Kinetics of inactivation of the F1F0 ATPase of Propionigenium modestum by dicyclohexylcarbodiimide in relationship to hydrogen ion and sodium concentration: Probing the binding site for the coupling ions

Abstract

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)

Formation of a functionally active sodium‐translocating hybrid F1F0 ATPase in Escherichia coli by homologous recombination

“…The hybrid was specifically activated by Na' in a pH-dependent manner in much the same way as the homologous l ? modestum enzyme (Kluge and Dimroth, 1993b). At pH9.0, the increase of ATPase activity in the presence of 1 mM NaCl was approximately 15-fold which indicates that a step involving Na' binding to the F,, moiety is rate limiting for ATP hydrolysis at F, and thus tight conformational coupling between events taking place at F,, and F,.…”

The Function of the Na + -Driven Flagellum of Vibrio cholerae Is Determined by Osmolality and pH

“…At very low Na ϩ concentrations, ATPase activity is drastically diminished and the enzyme translocates protons. Lowering the proton concentration from pH 7.0 to pH 9.0 decreases activity by 70% (38). To find out whether a similar dependence of the activity of the flagellum on the concentration of Na ϩ and H ϩ could be observed, we investigated the motility of V. cholerae ⌬pomAB coexpressing wild-type PomA and variants of PomB at pH 7.0, 8.0, and 9.0 in rich or minimal medium and at different Na ϩ concentrations.…”

Specific Modification of a Na + Binding Site in NADH:Quinone Oxidoreductase from Klebsiella pneumoniae with Dicyclohexylcarbodiimide

“…A likely target for DCCD modification is a carboxylic group located in the membraneembedded part of the complex. A hydrophobic environment shifts the pK of a carboxylic group to the alkaline range, which facilitates protonation and enhances its reactivity with the carbodiimide (21). In the Na ϩ -translocating F 1 F o ATPase from Propionigenium modestum, DCCD modifies the protonated carboxylic group of glutamate 65 in subunit c from the membranebound F o part.…”