Diethylpyrocarbonate inhibition of vacuolar H+-pyrophosphatase possibly involves a histidine residue.

摘要: Vacuolar proton pumping pyrophosphatase (H+-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at expense PPi hydrolysis. A histidine-specific modifier, diethylpyrocarbonate (DEPC), could substantially inhibit enzymic activity and H+-translocation H+-PPase concentration-dependent manner. Absorbance 240 nm was increased upon incubation with DEPC, demonstrating that an N-carbethoxyhistidine moiety probably formed. On other hand, hydroxylamine, reagent can deacylate N-carbethoxyhistidine, reverse absorption change partially restore hydrolysis as well. The pKa modified residues enzyme determined be 6.4, value close histidine. Thus, we speculate inhibition by DEPC possibly attributed modification histidyl on enzyme. Furthermore, follows pseudo-first-order rate kinetics. reaction order 0.85 calculated double logarithmic plot apparent constant against concentration, suggesting one single histidine residue suffices H+-PPase. Inhibition changes Vmax but not Km values. Moreover, protected its physiological substrate, Mg2+-PPi. These results indicated specifically competes substrate active site DEPC-labeled might locate or near catalytic domain Besides, pretreatment N-ethylmaleimide decreased degree subsequent labeling DEPC. Taken together, suggest likely contains substrate-protectable contributing this may located sensitive Cys-629 NEM.