title: Diethylpyrocarbonate inhibition of vacuolar H+-pyrophosphatase possibly involves a histidine residue.
abstract: 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 the expense of PPi hydrolysis. A histidine-specific modifier, diethylpyrocarbonate (DEPC), could substantially inhibit enzymic activity and H+-translocation of vacuolar H+-PPase in a concentration-dependent manner. Absorbance of vacuolar H+-PPase at 240 nm was increased upon incubation with DEPC, demonstrating that an N-carbethoxyhistidine moiety was probably formed. On the other hand, hydroxylamine, a reagent that can deacylate N-carbethoxyhistidine, could reverse the absorption change at 240 nm and partially restore PPi hydrolysis activity as well. The pKa of modified residues of the enzyme was determined to be 6.4, a value close to that of histidine. Thus, we speculate that inhibition of vacuolar H+-PPase by DEPC possibly could be attributed to the modification of histidyl residues on the enzyme. Furthermore, inhibition of vacuolar H+-PPase by DEPC follows pseudo-first-order rate kinetics. A reaction order of 0.85 was calculated from a double logarithmic plot of the apparent reaction constant against DEPC concentration, suggesting that the modification of one single histidine residue on the enzyme suffices to inhibit vacuolar H+-PPase. Inhibition of vacuolar H+-PPase by DEPC changes Vmax but not Km values. Moreover, DEPC inhibition of vacuolar H+-PPase could be substantially protected against by its physiological substrate, Mg2+-PPi. These results indicated that DEPC specifically competes with the substrate at the active site and the DEPC-labeled histidine residue might locate in or near the catalytic domain of the enzyme. Besides, pretreatment of the enzyme with N-ethylmaleimide decreased the degree of subsequent labeling of H+-PPase by DEPC. Taken together, we suggest that vacuolar H+-PPase likely contains a substrate-protectable histidine residue contributing to the inhibition of its activity by DEPC, and this histidine residue may located in a domain sensitive to the modification of Cys-629 by NEM.
authors.affiliation: Department of Life Science, College of Life Science, National Tsing Hua University, Hsin Chu, Taiwan, Republic of China.
authors.forename: Yi Yuong
authors.initials: YY
authors.lastname: Hsiao
authors.forename: Ru Chuan
authors.initials: RC
authors.lastname: Van
authors.forename: Hsiao Hui
authors.initials: HH
authors.lastname: Hung
authors.forename: Rong Long
authors.initials: RL
authors.lastname: Pan
mesh_terms: D004047:Diethyl Pyrocarbonate / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D007887:Fabaceae / Q000201:enzymology; D006639:Histidine / Q000378:metabolism*; D019811:Hydroxylamine / Q000378:metabolism; D043564:Inorganic Pyrophosphatase; D008024:Ligands; D010940:Plant Proteins / Q000378:metabolism; D011522:Protons; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012044:Regression Analysis; D014617:Vacuoles / Q000201:enzymology*
chemical_list: D004791:Enzyme Inhibitors; D008024:Ligands; D010940:Plant Proteins; D011522:Protons; D019811:Hydroxylamine; D006639:Histidine; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D004047:Diethyl Pyrocarbonate