Abstract
A purple acid phosphatase from sweet potato is the first reported example of a protein containing an enzymatically active binuclear Fe-Mn center. Multifield saturation magnetization data over a temperature range of 2 to 200 K indicates that this center is strongly antiferromagnetically coupled. Metal ion analysis shows an excess of iron over manganese. Low temperature EPR spectra reveal only resonances characteristic of high spin Fe(III) centers (Fe(III)-apo and Fe(III)-Zn(II)) and adventitious Cu(II) centers. There were no resonances from either Mn(II) or binuclear Fe-Mn centers. Together with a comparison of spectral properties and sequence homologies between known purple acid phosphatases, the enzymatic and spectroscopic data strongly indicate the presence of catalytic Fe(III)-Mn(II) centers in the active site of the sweet potato enzyme. Because of the strong antiferromagnetism it is likely that the metal ions in the sweet potato enzyme are linked via a mu-oxo bridge, in contrast to other known purple acid phosphatases in which a mu-hydroxo bridge is present. Differences in metal ion composition and bridging may affect substrate specificities leading to the biological function of different purple acid phosphatases.
Highlights
Purple acid phosphatases comprise a family of binuclear metal-containing enzymes, the members of which have been identified in plants, animals, and fungi
For the red kidney bean and soybean purple acid phosphatases, it could be shown that the specific activity correlates with the content of the divalent ion, Zn [4, 8]
A similar treatment with EDTA of the sweet potato enzyme characterized in our laboratory does not remove manganese, suggesting that the Fe-Mn binuclear cluster in sweet potato purple acid phosphatase is responsible for the catalytic activity
Summary
Purple acid phosphatases comprise a family of binuclear metal-containing enzymes, the members of which have been identified in plants, animals, and fungi. Metal ion analysis showed that the sample of enzyme used for magnetic susceptibility and EPR measurements contained 1.035 Ϯ 0.108 iron, 0.582 Ϯ 0.044 manganese, 0.183 Ϯ 0.019 zinc, and 0.11 Ϯ 0.028 copper/ subunit of 55 kDa. Oxidation and Reduction—Pig purple acid phosphatase was prepared as described previously [9].
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