Allosteric Regulation of Type I Hexokinase: A Site-Directed Mutational Study Indicating Location of the Functional Glucose 6-Phosphate Binding Site in the N-Terminal Half of the Enzyme

Abstract

The Type I isozyme of mammalian hexokinase has evolved by a gene duplication–fusion mechanism, with resulting internal duplication of sequence and ligand binding sites. However, 1:1 binding stoichiometry indicates that only one of these is available for binding the product inhibitor, Glc-6-P; the location of that site, in the N- or C-terminal half, remains under debate. Recent structural studies (Aleshinet al., Structure6, 39–50, 1998; Mulichaket al., Nature Struct. Biol.5, 555–560, 1998) implicated Asp 84 or its analog in the C-terminal half, Asp 532, in binding of Glc-6-P. Zenget al.(Biochemistry35, 13157–13164, 1996) demonstrated that mutation of Asp 532 to Lys or Glu did not affect inhibition by the Glc-6-P analog, 1,5-anhydroglucitol-6-P. These same mutations, as well as mutation to Ala, at the Asp 84 position are now shown to result in increasedKifor 1,5-anhydroglucitol-6-P. The ability of Pito antagonize inhibition by the Glc-6-P analog is severely diminished or abolished by these mutations, suggesting that antagonism is dependent on precise positioning of the inhibitory hexose 6-phosphate. The structure of the enzyme complexed with Glc and Pihas been determined, and shows that Pioccupies the same site as the 6-phosphate group in the complex with Glc-6-P. Thus, antagonism between these ligands results from competition for a common anion binding site in the N-terminal half.