COOPERATIVITY AND STOICHIOMETRY OF SUBSTRATE-BINDING TO THE CATALYTICSITES OF ESCHERICHIA-COLI F1-ATPASE EFFECTS OF MAGNESIUM, INHIBITORS,AND MUTATION

The fluorescence of residue Trp(beta 331) in beta Y331W mutant Escheri chia coli F-1-ATPase was used as reporter probe to investigate the eff ects of magnesium ions, inhibitors, and mutation on substrate (ATP) bi nding stoichiometry and cooperativity. It was found that Mg2+ is requi red for catalytic site binding cooperativity. In the absence of magnes ium, ATP bound to three independent catalytic sites, each with K-d = 7 6 mu M. In contrast, MgATP bound to three catalytic sites with K-d1 < 50 nM, K-d2 = 0.5 mu M, and K-d3 = 25 mu M. There was no significant A TPase activity in the absence of Mg2+. Catalysis is therefore correlat ed 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 beta-subunit nucleotide-binding site. The inhibi tors dicyclohexylcarbodiimide and N-ethylmaleimide (in alpha S373C/bet a Y331W mutant F-1) gave potent inhibition of multisite ATPase activit y without significantly affecting MgATP binding stoichiometry or coope rativity. Therefore each seems to selectively attenuate positive catal ytic cooperativity. The same conclusions held for the alpha S373F muta tion (in alpha S373F/beta Y331W mutant F-1). 7-Chloro-4-nitrobenzo-2-o xa-1,3-diazole, however, reduced the catalytic site MgATP binding stoi chiometry from three to two, and appears to inhibit catalysis by steri cally blocking catalytic site 3.