Glucose 6-phosphate release of wild-type and mutant human brain hexokinases from mitochondria.
One molecule of glucose 6-phosphate inhibits brain hexokinase (HKI) with high affinity by binding to either one of two sites located in distinct halves of the enzyme. In addition to potent inhibition, glucose 6-phosphate releases HKI from the outer leaflet of mitochondria; however, the site of glucose 6-phosphate association responsible for the release of HKI is unclear. The incorporation of a C-terminal polyhistidine tag on HKI facilitates the rapid purification of recombinant enzyme from Escherichia coli. The tagged construct has N-formyl methionine as its first residue and has mitochondrial association properties comparable with native brain hexokinases. Release of wild-type and mutant hexokinases from mitochondria by glucose 6-phosphate follow equilibrium models, which explain the release phenomenon as the repartitioning of ligand-bound HKI between solution and the membrane. Mutations that block the binding of glucose 6-phosphate to the C-terminal half of HKI have little or no effect on the glucose 6-phosphate release. In contrast, mutations that block glucose 6-phosphate binding to the N-terminal half require approximately 7-fold higher concentrations of glucose 6-phosphate for the release of HKI. Results here implicate a primary role for the glucose 6-phosphate binding site at the N-terminal half of HKI in the release mechanism.
Skaff
D Andrew
DA
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
Kim
Chang Sup
CS
Tsai
Henry J
HJ
Honzatko
Richard B
RB
Fromm
Herbert J
HJ
D000255:Adenosine Triphosphate / Q000737:chemistry; D000409:Alanine / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology* / Q000378:metabolism; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000378:metabolism*; D006593:Hexokinase / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology*; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry; D008928:Mitochondria / Q000378:metabolism*; D008956:Models, Chemical; D009154:Mutation; D009239:N-Formylmethionine / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013552:Swine; D013912:Threonine / Q000737:chemistry
D008024:Ligands; C033223:polyhistidine; D013912:Threonine; D009239:N-Formylmethionine; D006639:Histidine; D019298:Glucose-6-Phosphate; D000255:Adenosine Triphosphate; D006593:Hexokinase; D008274:Magnesium; D000409:Alanine