INACTIVATION OF THE KLUYVEROMYCES-LACTIS H-ATPASE BY DICYCLOHEXYLCARBODIIMIDE - BINDING STOICHIOMETRY AND EFFECT OF NUCLEOPHILES()

Dicyclohexylcarbodiimide (DCCD) inactivated the plasma membrane H+-ATP ase (EC 3.6.1.35) from Kluyveromyces lactis, with a second-order rate constant of 420 M-1 min(-1). The inhibition kinetics was apparently co mplex, due to degradation of DCCD with time. Neither Mg2+ nor Mg-ADP a ffected the inactivation of the ATPase by DCCD. In contrast, vanadate, a transition state analog of phosphate, partially protected the enzym e with a K-d Of 14 mu M, indicating a coupling between the DCCD-reacti ve site and the vanadate-binding site. The incubation of H+-ATPase wit h. C-14-DCCD showed that the incorporation of 1.2 mol of DCCD/mol ATPa se leads to complete inactivation. The hydrophobic carbodiimide reacte d with the protonated form of the carboxylic group, which displayed a pK(a) of 7.4, strongly suggesting that the residue is in the hydrophob ic environment of the membrane. Benzylamine increased the rate of inac tivation by DCCD. Ln this case, full inactivation of the enzyme was as sociated with the incorporation of 2.4 mol of DCCD/mol of enzyme, indi cating the opening of new reactive sites, resulting from a conformatio nal change induced by benzylamine. (C) 1997 Academic Press.