Catalytic properties of Escherichia coli F₁-ATPase depleted of endogenous nucleotides

Nucleotide-depleted Escherichia coli F₁ was prepared by the procedure of Wise et al. (1983, Biochem. J. 215, 343-350). This enzyme had high rates of steady-state ATPase and GTPase activity. When "unisite" ATP hydrolysis was measured using an F₁ ATP concentration ratio of 10, all of the substoichiometric ATP became bound to the high-affinity catalytic site and none became bound to noncatalytic sites. The association rate constant for ATP binding was 7 × 10⁵ m^-1 s^-1 and the K_dATP was 7.9 × 10^-10 m, as compared to values of 3.8 × 10⁵ m^-1 s^-1 and 1.9 × 10^-10 m, respectively, in native (i.e., nucleotide-replete) F₁. Rate constants for bound ATP hydrolysis, ATP resynthesis, and P_i release, and the reaction equilibrium constant, were similar in nucleotide-depleted and native F₁. Therefore, we conclude that occupancy of the noncatalytic sites is not required for formation of the high-affinity catalytic site of F₁ and has no significant effect on unisite catalysis. In further experiments we looked for the occurrence of inhibitory, catalytic-site-bound MgADP in E. coli F₁. Such an entity has been reported for chloroplast and mitochondrial F₁. However, our experiments gave no indication for inhibitory MgADP in E. coli f₁.