Crystal structures of mutant monomeric hexokinase I reveal multiple ADP binding sites and conformational changes relevant to allosteric regulation

Hexokinase I, the pacemaker of glycolysis in brain tissue, is composed of t wo structurally similar halves connected by an alpha-helix. The enzyme dime rizes at elevated protein concentrations in solution and in crystal structu res; however, almost all published data reflect the properties of a hexokin ase I monomer in solution. Crystal structures of mutant forms of recombinan t human hexokinase I, presented here, reveal the enzyme monomer for the fir st time. The mutant hexokinases bind both glucose 6-phosphate and glucose w ith high affinity to their N and C-terminal halves, and ADP, also with high affinity, to a site near the N terminus of the polypeptide chain. Exposure of the monomer crystals to ADP in the complete absence of glucose 6-phosph ate reveals a second binding site for adenine nucleotides at the putative a ctive site (C-half), with conformational changes extending 15 Angstrom to t he contact interface between the N and C-halves. The structures reveal dist inct conformational states for the C-half and a rigid-body rotation of the N-half, as possible elements of a structure-based mechanism for allosteric regulation of catalysis. (C) 2000 Academic Press.