A functional arginine residue in the vacuolar H(+)-ATPase of higher plants.
The arginine-specific reagent phenylglyoxal inactivated the vacuolar H(+)-ATPase of red beet. Inactivation by phenylglyoxal followed pseudo-first-order kinetics and a double log plot of the t1/2 of inactivation versus phenylglyoxal concentration yielded a slope of 1.18. Neither inorganic anions nor DIDS protected from phenylglyoxal-mediated inactivation of the H(+)-ATPase. Indeed, Cl- stimulated the rate of phenylglyoxal-mediated H(+)-ATPase inactivation relative to SO4(2-). ATP, but not MgATP or ADP, protected from phenylglyoxal-mediated inactivation and inactivation resulted in a decrease in the Vmax of the H(+)-ATPase with little effect on the Km. Collectively, these results are consistent with phenylglyoxal-mediated inactivation of the vacuolar H(+)-ATPase resulting from modification of a single arginine residue in the catalytic nucleotide binding site of the vacuolar H(+)-ATPase. Stimulation of phenylglyoxal-mediated inactivation by Cl- indicates that exposure of the phenylglyoxal-sensitive functional arginine residue is enhanced in the presence of Cl-. The failure of MgATP to protect from phenylglyoxal inactivation suggests that ATP, rather than MgATP, binds directly to the catalytic site and that Mg2+ may act to promote catalysis subsequent to ATP binding.
Bennett
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AB
Department of Vegetable Crops, University of California, Davis 95616.
Borcherts
K
K
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism*; D002712:Chlorides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006096:Gramicidin / Q000494:pharmacology; D006859:Hydrogen / Q000378:metabolism*; D007700:Kinetics; D009705:Nucleosides / Q000494:pharmacology; D009711:Nucleotides / Q000494:pharmacology; D010658:Phenylglyoxal / Q000494:pharmacology; D010944:Plants / Q000187:drug effects / Q000201:enzymology*; D013379:Substrate Specificity; D014617:Vacuoles / Q000201:enzymology
D002712:Chlorides; D009705:Nucleosides; D009711:Nucleotides; D006096:Gramicidin; D006859:Hydrogen; D001120:Arginine; D000251:Adenosine Triphosphatases; D010658:Phenylglyoxal