Effect of dicyclohexylcarbodiimide on unisite and multisite catalytic activities of the adenosine triphosphatase of Escherichia coli

Abstract

The inhibitory effect of dicyclohexylcarbodiimide (DCCD) on the activity of the adenosine-triphosphatase of Escherichia coli (ECF1) has been examined in detail. DCCD reacted with ECF1 predominantly in beta subunits with a maximum of 2 mol of reagent per mole of ECF1 being incorporated in these subunits. Ninety-five percent inhibition of steady-state or multistate ATPase activity required incorporation of 1 mol of DCCD per mole of enzyme into beta subunits. Seventy-five percent inhibition of the initial rate of unisite catalysis was only obtained after incorporation of 2 mol of DCCD per mole of ECF1 into beta subunits. Analyses of the kinetics of unisite catalysis and nucleotide binding experiments both indicate that DCCD binds outside the substrate ATP binding site. Inhibition by this reagent appears to be due in part to an effect on the catalytic sites but mainly to the blocking of cooperativity between these sites.

Effects of the Inhibitors Azide, Dicyclohexylcarbodiimide, and Aurovertin on Nucleotide Binding to the Three F1-ATPase Catalytic Sites Measured Using Specific Tryptophan Probes

“…In contrast to its potent inhibition of steady-state (multisite) ATP hydrolysis, single turnover hydrolysis (unisite catalysis) is inhibited to a significantly lesser extent (16). Both the ␤-Trp-331 and ␤-Trp-148 enzymes were fully inhibited by DCCD (Ն99%).…”

“…DCCD reacts with residue ␤-Glu-192 in wild-type E. coli F 1 (21), and at full inhibition of ATPase, the stoichiometry of labeling is 2 mol/mol (16). In contrast to its potent inhibition of steady-state (multisite) ATP hydrolysis, single turnover hydrolysis (unisite catalysis) is inhibited to a significantly lesser extent (16).…”

“…We first demonstrated that, when ␤-Trp-148 F 1 was modified by DCCD as described in Ref. 5, ATPase activity was inhibited by Ն99%, indicating incorporation of 2 mol of DCCD/ mol of F 1 (16). When 2.5 mM NaATP, 1.0 mM MgSO 4 was added to the modified enzyme the fluorescence spectrum indicated the following nucleotide distribution: bound MgATP ϭ 1.6 Ϯ 0.2 mol/mol of F 1 ; bound MgADP ϭ 1.4 Ϯ 0.3 mol/mol of F 1 (means of five experiments Ϯ S.E.).…”

ATP Synthase and the Actions of Inhibitors Utilized To Study Its Roles in Human Health, Disease, and Other Scientific Areas

“…DCCD and N-(2,2,6,6-tetramethylpeperidyl-1-oxyl)-N-(cyclohexyl)carbodiimide (NCCD) are lipid-soluble carbodiimides. DCCD binds to both F 1 and F 0 of ATP synthases from mitochondria and some bacteria (137,204,400,441) (Table 20). F 1 from some bacteria, such as Helicobacter pylori, are insensitive to DCCD (36).…”

“…In F 1 from E. coli, ␤Glu192 binds DCCD, while in bovine MF 1 , ␤Glu199, corresponding to E. coli ␤Glu192, is modified. In F 1 from thermophilic Bacillus, ␤Glu181 (E. coli sequence) rather than ␤Glu192 is modified (137,400,441). Incorporation of 1 mol of DCCD into 1 mol of F 1 results in 95% inhibition of the ATPase activity of EF 1 , and 2 mol of DCCD/mol F 1 leads to complete inhibition (400).…”