COVALENT MODIFICATION OF CATALYTIC SITES ON MEMBRANE-BOUND BEEF-HEARTMITOCHONDRIAL ATPASE BY 2-AZIDO-ADENINE NUCLEOTIDES

Incubation in the dark of P-32-labeled 2-azido-adenine nucleotides wit h submitochondrial particles from beef heart led to tight binding of t he label by membrane-bound F-1. That is, the label remained with the p articles following two passages through centrifuge columns. After remo val of free nucleotides and ultraviolet irradiation, the radioactive l abel was covalently bound exclusively to the beta subunit of the ATPas e. Extraction of the modified enzyme from the membrane with chloroform followed by tryptic digestion and separation of peptides by reverse-p hase high-pressure liquid chromatography indicated that the radioactiv e label had been inserted into a peptide fragment that included part o f the catalytic site. Covalent modification of catalytic sites by 2-az ido-ADP was accompanied by parallel inhibition of both ATP synthesis a nd ATP hydrolysis by submitochondrial particles. Estimation of the lik ely amount of F-1 participating in the reaction and extrapolation to c omplete inhibition suggested that modification of no more than a singl e site was sufficient to block both reactions. The results support sug gestions of cooperative interactions between catalytic sites as well a s a single catalytic pathway for both enzymic reactions.