Kinetics of inactivation of the F1Fo ATPase of Propionigenium modestum by dicyclohexylcarbodiimide in relationship to H+ and Na+ concentration: probing the binding site for the coupling ions.
Purified F1Fo ATPase of Propionigenium modestum was rapidly inactivated by dicyclohexylcarbodiimide (DCCD) with k2 = 1.2 x 10(5) M-1 min-1 at pH 5.6 and 0 degree C. Na+ ions provided specific protection from the modification by DCCD while protons stimulated the reaction. Plots of pseudo-first-order rate constants of inactivation (kobs) against pH yielded titration curves with pK(H+) = 7.0 in the absence of Na+ and pK(H+) = 6.2 in the presence of 0.5 mM Na+. From the dependencies of kobs on Na+, pK(Na+) of about 2.5 and 3.3 were obtained at pH 6.5 and 8.0, respectively. These results indicate that DCCD reacts with a protonated group of the enzyme that dissociates with pK(H+) = 7.0 in the absence of Na+, and that Na+ ions promote the dissociation of this group. Additionally, higher Na+ concentrations were required at more acidic pH values to yield half-maximal protection from inactivation. These effects fit a competitive binding model for Na+ or H+ at the DCCD-reactive conserved acidic amino acid of subunit c (Glu-65). The active-site carboxylate could either be protonated and modified by DCCD or bind Na+ which then provides protection. Complementary results were obtained from the effects of Na+ and H+ on ATPase activity. The pH-rate profile of numax (with saturating Na+) indicated an increase of activity with apparent pK = 6.8, an optimum around pH 7.5, and decreasing activity with apparent pK = 8.7.(ABSTRACT TRUNCATED AT 250 WORDS)
Mikrobiologisches Institut, Eidenössische Technische Hochschule, ETH-Zentrum, Zürich, Switzerland.
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D001421:Bacteria, Anaerobic / Q000201:enzymology*; D001665:Binding Sites; D001667:Binding, Competitive; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011522:Protons; D012964:Sodium / Q000494:pharmacology*
D011522:Protons; D004024:Dicyclohexylcarbodiimide; D000584:Amiloride; D000255:Adenosine Triphosphate; D012964:Sodium; D006180:Proton-Translocating ATPases; C039614:ethylisopropylamiloride