Single-site catalysis of F1-ATPase from thermophilic bacterium PS3 and its dominance in steady-state catalysis at low ATP concentration.
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 degrees 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 degrees C, whether in the presence or absence of phosphate ion, a small amount of bound [alpha, gamma-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 microM.
D000227:Adenine Nucleotides / Q000032:analysis; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001419:Bacteria / Q000201:enzymology*; D001665:Binding Sites; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006358:Hot Temperature; D010710:Phosphates / Q000032:analysis; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D000227:Adenine Nucleotides; D010710:Phosphates; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases