Single Site Catalysis of the F1-ATPase from Saccharomyces cerevisiae and the Effect of Inorganic Phosphate on It

The kinetical characteristics of ATP hydrolysis by mitochondrial F1-ATPase from Saccharomyces cerevisiae (yeast) have been studied under conditions where only a single catalytic site per enzyme molecule bound ATP. Four major features were observed, that is, fast ATP binding to the enzyme, slow product release from the enzyme, an equilibrium close to unity between ATP and products on the enzyme, and promotion of ATP hydrolysis on the second addition of a large excess of ATP (cold chase). These are essentially the same as the kinetical characteristics observed for beef heart mitochondrial F1-ATPase, which were called as unisite catalysis by Grubmeyer et al. (Grubmeyer, C. et al. (1982) J. Biol. Chem. 257, 12092–12100), although the release of a hydrolysis product, P1, from the yeast enzyme appeared to occur significantly faster than that from the beef enzyme, which resulted in a decreased extent of cold chase promotion of ATP hydrolysis of the yeast enzyme. The yeast F1-ATPase showed unisite catalysis even in the absence of P1 in the reaction mixtures, while it was reported for the beef F1-ATPase that the presence of P1 in the reaction mixture was essential for unisite catalysis (Penefsky, H.S. & Grubmeyer, C. (1984) in H+-ATPase (ATP Synthase) (Papa, S. et al., eds.) pp. 195–204, The ICSU Press). Another difference in the P1 effect on the kinetics was that ATP hydrolysis was initiated without a lag time in the absence of P1 in the case of the yeast enzyme when a 1, 000-fold molar excess of ATP per enzyme molecular was mixed with the enzyme. Thus the “P1-activation” observed for the beef enzyme may not be common to all F1-ATPases.