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

Journal of Protein Chemistry
Yi Yuong HsiaoRong-Long Pan

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 singl...Continue Reading

Citations

Feb 5, 2011·The Journal of Biological Chemistry·Chien-Hsien LeeRong-Long Pan
Feb 12, 2004·Biochimica Et Biophysica Acta·Yi Y HsiaoRong L Pan
Jun 5, 2004·Biochimica Et Biophysica Acta·Su J YangRong L Pan
Jun 5, 2004·Biochimica Et Biophysica Acta·Anders Schultz, Margareta Baltscheffsky
Sep 28, 2005·Archives of Biochemistry and Biophysics·Hsin Hung LinRong Long Pan
Jun 5, 2007·Biochimica Et Biophysica Acta·Yi-Yuong HsiaoRong-Long Pan

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