Further Studies on the Coupling of Mitochondrially Bound Hexokinase to Intramitochondrially Compartmented ATP, Generated by Oxidative Phosphorylation

Hexokinase, bound to nonphosphorylating rat brain mitochondria, exhibits Michaelis–Menten kinetic behavior, with an apparentKmfor ATP of 0.44 ± 0.08 mM. After initiation of oxidative phosphorylation, a steady-state rate of Glc phosphorylation is maintaineddespitethe fact that extramitochondrial [ATP] continues to increase but remains well below saturating levels (i.e., <0.4 mM). This independence from extramitochondrial [ATP] is taken to indicate that hexokinase is not utilizing extramitochondrial ATP as substrate, but rather draws substrate ATP from an intramitochondrial compartment supplied by oxidative phosphorylation. The steady-state rate of Glc phosphorylation by hexokinase bound to phosphorylating mitochondria isnotaltered by increase in total rate of ATP production resulting from addition of hexokinase-depleted mitochondria to the system. In contrast, the steady-state rate of Glc phosphorylation by yeast hexokinase, which does not bind to mitochondria, is directly related to the total rate of ATP production in the system. These results are also consistent with the view that, during oxidative phosphorylation, mitochondrially bound hexokinase is selectively using intramitochondrially compartmented ATP; such substrate selectivity would be expected to require physical association of hexokinase with the mitochondria and be dependent solely on the oxidative phosphorylation activity of the hexokinase-bearing organelles. TheKmfor Glc is only modestly affected by the binding of hexokinase to mitochondria and not further altered upon induction of active oxidative phosphorylation, suggesting that neither binding nor oxidative phosphorylation greatly affects the conformation of the Glc binding site. The reliance on intramitochondrial ATP is suggested to result from oxidative phosphorylation-dependent changes in the interaction between the mitochondrial surface and the regions of the hexokinase molecule involved in binding ATP.