Specific inhibition of cyclic AMP-dependent protein kinase, adenylate cyclase and phosphodiesterase by ATP and cyclic AMP analogs

Abstract

The catalytic subunit of cyclic AMP-dependent protein kinase was isolated from pigeon breast muscle. The enzyme was 11,000-fold purified and, according to the data of SDS polyacrylamide gel electrophoresis, the enzyme preparation obtained was homogeneous. The molecular weight of the enzyme was estimated to be equal to 42,000 daltons. The pH optimum for the phosphotransferase reaction catalyzed by the catalytic subunit was 8.0. The kinetic mechanism of the reaction catalyzed by the cyclic AMP-dependent pigeon breast muscle protein kinase was studied. The results obtained permitted us to draw the conclusion that the reaction proceeded by the random Bi-Bi mechanism. Using the ATP analogs containing various reactive groups, a study was carried out on the ATP-binding site of the catalytic subunit. Among the ATP analogs tested, the compounds which irreversibly modified the active site of the enzyme were found. On the basis of the data on the kinetics of inhibition of the catalytic subunit by these analogs and pH dependence of the inactivation reaction, an assumption was made about possible involvement of the histidine residue in the catalytic action of the enzyme. A preparation of the solubilized rabbit heart adenylate cyclase possessing distinct catalytic activity was obtained. The effect of various ATP analogs on the solubilized adenylate cyclase was studied. Despite the presence of alkylating groups in different positions of the analogs tested, no covalent blocking of the active site of the enzyme was observed. All the compounds appeared to be competitive inhibitors. The affinity of the analogs with haloidalkyl groups in the phosphate chain increased with the increase in the number of phosphorus atoms. The 8-substituted ATP analogs that were likely to have syn-conformation possessed low affinity for the enzyme. The absence of a nucleophylic group in the active site of adenylate cyclase in the immediate proximity to the binding region of the γ-phosphorus atom of the ATP molecule was postulated. A partially purified phosphodiesterase preparation from rat liver, sufficiently specific towards cyclic AMP and characterized by two Michaelis constants for the cyclic nucleotide, namely 360 and 2.5–5 μ m, was obtained. The interaction of the phosphodiesterase with the synthesized cyclic AMP analogs containing reactive groups of various types in certain positions of the nucleotide molecule was studied. All compounds tested were found to be phosphodiesterase inhibitors acting on the active site of the enzyme; a number of these analogs provided irreversible inhibition. On the basis of the inhibitory analysis data, a tentative topography of the ATP-binding sites of the active centers of adenylate cyclase and protein kinase was proposed. Based on the results obtained in the study on the interaction of phosphodiesterase with cyclic AMP analogs, structural and functional characteristics of cyclic AMP-binding site of phosphodiesterase were determined.