LOCATION AND PROPERTIES OF PYROPHOSPHATE-BINDING SITES IN ESCHERICHIA-COLI F1-ATPASE

Binding of pyrophosphate (PPi) to the three catalytic (''C'') and three noncatalytic (''NC'') nucleotide sites of Escherichia coli F-1-ATPase was determined by fluorescence spectroscopy using mutant enzymes with tryptophan inserted specifically in either C sites (beta Y331W) or NC sites (alpha R365W). Fluorescence of the tryptophan is quenched on binding of nucleotide; PPi binding parameters were determined by competition with ATP or adenyl-5'-yl imidodiphosphate. It was found that MgPPi binds to each NC site with K-d = 20 mu M. In contrast, even at millimolar concentration, neither MgPPi nor free PPi showed significant binding to C sites. We confirmed that free PPi displaces nucleotide from C sites, but this was shown to be due to complexation of Mg2+ ions rather than to occupancy of the sites. MgPPi bound at NC sites was found not to affect ATP hydrolysis rates. From the data we propose a two-phase model for nucleotide binding at NC sites. In phase one, NC sites recognize the pyrophosphate ''end'' of the nucleotide, which binds initially with K-d similar to MgPPi; in phase two, a slow conformational change occurs which tightly sequesters adenine nucleotide. Phase two does not occur with guanine nucleotide. This model explains the preference of NC sites for adenine nucleotides. P-i (5 mM) did not bind to either C or NC sites.