Previous work has shown that catalytic function is associated exclusively with the C-terminal half of the Type I isozyme of mammalian hexokinase. In contrast, we now demonstrate that both halves of the Type II isozyme possess comparable catalytic activities. Mutation of a catalytically important Ser residue to Ala at analogous positions in either the N- or the C-terminal halves (S155A or S603A, respectively) of the rat Type II isozyme resulted in approximately 60% reduction in specific activity of the enzyme, with more than 90% reduction in the doubly mutated enzyme (S155A/S603A). Catalytic activity was retained in a chimeric hexokinase comprising the N-terminal half of Type II hexokinase and catalytically inactive (by site-directed mutation) C-terminal half of the Type I isozyme. The N- and C-terminal catalytic sites of Type II hexokinase are similar in V(max) and K(m) (approximately equal to 130 microM) for glucose; however, the N-terminal site has a lower (0.45 vs 1.1 mM) K(m) for ATP, is slightly more sensitive to inhibition by the product analog 1,5-anhydroglucitol-6-P, and is much more sensitive to inhibition by P(i). It is suggested that the Type II isozyme most closely resembles the 100-kDa hexokinase which resulted from duplication and fusion of a gene encoding an ancestral 50-kDa hexokinase and which was the precursor for the contemporary Type I, Type II, and Type III mammalian isozymes. Subsequent evolutionary changes could then have led to functional differentiation of the N- and C-terminal halves, as seen with the Type I (and possibly the Type III) isozyme.