Inactivation of the Kluyveromyces lactis H+-ATPase by dicyclohexylcarbodiimide: binding stoichiometry and effect of nucleophiles.
Dicyclohexylcarbodiimide (DCCD) inactivated the plasma membrane H+-ATPase (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 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 a Kd of 14 microM, indicating a coupling between the DCCD-reactive site and the vanadate-binding site. The incubation of H+-ATPase with 14C-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 pKa of 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.
Velázquez
I
I
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, D.F.
Martínez
F
F
Pardo
J P
JP
D001596:Benzylamines / Q000494:pharmacology; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007716:Kluyveromyces / Q000201:enzymology*; D008565:Membrane Proteins / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D001596:Benzylamines; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D014638:Vanadates; D004024:Dicyclohexylcarbodiimide; C030796:benzylamine; D006180:Proton-Translocating ATPases