Binding of non-catalytic ATP to human hexokinase I highlights the structural components for enzyme-membrane association control

Background: Hexokinase I sets the pace of glycolysis in the brain, catalyzi ng the ATP-dependent phosphorylation of glucose. The catalytic properties o f hexokinase I are dependent on product inhibition as well as on the action of phosphate. In vivo, a large fraction of hexokinase I is bound to the mi tochondrial outer membrane, where the enzyme adopts a tetrameric assembly. The mitochondrion-bound hexokinase I is believed to optimize the ATP/ADP ex change between glucose phosphorylation and the mitochondrial oxidative phos phorylation reactions. Results: The crystal structure of human hexokinase I has been determined at 2.25 Angstrom resolution. The overall structure of the enzyme is in keepin g with the closed conformation previously observed in yeast hexokinase. One molecule of the ATP analogue AMP-PNP is bound to each N-terminal domain of the dimeric enzyme in a surface cleft, showing specific interactions with the nucleotide, and localized positive electrostatic potential. The molecul ar symmetry brings the two bound AMP-PNP molecules, at the centre of two ex tended surface regions, to a common side of the dimeric hexokinase I molecu le. Conclusions: The binding of AMP-PNP to a protein sire separated from the ca talytic centre of human hexokinase I can be related to the! role played by some nucleotides in dissociating the enzyme from the mitochondrial membrane , and helps in defining the molecular regions of hexokinase I that are expe cted to be in contact with the mitochondrion. The structural information pr esented here is in keeping with monoclonal antibody mapping of the free and mitochondrion-bound forms of the enzyme, and with sequence analysis of hex okinases that differ in their mitochondria binding properties.