INACTIVATION OF ESCHERICHIA-COLI BF1-ATPASE BY DICYCLOHEXYLCARBODIIMIDE - CHEMICAL MODIFICATION OF THE BETA-SUBUNIT

Addition of dicyclohexylcarbodiimide (DCCD) to BF1ATPase isolated from Escherichia coli resulted in irreversible loss of enzymic activity. The kinetics of inactivation were consistent with 1 mol of DCCD reacting with one active site of BF1ATPase to give an inactive complex. Inactivation was more rapid at acid pH than at alkaline pH, the half-maximum effect being obtained at about pH 7. The half-time of inactivation was increased 4 times by 10 mM MgCl2 and twice by 10 mM ADP or ATP. Inactivation of BF1ATPase was accompanied by the covalent binding of DCCD to the β subunit of BF1ATPase as shown by the use of (14C)DCCD. The loss of enzymic activity was proportional to the incorporation of (14C)DCCD. Linear extrapolation to zero activity corresponded to the binding of 1 mol of (l4C)-DCCD to 1 mol of BF1ATPase. Glycine ethyl ester, a nucleophilic reagent, which reacts with the DCCD-activated carboxyl group to form an amide bond, did not relieve the inactivation brought about by DCCD but prevented the binding of (14C)DCCD, indicating that DCCD binds to a carboxyl group. Further evidence for a modification of a carboxyl group by DCCD was provided by isoelectrofocusing assays which suggested the disappearance of a negative charge in DCCD-modified BF1ATPase. The formation of the fluorescent aurovertin-BF1ATPase complex was not prevented by DCCD, neither was the enhancement of fluorescence caused by ADP. In contrast, the quenching of aurovertin fluorescence due to ATP was prevented by DCCD. DCCD had no apparent effect on the binding affinity of BF1ATPase for (14C)ADP. The presence of a carboxyl group at the active site of BF,-ATPase is supported by the nature of the data presented in this paper. © 1979, American Chemical Society. All rights reserved.