Abstract
It had previously been suggested that Vmax hydrolysis 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 Vmax 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 betaY331W mutant Escherichia coli F1-ATPase, in which the genetically engineered tryptophan provides a direct fluorescent probe of catalytic site occupancy. We found that MgTNP-ATP hydrolysis at Vmax 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 (approximately 40%) of Vmax. Binding of MgTNP-ATP to the three catalytic sites showed strong binding cooperativity (Kd1 < 1 nm, Kd2 = 23 nm, Kd3 = 1.4 microM). 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 F1-ATPases and that these three properties are strongly correlated.
Highlights
ATP synthesis by oxidative phosphorylation is catalyzed by ATP synthase
One point that is evident from these data is that E. coli F1 hydrolyzes MgTNP-ATP relatively better than does mitochondrial F1, e.g. the ratio Vmax(MgTNP-ATP)/Vmax(MgATP) is about 10-fold higher in E. coli F1 as compared to mitochondrial F1
The major objective of this study was to determine whether maximal rates of MgTNP-ATP hydrolysis by F1-ATPase are achieved when just two of the three catalytic sites on the enzyme are occupied by substrate, as had been suggested previously (Grubmeyer and Penefsky, 1981b), or whether maximal rates are achieved only when all three catalytic sites are filled, as had been demonstrated to be the case for MgATP hydrolysis (Weber et al, 1993, 1994a)
Summary
ATP synthesis by oxidative phosphorylation is catalyzed by ATP synthase. The F1 sector of this enzyme contains three catalytic nucleotide binding sites, located on the three -subunits (Senior, 1988; Fillingame, 1990; Allison et al, 1992; Capaldi et al, 1994; Abrahams et al, 1994). F1 may be isolated in soluble form; it is an active ATPase (F1-ATPase), which has proved valuable for studies of catalytic mechanism Since their introduction (Hiratsuka and Uchida, 1973), the trinitrophenyl (TNP) derivatives of adenine nucleotides have been widely used to characterize nucleotide binding sites of proteins and enzymes. For hydrolysis of MgTNP-ATP by mitochondrial F1, it had been suggested previously that Vmax was reached upon occupation of only two of the three catalytic sites (Grubmeyer and Penefsky, 1981b). If this were the case, it would imply that the enzyme utilizes all three catalytic sites for ATP hydrolysis but only two sites for TNP-ATP hydrolysis. In the study presented here, we establish the relationship between catalytic site occupancy and MgTNP-ATP hydrolytic activity, and we report the Kd values for binding of MgTNP-ATP and free TNP-ATP to F1 catalytic sites
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