Hexokinases are comprised of two highly homologous approximately 50-kDa halves and are product-inhibited by glucose-6-P. Four amino acid residues, Ser603, Asp657, Glu708, and Glu742, located in the C-terminal half 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 mutagenesis of residues predicted to interact with glucose in the N-terminal half, i.e. Ser155, Asp209, and Glu260, to Ala, have no effect on hexokinase activity. In addition, inhibition by hexose mono- and bisphosphates is unchanged for each of the mutant enzymes. Significantly, the overexpressed N-terminal polypeptide is devoid of catalytic activity but does have the capacity to bind ATP-agarose and be released with ATP and glucose-6-P. In contrast, the overexpressed C-terminal polypeptide is catalytically active and shows the same product inhibition pattern as the complete 100-kDa parent enzyme. These results emphasize that the N-terminal half of tumor hexokinase is essential neither for catalysis 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 the outer mitochondrial membrane and the C-terminal catalytic unit.