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To study the stator function in ATP synthase, a fluorimetric assay has been devised for quantitative determination of binding affinity of δ-subunit to Escherichia coliF1-ATPase. The signal used is that of the natural tryptophan at residue δ28, which is enhanced by 50% upon binding of δ-subunit to α3β3γε complex.Kdfor δ binding is 1.4 nm, which is energetically equivalent (50.2 kJ/mol) to that required to resist the rotor strain. Only one site for δ binding was detected. The δW28L mutation increased Kdto 4.6 nm, equivalent to a loss of 2.9 kJ/mol binding energy. While this was insufficient to cause detectable functional impairment, it did facilitate preparation of δ-depleted F1. The αG29D mutation reduced Kdto 26 nm, equivalent to a loss of 7.2 kJ/mol binding energy. This mutation did cause serious functional impairment, referable to interruption of binding of δ to F1. Results with the two mutants illuminate how finely balanced is the stator resistance function. δ′ fragment, consisting of residues δ1–134, bound with the same Kdas intact δ, showing that, at least in absence of Fosubunits, the C-terminal domain of δ contributes zero binding energy. Mg2+ions had a strong effect on increasing δ binding affinity, supporting the possibility of bridging metal ion involvement in stator function. High pH environment greatly reduced δ binding affinity, suggesting the involvement of protonatable side-chains in the binding site.

Binding of pyrophosphate (PPi) to the three catalytic (“C”) and three noncatalytic (“NC”) nucleotide sites of Escherichia coliF1-ATPase was determined by fluorescence spectroscopy using mutant enzymes with tryptophan inserted specifically in either C sites (βY331W) or NC sites (αR365W). Fluorescence of the tryptophan is quenched on binding of nucleotide; PPibinding parameters were determined by competition with ATP or adenyl-5′-yl imidodiphosphate. It was found that MgPPibinds to each NC site with Kd= 20 μM. In contrast, even at millimolar concentration, neither MgPPinor free PPishowed significant binding to C sites. We confirmed that free PPidisplaces nucleotide from C sites, but this was shown to be due to complexation of Mg2+ions rather than to occupancy of the sites. MgPPibound 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 Kdsimilar 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.

Tryptophan was specifically inserted as the residue immediately preceding the P-loop sequence in F1-ATPase catalytic sites. The mutant enzyme (βF148W) showed normal enzymatic characteristics. The fluorescence responses of β-tryptophan 148 enabled us to differentiate between nucleoside di- and triphosphate bound in catalytic sites; MgADP quenched at 350 nm, whereas MgAMPPNP and MgADP·BeFxcomplex enhanced the fluorescence at 325 nm. With MgATP, both effects were seen simultaneously. This allowed analysis of bound catalytic site nucleotides directly under steady-state MgATP hydrolysis conditions. At mMconcentration of MgATP (Vmaxconditions) one of the three catalytic sites was filled with substrate MgATP and the other two sites were filled with product MgADP. A model for F1-ATPase steady-state turnover is presented that encompasses these findings.

It had previously been suggested that Vmaxhydrolysis rate of 2′,3′-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate (TNP-ATP) by F1-ATPase required filling of only two catalytic sites on the enzyme (Grubmeyer, C., and Penefsky, H. S. (1981) J. Biol. Chem.256, 3718-3727), whereas recently it was shown that Vmaxrate of ATP hydrolysis requires that all three catalytic sites are filled (Weber, J., Wilke-Mounts, S., Lee, R. S. F., Grell, E., and Senior, A. E.(1993) J. Biol. Chem.268, 20126-20133). To resolve this apparent discrepancy, we measured equilibrium binding and hydrolysis of MgTNP-ATP under identical conditions, using βY331W mutant Escherichia coliF1-ATPase, in which the genetically engineered tryptophan provides a direct fluorescent probe of catalytic site occupancy. We found that MgTNP-ATP hydrolysis at Vmaxrate did require filling of all three catalytic sites, but in contrast to the situation with MgATP, “bisite hydrolysis” of MgTNP-ATP amounted to a substantial fraction (∼40%) of Vmax.

X-ray structure analysis of the noncatalytic sites of F1-ATPase revealed that residue α-Asp261lies close to the Mg of bound Mg-5′-adenylyl-β,γ-imidodiphosphate. Here, the mutation αD261N was generated in Escherichia coliand combined with the αR365W mutation, allowing nucleotide binding at F1noncatalytic sites to be specifically monitored by tryptophan fluorescence spectroscopy. Purified αD261N/αR365W F1-ATPase showed catalytic activity similar to wild-type. An important feature was that, without any resort to nucleotide-depletion procedures, the noncatalytic sites in purified native enzyme were already empty. Binding studies with MgATP, MgADP, and the corresponding free nucleotides led to the following conclusions. Residue α-Asp261interacts with the Mg of Mg-nucleotide in noncatalytic sites and provides a large component of the binding energy (∼3 kcal/mol). It is the primary determinant of the preference of noncatalytic sites for Mg-nucleotide. The natural ligands at these sites in wild-type enzyme are the Mg-nucleotides and free nucleotides bind poorly.