Cooperativity and stoichiometry of substrate binding to the catalytic sites of Escherichia coli F1-ATPase. Effects of magnesium, inhibitors, and mutation.
The fluorescence of residue Trp beta 331 in beta Y331W 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 Kd = 76 microM. In contrast, MgATP bound to three catalytic sites with Kd1 < 50 nM, Kd2 = 0.5 microM, and Kd3 = 25 microM. 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 beta-subunit nucleotide-binding site. The inhibitors dicyclohexylcarbodiimide and N-ethylmaleimide (in alpha S373C/beta Y331W 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 alpha S373F mutation (in alpha S373F/beta Y331W 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.
Weber
J
J
Department of Biochemistry, University of Rochester Medical Center, New York 14642.
Wilke-Mounts
S
S
Senior
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AE
D000255:Adenosine Triphosphate / Q000378:metabolism; D002384:Catalysis; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology*; D009154:Mutation; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D008274:Magnesium; D005033:Ethylmaleimide