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

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