PRE-STEADY-STATE AND STEADY-STATE KINETICS OF BEEF HEART MITOCHONDRIAL ATPASE: INFORMATION THEORY RELATED TO ENZYME REGULATION

The pre-steady-state kinetics of beef heart mitochondrial ATPase (F(,1)) were examined. F(,1)was found to exhibit hysteretic behavior when hydrolyzing ATP. The hysteretic property was expressed as an activation process which occurred when the enzyme was mixed with its substrate. Many catalytic turnovers were required before the activation was complete. The effects of various inhibitors on the lag were monitored. In addition, the F(,1) was allowed to reach steady-state velocity, the reaction terminated and then monitored after re-initiation with the stopped-flow technique. Further studies were conducted where the effects of inhibitors were monitored on catalysis with sub-stoichiometric substrate concentrations. An information theoretic model is proposed for describing the regulation of F(,1). The model incorporates the concept of a communication system in which catalytic activity is controlled. This model makes testable predictions about the behavior of allosteric enzymes under various definable conditions. The model is applied in detail to other allosteric enzymes. A transient release of protons was observed with nucleotide depleted F(,1). It is shown that proton release is due to Mg(\u272+), AMP-PNP, or ADP binding. Maximum proton release per free F(,1) is observed with metal-nucleotide complex. Modification of nucleotide free F(,1) with NBD-Cl eliminated the proton burst which suggests that the modified tyrosine is directly involved in the release of protons. The effect of azide on F(,1) activity was studied. Azide was shown to elicit a slow hysteretic effect on ATP hydrolysis by F(,1). The hysteretic effect was not due to turnover of the enzyme but to the binding of azide and magnesium. The results indicate that azide and magnesium inhibition is synergistic. It is also shown that azide and HCO(,3)(\u27-) bind at separate sites on the F(,1) complex. The results presented indicate that metal binding is important in the regulation of F(,1) activity and in the inhibition of this activity by azide. A model is presented which is consistent with the hysteretic inhibition of F(,1) by azide, in which there is a slow equilibration between free enzyme and the enzyme-magnesium-azide complex