Purification and characterization of sorbitol-6-phosphate phosphatase from apple leaves

Sorbitol-6-phosphate phosphatase (SorPP; EC 3.1.3.50) catalyzes the final step in sorbitol biosynthesis in sorbitol-synthesizing plant species, but its kinetic and regulatory properties have not been characterized. In this study, the enzyme was purified 1727-fold to apparent homogeneity from apple leaves with a maximal specific activity of 89.8 mumol min(-1) mg(-1) protein measured at 2 mM sorbitol-6-phosphate (sorbitol-6-P). The enzyme is a monomer with a molecular mass of 61 kDa. The enzyme is highly specific for sorbitol-6-P with a Kin of 0.85 mM and is unable to cleave other phosphate esters at a significant rate. The activity is absolutely dependent on Mg2+ with a Km of 0.29 mM at an optimal pH of 6.8. Fluoride, vanadate, molybdate, and inorganic phosphate inhibit SorPP activity. Sorbitol is a competitive inhibitor for SorPP with a Ki of 109 mM. The possible feedback mechanism for the regulation of sorbitol biosynthesis is also discussed. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.