| Adenosine 5'--triphosphate (ATP) can release hexokinase I (HKI) from its binding site on the outer mitochondrial membrane, but the mechanism of ATP release of HKI from mitochondria is unclear. ATP binds to the C--terminal half of HKI as a substrate and to the N--terminal half near the membrane binding element. ATP may also bind to the voltage dependent anion channel (VDAC), the integral membrane component that putatively targets HKI to the mitochondrion. The fluorescent nucleotide analogue 2',3'--O--(2,4,6--trinitrophenyl) adenosine 5'--diphosphate (TNP--ADP) binds with high affinity to the C--terminal half of HKI (Kd = 0.79 +/- 0.09 muM) and to VDAC (Kd = 1.3 +/- 0.1 muM), but not to the N--terminal half of HKI (Kd > 50 muM). ATP competes with TNP--ADP for binding sites on HKI (KA = 190 +/- 20 muM) and VDAC (K A = 550 +/- 40 muM), but CTP does not displace TNP--ADP from either HKI or VDAC. Other trinitophenyl nucleotides (TNP--ATP, TNP--AMP, and TNP--CTP) bind with high affinity to HKI and VDAC. ATP and trinitrophenyl nucleotides individually release wild--type HKI from mitochondria; however, CTP is ineffective as an agent of release. ATP and ADP--TNP release the truncated N--domain of HKI from mitochondria, excluding nucleotide binding to either the N-- or C--half of HKI in the release mechanism. Results here are consistent with a release mechanism caused by the binding of ATP or TNP--ADP to VDAC. |
