Cooperatively between catalytic sites in the mechanism of action of beef heart mitochondrial adenosine triphosphatase.

摘要: Occupancy of only one two hydrolytic sites on beef heart mitochondrial ATPase (F1) by the radioactive ATP analog, 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-[gamma-32P]-triphosphate (TNP-[gamma-32P]ATP) is associated with a low rate hydrolysis substrate even under conditions otherwise favoring catalysis. Addition excess nonradioactive TNP-ATP, in concentrations sufficient to fill catalytic Site 2 enzyme (Grubmeyer, C., and Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718-3727), accelerates 15- 20-fold. Since serves as an effective isotope trap, involvement medium TNP-[gamma-32P]-ATP intermediate ruled out. These observations constitute direct evidence for cooperativity between active F1. It proposed that use high binding affinity substrates or analogs, combined trap technique, offers new approach detection study site enzymes. The hydrolyzable nucleotides GTP, ITP, are excellent promoters previously bound TNP-[gamma-32P]ATP whereas addition nonhydrolyzable such TNP-ADP, ADP, adenylyl imidodiphosphate result lower extent hydrolysis. AMP without effect. Studies [gamma-32P]ATP TNP-[gamma-32P]ITP, appropriate conditions, also provide consistent promoted Based upon these findings, model presented mechanism action F1 which site-site reflects promoter-dependent substrate.