FUNCTIONAL-ORGANIZATION OF MAMMALIAN HEXOKINASES - BOTH N-TERMINAL AND C-TERMINAL HALVES OF THE RAT TYPE-II ISOZYME POSSESS CATALYTIC SITES

Previous work has shown that catalytic function is associated exclusiv ely with the C-terminal half of the Type I isozyme of mammalian hexoki nase. In contrast, we now demonstrate that both halves of the Type II isozyme possess comparable catalytic activities. Mutation of a catalyt ically important Ser residue to Ala at analogous positions in either t he N- or the C-terminal halves (S155A or S603A, respectively) of the r at Type II isozyme resulted in approximately 60% reduction in specific activity of the enzyme, with more than 90% reduction in the doubly mu tated enzyme (S155A/S603A). Catalytic activity was retained in a chime ric hexokinase comprising the N-terminal half of Type II hexokinase an d catalytically inactive (by site-directed mutation) C-terminal half o f the Type I isozyme. The N- and C-terminal catalytic sites of Type II hexokinase are similar in V-max and K-m (approximate to 130 mu M) 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 ana log 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 resemble s the 100-kDa hexokinase which resulted from duplication and fusion of a gene encoding an ancestral 50-kDa hexokinase and which was the prec ursor for the contemporary Type I, Type II, and Type III mammalian iso zymes. Subsequent evolutionary changes could then have led to function al differentiation of the N- and C-terminal halves, as seen with the T ype I (and possibly the Type III) isozyme. (C) 1996 Academic Press, In c.