REACTIVITY OF MITOCHONDRIAL F1-ATPASE TO DICYCLOHEXYLCARBODIIMIDE - INACTIVATION AND BINDING-STUDIES

There are two sites of action of DCCD on the mitochondrial ATPase complex, the membrane sector and the F1ATPase. Incubation of F1-ATPase isolated from beef heart mitochondria with dicyclohexylcarbodiimide (DCCD) resulted in inactivation of the enzyme. The DCCD inactivation of F1-ATPase was both time and concentration dependent. Kinetic data indicated that 1 mol of DCCD binds to 1 mol of active site. Inactivation of F1-ATPase by DCCD was pH dependent, being more marked at acid pH. Half-maximal effect was around pH 7.5, no inactivation occurring above pH 8.5. The half-time of inactivation was decreased by 20% by 10 mM ATP and ADP and increased by 50% by 10 mM MgCl2. Complete inactivation of F1-ATPase required the binding of 2 mol of [14C]DCCD per mol of F1-ATPase. The binding sites of [14C]DCCD were located on the β subunit of F1-ATPase. Two other carboxyl reagents which inhibit the ATPase activity, N-cyclohexyl-N'-β-(4-methylmorpholine)-ethylcarbodiimide and N-ethoxycarbonyl-2-ethoxy-1, 2-di-hydroquinoline, did not interfere with DCCD binding. Glycine ethyl ester, a nucleophile compound which reacts selectively with carbodiimide-activated carboxyl groups, removed half of the bound [14C]DCCD. The DCCD-modified F1-ATPase gave a fluorescent complex with aurovertin similar to the unmodified F1-ATPase. However, in contrast to the F1-ATPase-aurovertin complex, the fluorescent intensity of the DCCD-modified F1-ATPase-aurovertin complex was no longer quenched by ATP and MgCl2 and no longer enhanced by ADP. © 1979, American Chemical Society. All rights reserved.