Differential Effects of Adenylyl Imidodiphosphate on Adenosine Triphosphate Synthesis and the Partial Reactions of Oxidative Phosphorylation

Abstract

Abstract A study of the interaction of the ATP analog, adenylyl imidodiphosphate (AMP-PNP), with mitochondrial ATPase revealed that the analog was a strong competitive inhibitor of both the soluble, purified enzyme, Ki = 0.33 µm, and of the membrane-bound enzyme, Ki = 0.16 µm. AMP-PNP also was a strong inhibitor of the partial reactions of oxidative phosphorylation catalyzed by phosphorylating submitochondrial particles. Thus, the ATP-dependent reduction of NAD by succinate was inhibited competitively with Ki = 13 µm and the ATP-dependent enhancement of fluorescence of 1,8-anilinonaphthalene sulfonate and the exchange between 32Pi and ATP also were inhibited. However, AMP-PNP did not affect the forward reactions of oxidative phosphorylation, that is, the synthesis of ATP from ADP and Pi linked to the oxidation of succinate. It is proposed that the failure of AMP-PNP to inhibit oxidative phosphorylation may be traceable to a high affinity for ADP of the phosphorylating sites on F1 or may reflect two catalytic sites on the enzyme which are specialized, respectively, for ATP synthesis and ATP utilization.