Adenine Nucleotide Binding Sites on Beef Heart F1ATPase: Photoaffinity Labeling of β -subunit Tyr-368 at a Noncatalytic Site and β Tyr-345 at a Catalytic Site

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 84; no. 16; pp. 5715 - 5719
• Main Authors: Cross, Richard L., Cunningham, David, Miller, Chad G., Xue, Zhixiong, Zhou, Jun-Mei, Boyer, Paul D.
• Format: Journal Article
• Language: English
• Published: National Academy of Sciences of the United States of America 15-08-1987
• Subjects: Active sites; Adenine nucleotides; Adenosine triphosphatases; Amino acids; Binding sites; Catalysis; Centrifugation; Enzymes; Nucleotides; Product labeling
• ISSN: 0027-8424; 1091-6490

2-Azidoadenine [32P]nucleotide was bound specifically at catalytic or noncatalytic nucleotide binding sites on beef heart mitochondrial F1 ATPase. In both cases, photolysis resulted in nearly exclusive labeling of the β subunit. The modified enzyme was digested with trypsin, and labeled peptides were purified by reversed-phase high-pressure liquid chromatography. Amino acid sequence analysis of the major 32P-labeled tryptic fragments showed β -subunit Tyr-368 to be present at noncatalytic sites and β Tyr-345 to be present at catalytic sites. From the relationship between the degree of inhibition and extent of modification, it is estimated that one-third of the catalytic sites or two-thirds of the noncatalytic sites must be modified to give near-complete inhibition of catalytic activity.