COMPARTMENTATION AND METABOLIC PARAMETERS OF MITOCHONDRIAL HEXOKINASE AND CREATINE-KINASE DEPEND ON THE RATE OF OXIDATIVE-PHOSPHORYLATION

Oxidative phosphorylation in rat heart mitochondria was stimulated by the presence of hexokinase, by simultaneous operation of mitochondrial hexokinase and creatine kinase, or by mitochondrial hexokinase plus exogenously added phosphofructokinase. Under these conditions, P-32(i) studies were conducted to estimate the extent of ATP compartmentation in the mitochondria in the vicinity of the active sites of hexokinase and creatine kinase. In all cases studied the extent of ATP compartmentation at 500 mu M ATP concentration was no more than 12%. Within the same experimental design, the extent of ATP compartmentation increased with an increase in the rate of oxidative phosphorylation, The degree of ATP compartmentation depended on the relative location of the enzyme and inner mitochondrial membrane: it was maximal in the vicinity of the creatine kinase active sites and minimal for that of phosphofructokinase, The difference in the extent of ATP compartmentation in the neighborhood of the active sites of hexokinase and creatine kinase diminished with an increase in the rate of oxidative phosphorylation, We conclude that there is an ATP concentration gradient in the mitochondrial intermembrane space during oxidative phosphorylation, the minimum concentration being at the surface of the inner membrane, It was found that stimulation of oxidative phosphorylation led to a decrease in the apparent constants, K-m(MgATP) and V-max for the two enzymes, however, to different degrees. Possible reasons for the change in kinetic parameters of the above enzyme's are discussed, (C) 1995 Academic Press, Inc.