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, catalyzing the ATP-dependent phosphorylation of glucose. The catalytic properties of 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 mitochondrial outer membrane, where the enzyme adopts a tetrameric assembly. The mitochondrion-bound hexokinase I is believed to optimize the ATP/ADP exchange between glucose phosphorylation and the mitochondrial oxidative phosphorylation reactions.


RESULTS
The crystal structure of human hexokinase I has been determined at 2.25 A resolution. The overall structure of the enzyme is in keeping 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 molecular symmetry brings the two bound AMP-PNP molecules, at the centre of two extended surface regions, to a common side of the dimeric hexokinase I molecule.


CONCLUSIONS
The binding of AMP-PNP to a protein site separated from the catalytic 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 expected to be in contact with the mitochondrion. The structural information presented here is in keeping with monoclonal antibody mapping of the free and mitochondrion-bound forms of the enzyme, and with sequence analysis of hexokinases that differ in their mitochondria binding properties.
Rosano
C
C
Dipartimento di Fisica - INFM, Centro Biotecnologie Avanzate - IST, Universita' di Genova, Genova, 10. I-16132, Italy.
Sabini
E
E
Rizzi
M
M
Deriu
D
D
Murshudov
G
G
Bianchi
M
M
Serafini
G
G
Magnani
M
M
Bolognesi
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D018360:Crystallography, X-Ray; D005947:Glucose / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000378:metabolism; D006593:Hexokinase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D019298:Glucose-6-Phosphate; D000255:Adenosine Triphosphate; D006593:Hexokinase; D005947:Glucose