Purification and characterization of a glucokinase from young tomato (Lycopersicon esculentum L. Mill.) fruit.

In order to clearly establish the properties of the enzymes responsible for hexose phosphorylation we have undertaken the separation and characterization of these enzymes present in tomato fruit (Martinez-Barajas and Randall 1996). This report describes the partial purification and characterization of glucokinase (EC. 2.7.1.1) from young green tomato fruit. The procedure yielded a 360-fold enrichment of glucokinase. Tomato fruit glucokinase is a monomer with a molecular mass of 53 kDa. Glucokinase activity was optimal between pH 7.5 and 8.5, preferred ATP as the phosphate donor (K(m) = 0.223 mM) and exhibited low activity with GTP or UTP. The tomato fruit glucokinase showed highest affinity for glucose (K(m) = 65 microM). Activity observed with glucose was 4-fold greater than with mannose and 50-fold greater than with fructose. The tomato fruit glucokinase was sensitive to product inhibition by ADP (Ki = 36 microM). Little inhibition was observed with glucose 6-phosphate (up to 15 mM) at pH 8.0; however, at pH 7.0 glucokinase activity was inhibited 30-50% by physiological concentrations of glucose 6-phosphate.