Single-Site Catalysis of F1-ATPase from Thennophilic Bacterium PS3 and Its Dominance in Steady-State Catalysis at Low ATP Concentration1

Abstract

Single-site catalysis by F₁-ATPase from a thermophilic bacterium PS3 (TF₁) was examined by incubating the enzyme with a submolar amount of radioactive ATP. The profile of single-site catalysis by TF₁ at 23°C was different from that of beef heart mitochondrial F₁-ATPase (MF₁). ATP hydrolysis on the enzyme and release of the products was rapid, and subsequent addition of non-radioactive ATP (cold chase) did not promote the hydrolysis of radioactive ATP, indicating that the rate-limiting step was not the step of product release but the step of ATP binding to the enzyme. Thus, the characteristic features of so-called uni-site catalysis were not observed. At 60°C, whether in the presence or absence of phosphate ion, a small amount of bound [α,γ-³²P]ATP and cold chase promotion were observed. However, since bound ³²P₁ was not detected by centrifugal gel filtration, it is not yet certain whether TF₁ has typical uni-site characteristics. Based on the hydrolytic turnover rate for single-site catalysis and analysis of the kinetics of steady-state catalysis, it is proposed that single-site catalysis is dominant even in steady-state catalysis at ATP concentrations of less than about 20 μM.