
D001051:Apoenzymes; D001149:Arsenates; D005290:Ferric Compounds; D006023:Glycoproteins; D010710:Phosphates; D017973:Tungsten Compounds; C044659:molybdate; D008982:Molybdenum; D004492:Edetic Acid; C091137:purple acid phosphatase; D000135:Acid Phosphatase; D015032:Zinc; C025657:arsenic acid; C045951:tungstate