Covalent modification of catalytic sites on membrane-bound beef heart mitochondrial ATPase by 2-azido-adenine nucleotides.
Incubation in the dark of 32P-labeled 2-azido-adenine nucleotides with submitochondrial particles from beef heart led to tight binding of the label by membrane-bound F1. That is, the label remained with the particles following two passages through centrifuge columns. After removal of free nucleotides and ultraviolet irradiation, the radioactive label was covalently bound exclusively to the beta subunit of the ATPase. Extraction of the modified enzyme from the membrane with chloroform followed by tryptic digestion and separation of peptides by reverse-phase high-pressure liquid chromatography indicated that the radioactive label had been inserted into a peptide fragment that included part of the catalytic site. Covalent modification of catalytic sites by 2-azido-ADP was accompanied by parallel inhibition of both ATP synthesis and ATP hydrolysis by submitochondrial particles. Estimation of the likely amount of F1 participating in the reaction and extrapolation to complete inhibition suggested that modification of no more than a single site was sufficient to block both reactions. The results support suggestions of cooperative interactions between catalytic sites as well as a single catalytic pathway for both enzymic reactions.
Martins
I S
IS
Department of Biochemistry and Molecular Biology, State University of New York, Health Science Center at Syracuse 13210.
Penefsky
H S
HS
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000737:chemistry*; D002384:Catalysis; D002417:Cattle; D006868:Hydrolysis; D008929:Mitochondria, Heart / Q000201:enzymology*; D010777:Photochemistry; D013367:Submitochondrial Particles / Q000378:metabolism
D001386:Azides; D000244:Adenosine Diphosphate; C022295:2-azidoadenosine 3',5'-diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases