Rat muscle 5'-adenylic acid aminohydrolase. Role of K+ and adenylate energy charge in expression of kinetic and regulatory properties.

Abstract

The kinetic and regulatory properties of homogeneous AMP deaminase from rat skeletal muscle have ben examined with particular emphasis on (a) the role of potassium ions in the expression of these properties and (b) the role of the adenylate energy charge in the regulation of AMP deaminase activity. Although the enzyme has an absolute requirement for K+, the concentration required for maximum activation is dependent on the concentration of substrate. At saturating levels of AMP (greater than or equal 2.0 mM) maximum activation is observed with 25 mM KCl, whereas at lower substrate concentrations (0.2 mM) approximately 50 mM KCl is needed for maximum activation. Conversely, the response of enzyme activity ot increasing levels of substrate is dependent on the level of potassium ions. At substrating concentrations of K+, the saturation curve for AMP is highly sigmoidal (nh=3.2) whereas at higher KCl concentrations, the apparent cooperativity between AMP sites is almost completely abolished (nh=1.5). The inhibition by a number of phosphorylated metabolites, including ATP, GTP, creatine-P, and P1, is likewise sensitive to the concentration of K+. These results suggest that a significant amount of interaction between K+ sites and both substrate and effector sites is required for the expression of the catalytic and regulatory properties of the enzyme. The specific effects of ATP, creatine-P, and P1 on the parameters of Km and Vmax indicate that each of these profile of AMP deaminase activity generated in response to variations in the adenylate energy charge shows that within the physiological range of energy charge (0.75 to 0.95), the activity increases linearly with decreasesing energy charge and is insensitive to both the total adenylate pool size and the presence of P1 and creatine-P. These data suggest that the most important factor in the regulation of AMP deaminase activity is the state of the energy charge rather than the absolute concentrations of the individual effectors.