Specific protection by Na+ or Li+ of the F1F0-ATPase of Propionigenium modestum from the reaction with dicyclohexylcarbodiimide.
Incubation of the purified F1F0-ATPase of Propionigenium modestum with dicyclohexylcarbodiimide (DCCD) led to inactivation of the enzyme in a strongly pH-dependent manner. Rapid inactivation occurred at pH 5-7, while the increase of the pH from 7 to 9 resulted in a continuous reduction of the inactivation rate. In the presence of Na+ ions, the ATPase was specifically protected from inactivation by DCCD. The protective effect of Na+ was most pronounced at pH 9.0 and less significant at pH 7.0. In addition to Na+, Li+ also protected the ATPase from inactivation by DCCD, but approximately 10 times higher concentrations were required for the same effect. Similarly, the Na+ concentration causing half-maximal stimulation of ATPase activity was about 10 times below the Li+ concentration required for the same activation. It is concluded from these results that a binding site is present for Na+ or Li+ on the enzyme with an about 10 times lower affinity for the latter alkali ion, which when occupied stimulates ATPase activity and protects it from inactivation by DCCD. Inactivation of ATPase activity by DCCD correlated well with a specific labeling of subunit c of the enzyme in the presence of the [14C]DCCD derivative. Like ATPase inactivation, the labeling was promoted by more acidic pH values and inhibited by Na+ ions. We suggest from these data that the DCCD-reactive amino acid residue of subunit c (most likely Glu-65) must be protonated for the reaction with the carbodiimide and provides the Na(+)-binding site in its deprotonated state. Dissociation of the carboxylic acid (at high pH) and binding of Na+ ions to the carboxylate thus abolish the reactivity toward DCCD.
Kluge
C
C
Mikrobiologisches Institut, Eidgenössischen Technischen Hochschule, ETH-Zentrum, Zürich, Switzerland.
Dimroth
P
P
D001421:Bacteria, Anaerobic / Q000201:enzymology*; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism*; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D008094:Lithium / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*
D004024:Dicyclohexylcarbodiimide; D008094:Lithium; D012964:Sodium; D006180:Proton-Translocating ATPases