Abstract
Binding of pyrophosphate (PPi) to the three catalytic ("C") and three noncatalytic ("NC") nucleotide sites of Escherichia coli F1-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 Kd = 20 microM. 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 Kd 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. Pi (5 mM) did not bind to either C or NC sites.
Highlights
General Comments-The use of mutant E. coli F 1-ATPase enzymes containing tryptophans substituted in catalytic sites (I3Y331W) or noncatalytic sites has enabled us to determine at which of these sites PPi or Pi compete with nucleoside triphosphate for binding, and to determine apparent K d values
It is seen that MgPPi binds at noncatalytic sites, and the apparent Kd(MgPP i) of 20 JLM is surprisingly similar to that for MgAMP-PNP, MgATP, and MgADP, as measured by the tryptophan fluorescence signal
Occupancy of the noncatalytic sites by MgPPi had no effect on ATP hydrolysis
Summary
Vol 270, No 21, Issue of May 26, pp. 12653-12658, 1995 Printed in U.S.A. Location and Properties of Pyrophosphate-binding Sites in Escherichia coli Fl-ATPase*. F 1 obtained from the aR365W mutant was found to retain catalytic activity similar to wild-type, and in this enzyme the newly inserted tryptophan provided a direct and specific fluorescent probe of nucleotide binding at noncatalytic sites (Weber et al., 1994b). Using this signal we established the Mg dependence, nucleotide specificity, and nucleotide-binding parameters at noncatalytic sites and demonstrated that occupancy of noncatalytic sites is not required for ATP hydrolysis. The availability of mutant E. coli enzymes with reporter tryptophans substituted at the catalytic or the noncatalytic sites provides a new technique which can be used to study PPi binding. We offer an explanation for the effect of PPi to promote net loss of nucleotide from catalytic sites, based on its ability to complex Mg2+ ions rather than by direct competition for occupancy of the sites
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