Purification, Primary Structure, and Properties of Euphorbia characias Latex Purple Acid Phosphatase

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F. Pintus^#, D. Spanò^#, S. Corongiu, G. Floris, and R. Medda^*

Department of Applied Sciences in Biosystems, University of Cagliari, Cagliari, Italy; fax: +39 (070) 675-4523; E-mail: rmedda@unica.it

^# These authors contributed equally to this work.

^* To whom correspondence should be addressed.

Received November 25, 2010; Revision received January 22, 2011

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A purple acid phosphatase was purified to homogeneity from Euphorbia characias latex. The native protein has a molecular mass of 130 ± 10 kDa and is formed by two apparently identical subunits, each containing one Fe(III) and one Zn(II) ion. The two subunits are connected by a disulfide bridge. The enzyme has an absorbance maximum at 540 nm, conferring a characteristic purple color due to a charge-transfer transition caused by a tyrosine residue (Tyr172) coordinated to the ferric ion. The cDNA nucleotide sequence contains an open reading frame of 1392 bp, and the deduced sequence of 463 amino acids shares a very high degree of identity (92-99%) to other purple acid phosphatases isolated from several higher plants. The enzyme hydrolyzes well p-nitrophenyl phosphate, a typical artificial substrate, and a broad range of natural phosphorylated substrates, such as ATP, ADP, glucose-6-phosphate, and phosphoenolpyruvate. The enzyme displays a pH optimum of 5.75 and is inhibited by molybdate, vanadate, and Zn²⁺, which are typical acid phosphatase inhibitors.

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KEY WORDS: acid phosphatase, Euphorbia characias, iron ion, metalloenzymes, purple phosphatase, zinc ion

DOI: 10.1134/S0006297911060101

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