TY - JOUR
T1 - Cooperativity and stoichiometry of substrate binding to the catalytic sites of Escherichia coli F1-ATPase
T2 - Effects of magnesium, inhibitors, and mutation
AU - Weber, Joachim
AU - Wilke-Mounts, Susan
AU - Senior, Alan E.
PY - 1994/8/12
Y1 - 1994/8/12
N2 - The fluorescence of residue Trpβ331 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 Kd = 76 μM. In contrast, MgATP bound to three catalytic sites with Kd1 < 50 nM, Kd2 = 0.5 μM, and Kd3 = 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-ethylmaleimide (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.
AB - The fluorescence of residue Trpβ331 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 Kd = 76 μM. In contrast, MgATP bound to three catalytic sites with Kd1 < 50 nM, Kd2 = 0.5 μM, and Kd3 = 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-ethylmaleimide (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.
UR - http://www.scopus.com/inward/record.url?scp=0028033547&partnerID=8YFLogxK
M3 - Article
C2 - 8051144
AN - SCOPUS:0028033547
SN - 0021-9258
VL - 269
SP - 20462
EP - 20467
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 32
ER -