Studies of energy transport in heart cells. The importance of creatine kinase localization for the coupling of mitochondrial phosphorylcreatine production to oxidative phosphorylation.

Abstract

To define further the importance of enzyme compartmentation for phosphorylcreatine production coupled to oxidative phosphorylation, we studied the kinetics of phosphorylcreatine synthesis by the heart mitochondrial isoenzyme of creatine kinase.The kinetic constants for the formation of the binary and ternary enzyme.substrate complexes during the forward reaction were similar for bound and soluble enzyme.These constants were measured under nonrespiring conditions with pyruvate kinase employed as the exogenous ATP regenerating system.The values at pH 7.4 and 30°C are as follows: bound = 0.73 m; K,A,, 0.73 mnt; Kicn 5.0 m; Kmcn 5.0 m; and soluble = 0.60 mnt; K,ATP, 0.35 mnt; Kit, 8.5 mnt; Kmcr, 5.0 111~.Four other assay systems were used to examine the kinetics of phosphorylcreatine production: 1) native-bound enzyme with ATP supplied by oxidative phosphorylation; 2) bound enzyme with ATP from the pyruvate kinase reaction; 3) soluble mitochondrial enzyme plus liver mitochondria with ATP from oxidative phosphorylation; and 4) soluble mitochondrial enzyme with pyruvate kinase-generated ATP.Under these four conditions, with constant 1 1 1 1 ~ ATP, the apparent K , for creatine was not influenced by either enzyme solubilization or the source of ATP.However, when creatine was held constant at 25 mM, a 5-to 7-fold decrease was noted for the apparent K , value for ATP when it was supplied by oxidative phosphorylation to the bound enzyme.The values for the apparent K , constants under these conditions were: 1) K, = 37 CM; 2) K,,, = 200 p i ; 3) K,,, = 145 CM; and 4) K,,, = 200 PM, respectively.Phosphorylcreatine product inhibition studies also demonstrated major differences for the apparent K , for ATP which were related to enzyme location.During oxidative phosphorylation, competitive inhibition between phosphorylcreatine and ATP was observed for both bound and soluble mitochondrial creatine kinase