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Specificity of the proton atpase of escherichia coli for adenine guanine and inosine nucleotides in catalysis and binding


, : Specificity of the proton atpase of escherichia coli for adenine guanine and inosine nucleotides in catalysis and binding. Biochemistry 23(21): 4998-5003

Specificity of the E. 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. No nucleotide was tightly bound in the nonexchangeable sites of F1 during proton pumping driven by MgGTP in these reconstituted membranes; 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. Thus, inosine and guanine nucleotides do not bind to soluble F1 in nonexchangeable fashion, in contrast to adenine nucleotides. Purified .alpha.-subunit from F1 bound ATP at a single site but showed no binding of GTP or ITP, supporting previous suggestions that the nonexchangeable sites in intact F1 are on .alpha.-subunits.

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