Specificity of the Proton Adenosinetriphosphatase of Escherichia coli for Adenine, Guanine, and Inosine Nucleotides in Catalysis and Binding

Specificity of the Escherichia coli proton ATPase for adenine, guanine, and inosine nucleotides in catalysis and binding was studied. MgADP, CaADP, MgGDP, and MgIDP were each good substrates for oxidative phosphorylation. The corresponding triphosphates were each substrates for hydrolysis and proton pumping. At 1 mM concentration, MgATP, MgGTP, and MgITP drove proton pumping with equal efficiency. At 0.1 mM concentration, MgATP was 4-fold more efficient than MgITP or MgGTP. Nucleotide-depleted soluble F₁ could rebind to F₁-depleted membranes and block proton conductivity through F₀; rebound nucleotide-depleted F₁ catalyzed pH gradient formation with MgATP, MgGTP, or MgITP. This showed that the nonexchangeable nucleotide sites on F₁ need not be occupied by adenine nucleotide for proton pumping to occur. It was further shown that no nu cleotide was tightly bound in the nonexchangeable sites of F₁ during proton pumping driven by MgGTP in these reconstituted membranes, whereas adenine nucleotide was tightly bound when MgATP was the substrate. Nucleotide-depleted soluble F₁ bound maximally 5.9 ATP, 3.2 GTP, and 3.6 ITP of which half the ATP and almost all of the GTP and ITP exchanged over a period of 30-240 min with medium ADP or ATP. Also, half of the bound ATP exchanged with medium GTP or ITP. These data showed that inosine and guanine nucleotides do not bind to soluble F₁ in nonexchangeable fashion, in contrast to adenine nucleotides. Purified α-subunit from F₁ bound ATP at a single site but showed no binding of GTP nor ITP, supporting previous suggestions that the nonexchangeable sites in intact F₁ are on α-subunits.