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

The Type I isozyme of mammalian hexokinase has evolved by a gene duplicatio n-fusion mechanism, with resulting internal duplication of sequence and lig and binding sites. However, 1:1 binding stoichiometry indicates that only o ne of these is available for binding the product inhibitor, Glc-6-P; the lo cation of that site, in the N- or C-terminal half, remains under debate. Re cent structural studies (Aleshin et al, Structure 6, 39-50, 1998; Mulichak et al., Nature Struct. Biol. 5, 555-560, 1998) implicated Asp 84 or its ana log in the C-terminal half, Asp 532, in binding of Glc-6-P. Zeng et al. (Bi ochemistry 35, 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-anhydrogluc itol-6-P. These same mutations, as well as mutation to Ala, at the Asp 84 p osition are now shown to result in increased Ki for 1,5-anhydroglucitol-6-P . The ability of Pi to antagonize inhibition by the Glc-6-P analog is sever ely diminished or abolished by these mutations, suggesting that antagonism is dependent on precise positioning of the inhibitory hexose B-phosphate. T he structure of the enzyme complexed with Glc and Pi has been determined, a nd shows that Pi occupies the same site as the 6-phosphate group in the com plex with Glc-6-P. Thus, antagonism between these ligands results from comp etition for a common anion binding site in the N-terminal half. (C) 1999 Ac ademic Press.