The presence of functional arginine residues in phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae.
Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) is completely inactivated by phenylglyoxal and 2,3-butanedione in borate buffer at pH 8.4, with pseudo-first-order kinetics and a second-order rate constant of 144 min-1 X M-1 and 21.6 min-1 X M-1, respectively. Phosphoenolpyruvate, ADP and Mn2+ (alone or in combination) protect the enzyme against inactivation, suggesting that the modification occurs at or near to the substrate-binding site. Almost complete restoration of activity was obtained when a sample of 2,3-butanedione-inactivated enzyme was freed of excess modifier and borate ions, suggesting that only arginyl groups are modified. The changes in the rate of inactivation in the presence of substrates and Mn2+ were used to determine the dissociation constants for enzyme-ligand complexes, and values of 23 +/- 3 microM, 168 +/- 44 microM and 244 +/- 54 microM were found for the dissociation constants for the enzyme-Mn2+, enzyme-ADP and enzyme-phosphoenolpyruvate complexes, respectively. Based on kinetic data, it is shown that 1 mol of reagent must combine per enzyme active unit in order to inactivate the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of 3-4 mol [7-14C]phenylglyoxal per mol of enzyme subunit. Assuming a stoichiometry of 1:1 between phenylglyoxal incorporation and arginine modification, our results suggest that the modification of only two of the three to four reactive arginine residues per phosphoenolpyruvate carboxykinase subunit is responsible for inactivation.
Malebrán
L P
LP
Departamento de Química, Facultad de Ciencia, Universidad de Santiago de Chile.
Cardemil
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D000244:Adenosine Diphosphate / Q000494:pharmacology; D000447:Aldehydes / Q000494:pharmacology*; D001120:Arginine; D002074:Butanones / Q000494:pharmacology*; D055598:Chemical Phenomena; D002621:Chemistry; D003931:Diacetyl / Q000494:pharmacology*; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D010658:Phenylglyoxal / Q000378:metabolism / Q000494:pharmacology*; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000447:Aldehydes; D002074:Butanones; D008345:Manganese; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D001120:Arginine; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D003931:Diacetyl; D010658:Phenylglyoxal