ASYMMETRY OF THE 3 CATALYTIC SITES ON BETA-SUBUNITS OF TF1 FROM A THERMOPHILIC BACILLUS STRAIN PS3

F-1-ATPase isolated from plasma membrane of a thermophilic Bacillus st rain PS3 (TF1) has very little or no endogenously bound adenine nucleo tides. However, it can bind one ADP per mol of the enzyme on one of th ree beta subunits to form a stable TF1 ADP complex when incubated with a high concentration of ADP [Yoshida, M. and Allison, W.S. (1986) J. Biol. Chem. 261, 5714-5721]. The same TF1.ADP complex was recovered af ter filling all ADP binding sites with [H-3]ADP and repeated gel filtr ation. Direct binding assay revealed that the TF1 ADP complex had lost the highest affinity site for TNP-ADP. When a substoichiometric amoun t of TNP-ATP was added, the complex hydrolyzed TNP-ATP slowly (single site hydrolysis), like native TF1. However, this hydrolysis was not pr omoted by chase-addition of excess ATP. The optimal pH of the ATPase a ctivity of TF1 or the TF1 ADP complex measured with a short reaction p eriod, 6.5, was lower than the reported value, 9.0, under the steady-s tate condition. Although the bound ADP was released from the complex o nly when the enzyme underwent multiple catalytic turnover, the rate of this release was much slower than the turnover. These results suggest that when one ADP binds to a site on one of the beta subunits and sta ys there for a long time, the enzyme will change form and the bound AD P will become a special species which is not able to be directly invol ved in the enzyme catalysis. This binding site for ADP appears to be t he first site responsible for the single-site catalysis reaction obser ved for native TF1.