FURTHER-STUDIES ON THE COUPLING OF MITOCHONDRIALLY BOUND HEXOKINASE TO INTRAMITOCHONDRIALLY COMPARTMENTED ATP, GENERATED BY OXIDATIVE-PHOSPHORYLATION

Hexokinase, bound to nonphosphorylating rat brain mitochondria, exhibi ts Michaelis-Menten kinetic behavior, with an apparent K-m for ATP of 0.44 +/- 0.08 mM, After initiation of oxidative phosphorylation, a ste ady-state rate of Glc phosphorylation is maintained despite the fact t hat extramitochondrial [ATP] continues to increase but remains well be low saturating levels (i.e., <0.4 mM). This independence from extramit ochondrial [ATP] is taken to indicate that hexokinase is not utilizing extramitochondrial ATP as substrate, but rather draws substrate ATP f rom an intramitochondrial compartment supplied by oxidative phosphoryl ation. The steady-state rate of Glc phosphorylation by hexokinase boun d to phosphorylating mitochondria is not altered 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 mitochondr ia, is directly related to the total rate of ATP production in the sys tem. These results are also consistent with the view that, during oxid ative phosphorylation, mitochondrially bound hexokinase is selectively using intramitochondrially compartmented ATP; such substrate selectiv ity would be expected to require physical association of hexokinase wi th the mitochondria and be dependent solely on the oxidative phosphory lation activity of the hexokinase-bearing organelles. The K-m for Glc is only modestly affected by the binding of hexokinase to mitochondria and not further altered upon induction of active oxidative phosphoryl ation, suggesting that neither binding nor oxidative phosphorylation g reatly affects the conformation of the Glc binding site. The reliance on intramitochondrial ATP is suggested to result from oxidative phosph orylation-dependent changes in the interaction between the mitochondri al surface and the regions of the hexokinase molecule involved in bind ing ATP. (C) 1998 Academic Press.