Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures

An intracellular acid phosphatase (IAP) from P_i-starved (−P_i) tomato (Lycopersicon esculentum) suspension cells has been purified to homogeneity. IAP is a purple acid phosphatase (PAP), as the purified protein was violet in colour (λ_max=546 nm) and was insensitive to l-tartrate. PAGE, periodic acid–Schiff staining and peptide mapping demonstrated that the enzyme exists as a 142 kDa heterodimer composed of an equivalent ratio of glycosylated and structurally dissimilar 63 (α-subunit) and 57 kDa (β-subunit) polypeptides. However, the nine N-terminal amino acids of the α- and β-subunits were identical, exhibiting similarity to the deduced N-terminal portions of several putative plant PAPs. Quantification of immunoblots probed with rabbit anti-(tomato acid phosphatase) immune serum revealed that the 4-fold increase in IAP activity due to P_i-deprivation was correlated with similar increases in the amount of antigenic IAP α- and β-subunits. IAP displayed optimal activity at pH 5.1, was activated 150% by 10 mM Mg²⁺, but was potently inhibited by Zn²⁺, Cu²⁺, Fe³⁺, molybdate, vanadate, fluoride and P_i. Although IAP demonstrated broad substrate selectivity, its specificity constant (V_max/K_m) with phosphoenolpyruvate was >250% greater than that obtained with any other substrate. IAP exhibited significant peroxidase activity, which was optimal at pH 9.0 and insensitive to Mg²⁺ or molybdate. This IAP is proposed to scavenge P_i from intracellular phosphate esters in −P_i tomato. A possible secondary IAP role in the metabolism of reactive oxygen species is discussed. IAP properties are compared with those of two extracellular PAP isoenzymes that are secreted into the medium of −P_i tomato cells [Bozzo, Raghothama and Plaxton (2002) Eur. J. Biochem. 269, 6278–6286].