Binding and hydrolysis of TNP-ATP by Escherichia coli F-1-ATPase

It had previously been suggested that V-max hydrolysis rate of 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) by F-1-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 V-max rate 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 beta Y331W mutant Escherichia coli F-1-ATPase, in which the genetically engineered tryptophan provides a direct fluorescent probe of catalytic site occupancy, We found that MgTNP-ATP hydrolysis at V-max rate 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 (similar to 40%) of V-max. Binding of MgTNP-ATP to the three catalytic sites showed strong binding cooperativity (K-d1 < 1 nM, K-d2 = 23 nM, K-d3 = 1.4 mu M). Free TNP-ATP (i.e. in presence of EDTA) bound to all three catalytic sites with lower affinity but was not hydrolyzed, These data emphasize that the presence of Mg2+ is critical for cooperativity of substrate binding, formation of the very high affinity first catalytic site, and hydrolytic activity in F-1-ATPases and that these three properties are strongly correlated.