STRUCTURE-FUNCTION-RELATIONSHIPS IN HEXOKINASE - SITE-DIRECTED MUTATIONAL ANALYSES AND CHARACTERIZATION OF OVEREXPRESSED FRAGMENTS IMPLICATE DIFFERENT FUNCTIONS FOR THE N-TERMINAL AND C-TERMINAL HALVES OF THE ENZYME

Hexokinases are comprised of two highly homologous approximately 50-kD a halves and are product-inhibited by glucose-6-P. Four amino acid res idues, Ser603, Asp657, Glu708, and Glu742, located in the C-terminal h alf of the tumor mitochondrial enzyme have been shown to be essential for enzyme function (Arora, K. K., Filburn, C. R., and Pedersen, P. L. (1991) J. Biol. Chem. 266, 5359-5362). Here we have assessed also the role of the N-terminal half of the same enzyme.Site-directed mutagene sis of residues predicted to interact with glucose in the N-terminal h alf, i.e. Ser155, Asp209, and Glu260, to Ala, have no effect on hexoki nase activity. In addition, inhibition by hexose mono- and bisphosphat es is unchanged for each of the mutant enzymes. Significantly, the ove rexpressed N-terminal polypeptide is devoid of catalytic activity but does have the capacity to bind ATP-agarose and be released with ATP an d glucose-6-P. In contrast, the overexpressed C-terminal polypeptide i s catalytically active and shows the same-product inhibition pattern a s the complete 100-kDa parent enzyme. These results emphasize that the N-terminal half of tumor hexokinase is essential neither for catalysi s nor product modulation. Rather, the N-terminal half may play another role, perhaps in modulation of the ATP/glucose-6-P-dependent binding of the enzyme to tumor mitochondria or by acting as a spacer between t he outer mitochondrial membrane and the C-terminal catalytic unit.