Localization of a carboxylic residue possibly involved in the inhibition of vacuolar H+-pyrophosphatase by N, N'-dicyclohexylcarbodi-imide.
A vacuolar H(+)-pyrophosphatase (EC 3.6.1.1) that catalyses PP(i) hydrolysis and the electrogenic translocation of protons from the cytosol to the vacuole lumen, was purified from etiolated hypocotyls of mung bean seedlings (Vigna radiata L.). Group-specific modification was used to identify a carboxylic residue involved in the inhibition of vacuolar H(+)-pyrophosphatase. Carbodi-imides, such as N,N'-dicyclohexylcarbodi-imide (DCCD) and 1-ethyl-3-(3-dimethylamino-propyl)carbodi-imide, and Woodward's reagent K caused a progressive decline in the enzymic activity of vacuolar H(+)-pyrophosphatase in a time- and concentration-dependent manner. The stoichiometry of labelling of the vacuolar H(+)-pyrophosphatase by [(14)C]DCCD determined that DCCD modifies one carboxylic residue per subunit of the enzyme. Protection studies suggest that the DCCD-reactive carboxylic residue resides at or near the substrate-binding site. Furthermore, peptide mapping analysis reveals that Asp(283), located in the putative loop V of a tentative topological model of vacuolar H(+)-pyrophosphatase on the cytosolic side, was labelled by radioactive [(14)C]DCCD. Cytosolic loop V contains both DCCD-sensitive Asp(283) and a conserved motif sequence, rendering it a candidate for the catalytic site of vacuolar H(+)-pyrophosphatase. A topological picture of the active domain of vacuolar H(+)-pyrophosphatase is tentatively proposed.
Yang
S J
SJ
Department of Radiological Technology, Chungtai Institute of Health Science and Technology, Taichung 40605, Taiwan, Republic of China.
Jiang
S S
SS
Kuo
S Y
SY
Hung
S H
SH
Tam
M F
MF
Pan
R L
RL
D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007887:Fabaceae; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010946:Plants, Medicinal; D017433:Protein Structure, Secondary; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors*; D013329:Structure-Activity Relationship; D014617:Vacuoles / Q000187:drug effects / Q000201:enzymology*
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D004024:Dicyclohexylcarbodiimide; D001216:Asparagine; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase