STRUCTURAL STUDIES OF PURPLE ACID PHOSPHATASE FROM RAT BONE

Jia Jia¹, Eva Johansson¹, Helena Kaija², Pirkko Vihko², Gunter Schneider¹ and Ylva Lindqvist¹

¹Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Doktorsringen 9A1, S-171 77 Stockholm, Sweden
² Biocenter Oulu and WHO Collaborating Centre for Research on Reproductive Health, University of Oulu, Kajaanintie 50, FIN-90220 Oulu, Finland

Purple acid phosphatases (PAPs) are enzymes which catalyse the hydrolysis of monophosphate esters in an acidic environment. These enzymes contain a dinuclear metal centre and a charge transfer transition from a tyrosine to a ferric ion gives PAP its characteristic purple colour [1]. A second property has given the enzyme another name, tartrate resistant acid phosphatase (TRAP), due to the fact that tartrate does not inhibit the enzyme.

PAPs are expressed in bone-resorbing osteoclasts and certain tissue macrophages. The enzyme has been found in large quantities in patients with hairy cell leukemia and it is a cytochemical marker for this disease. Increased serum concentrations and activity of PAP have been demonstrated in children, in postmenopausal women and in patients with metabolic bone diseases.

The physiological function of PAP is unclear, but there are some suggested functions:

* It has been shown that PAP works as a protein tyrosine phosphatase in vitro and it might also do so in vivo [2].

* PAP can dephosphorylate the bone matrix protein osteopontin to which osteoclasts bind. After dephosphorylation the osteoclasts can no longer bind and in this way PAP might regulate the attachment of osteoclasts to bone matrix [3].

* Being a di-iron protein PAP can be involved in iron transport and iron metabolism [4].

* PAP is a ferric ion containing protein it can be suitable for generation of free oxygen radicals by Fenton's reaction (Fe²⁺ + H₂O₂ -> Fe³⁺ + OH^- + .OH). The free oxygen radical is involved in bone resorption [5].

The protein used in this structural study is rat bone PAP expressed in a baculovirus system. The structure of PAP from red kidney bean has been determined [6] and the sequence identity between mammalian and plant PAP is 20 % [7].

Only small crystals have been grown so far and data have been collected on a 30 mm crystal at ID13, ESRF. The crystals are orthorombic, space group P2₁2₁2₁ or possibly P2₁2₁2 with cell dimensions a=57.2 A, b=69.5 A and c=87.2 A. The crystals diffract to 2.2 A (Rmerge of 9.8 %).

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