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.[1]References
- Glucose 6-phosphate release of wild-type and mutant human brain hexokinases from mitochondria. Skaff, D.A., Kim, C.S., Tsai, H.J., Honzatko, R.B., Fromm, H.J. J. Biol. Chem. (2005) [Pubmed]
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