Essential arginyl residues in the H+-translocating ATPase of plasma membrane from the yeast Schizosaccharomyces pombe.
The H+-translocating adenosine-5'-triphosphatase (ATPase) purified from the yeast Schizosaccharomyces pombe is inactivated upon incubation with the arginine modifier 2,3-butanedione. The inactivation of the enzyme is maximal at pH values above 8.5. The modified enzyme is reactivated when incubated in the absence of borate after removal of 2,3-butanedione. The extent of inactivation is half maximal at 10 mM 2,3-butanedione for an incubation of 30 min at 30 degrees C at pH 7.0. Under the same conditions, the time-dependence of inactivation is biphasic in a semi-logarithmic plot with half-lives of 10.9 min and 65.9 min. Incubation with 2,3-butanedione lowering markedly the maximal rate of ATPase activity does not modify the Km for MgATP. These data suggest that two classes of arginyl residues play essential role in the plasma membrane ATPase activity. Magnesium adenosine 5'-triphosphate (MgATP) and magnesium adenosine 5'-diphosphate (MgADP), the specific substrate and product, protect partially against enzyme inactivation by 2,3-butanedione. Free ATP or MgGTP which are not enzyme substrates do not protect. Free magnesium, another effector of enzyme activity, exhibits partial protection at magnesium concentrations up to 0.5 mM, while increased inactivation is observed at higher Mg2+ concentrations. These protections indicate either the existence of at least one reactive arginyl in the substrate binding site or a general change of enzyme conformation induced by MgATP, MgADP or free magnesium.
Di Pietro
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Goffeau
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D001120:Arginine / Q000032:analysis*; D001203:Ascomycota / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D055598:Chemical Phenomena; D002621:Chemistry; D003931:Diacetyl / Q000037:antagonists & inhibitors / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000201:enzymology*
D001120:Arginine; D006180:Proton-Translocating ATPases; D008274:Magnesium; D003931:Diacetyl