Inactivation of theKluyveromyces lactisH+-ATPase by Dicyclohexylcarbodiimide: Binding Stoichiometry and Effect of Nucleophiles

Dicyclohexylcarbodiimide (DCCD) inactivated the plasma membrane H+-ATPase (EC 3.6.1.35) fromKluyveromyces lactis,with a second-order rate constant of 420m−1min−1. The inhibition kinetics was apparently complex, due to degradation of DCCD with time. Neither Mg2+nor Mg-ADP affected the inactivation of the ATPase by DCCD. In contrast, vanadate, a transition state analog of phosphate, partially protected the enzyme with aKdof 14 μm, indicating a coupling between the DCCD-reactive site and the vanadate-binding site. The incubation of H+-ATPase with14C-DCCD showed that the incorporation of 1.2 mol of DCCD/mol ATPase leads to complete inactivation. The hydrophobic carbodiimide reacted with the protonated form of the carboxylic group, which displayed a pKaof 7.4, strongly suggesting that the residue is in the hydrophobic environment of the membrane. Benzylamine increased the rate of inactivation by DCCD. In this case, full inactivation of the enzyme was associated with the incorporation of 2.4 mol of DCCD/mol of enzyme, indicating the opening of new reactive sites, resulting from a conformational change induced by benzylamine.