Studies of Energy Transport in Heart Cells. Mitochondrial Isoenzyme of Creatine Phosphokinase: Kinetic Properties and Regulatory Action of Mg2+ Ions

Abstract

1 The kinetic properties of mitochondrial creatine phosphokinase (K_m for all substrates and maximal rates of the forward and reverse reaction) have been studied. Since (a) K_m value for MgADP⁻ (0.05 mM) and creatine phosphate (0.5 mM) are significantly lower than K_m for MgATP^2- (0.7 mM) and creatine (5.0 mM) and (b) maximal rate of the reverse reaction (creatine phosphate + ADP → ATP + creatine) equal to 3.5 μmol × min⁻¹× mg⁻¹ is essentially higher than maximal rate of the forward reaction (0.8 μmol × min⁻¹× mg⁻¹), ATP synthesis from ADP and creatine phosphate is kinetically preferable over the forward reaction. 2 A possible regulatory role of Mg²⁺ ions in the creatine phosphokinase reaction has been tested. It has been shown that in the presence of all substrates and products of the reaction the ratio of the rates of forward and reverse reactions can be effectively regulated by the concentration of Mg²⁺ ions. At limited Mg²⁺ concentrations creatine phosphate is preferably synthesized while at high Mg²⁺ concentrations (more ATP in the reaction medium) ATP synthesis takes place. 3 The kinetic (mathematical) model of the mitochondrial creatine phosphokinase reaction has been developed. This model accounts for the existence of a variety of molecular forms of adenine nucleotides in solution and the formation of their complexes with magnesium. It is based on the assumption that the mitochondrial creatine phosphokinase reaction mechanism is analogous to that for soluble isoenzymes. 4 The dependence of the overall rate of the creatine phosphokinase reaction on the concentration of total Mg²⁺ ions calculated from the kinetic model quantitatively correlates with the experimentally determined dependence through a wide range of substrates (ATP, ADP, creatine and creatine phosphate) concentration. The analysis of the kinetic model demonstrates that the observed regulatory effect of Mg²⁺ on the overall reaction rate can be explained by (a) the sigmoidal variation in the concentration of the MgADP⁻ complex resulting from the competition between ATP and ADP for Mg²⁺ and (b) the high affinity of the enzyme to MgADP⁻. 5 The results predicted by the model for the behaviour of mitochondrial creatine phosphokinase under conditions of oxidative phosphorylation point to an intimate functional interaction of mitochondrial creatine phosphokinase and ATP-ADP translocase.