3′ Esters of ADP as Energy‐Transfer Inhibitors and Probes of the Catalytic Site of Oxidative Phosphorylation

Abstract

A large series of 3'' esters of ADP was synthesized. Several of these can serve as photoaffinity labels; other exhibit fluorescent properties. The corresponding AMP and ATP derivatives were also synthesized in some cases. The influence of the 3''-O-acyl nucleotides on energy-linked functions of beef heart submitochondrial particles was investigated. 3'' Esters of ADP are powerful and highly specific inhibitors of oxidative phosphorylation. The inhibition is competive to ADP and Ki values as low as 0.05 .mu.M (for the 3''-O-(1)naphthoyl ester of ADP) could be observed. The inhibition of oxidative phosphorylation by 3'' esters of ADP appears to be non-competitive vs. Pi. The nucleotide analogs are not phosphorylated themselves. The corresponding ATP analogs cannot drive energy-linked processes. The 3'' esters of AMP are ineffective as inhibitors; the ATP derivatives are only comparatively weak inhibitors. Uncoupled or solubilized ATPase is almost 2 orders of magnitude less sensitive to inhibition by 3'' esters than coupled systems. The analogs exert maximal inhibition specifically in systems involving an energized state of the coupling device. Azido-group-bearing analogs can be used for irreversible photoinactivation of the coupling ATPase. Photoinactivation also is most efficient when carried out with energized particles. The inhibitory properties are similar in ATP-driven NAD+ reduction by succinate, and in the uncoupler-sensitive ATP .dblarw. Pi exchange. The required concentrations for half-maximal inhibition are somewhat higher than in oxidative phosphorylation, but lower than with uncoupled ATPase. Molecular models, substituent properties and the conditions required for inhibition suggest that these highly effective analgos of ADP may act as conformation-specific probes at the catalytic site of oxidative phosphorylation. The results are interpreted in terms of a model suggesting that, in the process of ATP synthesis, a hydrophobic cavity on the enzyme is exposed only in the energized state, accepting the large 3'' substituent. The substituent apparently inhibits phosphoryl transfer and/or conformational transitions inherent in the process of ADP phosphorylation by steric hinderance.