title: 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 (Aleshin et al., Structure 6, 39-50, 1998; Mulichak et 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. Zeng et al. (Biochemistry 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-anhydroglucitol-6-P. These same mutations, as well as mutation to Ala, at the Asp 84 position 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 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 Pi has been determined, and shows that Pi occupies 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.
authors.affiliation: Department of Biochemistry, Michigan State University, East Lansing, Michigan, 48824-1319, USA.
authors.forename: S
authors.initials: S
authors.lastname: Sebastian
authors.forename: J E
authors.initials: JE
authors.lastname: Wilson
authors.forename: A
authors.initials: A
authors.lastname: Mulichak
authors.forename: R M
authors.initials: RM
authors.lastname: Garavito
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D003460:Crystallization; D019281:Dimerization; D005947:Glucose / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D006593:Hexokinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010710:Phosphates / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection