Diethylpyrocarbonate inhibition of vacuolar H+-pyrophosphatase possibly involves a histidine residue.
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.
Hsiao
Yi Yuong
YY
Department of Life Science, College of Life Science, National Tsing Hua University, Hsin Chu, Taiwan, Republic of China.
Van
Ru Chuan
RC
Hung
Hsiao Hui
HH
Pan
Rong Long
RL
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*
D004791:Enzyme Inhibitors; D008024:Ligands; D010940:Plant Proteins; D011522:Protons; D019811:Hydroxylamine; D006639:Histidine; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D004047:Diethyl Pyrocarbonate