The fluorescence of residue Trp(β331) in βY331W mutant Escherichia coli F1-ATPase was used as reporter probe to investigate the effects of magnesium ions, inhibitors, and mutation on substrate (ATP) binding stoichiometry and cooperativity. It was found that Mg2+ is required for catalytic site binding cooperativity. In the absence of magnesium, ATP bound to three independent catalytic sites, each with K(d) = 76 μM. In contrast, MgATP bound to three catalytic sites with K(d1) < 50 nM, K(d2) = 0.5 μM, and K(d3) = 25 μM. There was no significant ATPase activity in the absence of Mg2+. Catalysis is therefore correlated with substrate binding cooperativity and the formation of the high-affinity catalytic site 1. Catalytic site 3 had properties similar to those of the isolated β-subunit nucleotide-binding site. The inhibitors dicyclohexylcarbodiimide and N-ethyl-maleimide (in αS373C/βY331W mutant F1) gave potent inhibition of multisite ATPase activity without significantly affecting MgATP binding stoichiometry or cooperativity. Therefore each seems to selectively attenuate positive catalytic cooperativity. The same conclusions held for the αS373F mutation (in αS373F/βY331W mutant F1). 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole, however, reduced the catalytic site MgATP binding stoichiometry from three to two, and appears to inhibit catalysis by sterically blocking catalytic site 3.
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - 1994|