Reactivity of cysteinyl, arginyl, and lysyl residues of Escherichia coli phosphoenolpyruvate carboxykinase against group-specific chemical reagents.
Calcium-activated phosphoenolpyruvate carboxykinase from Escherichia coli is not inactivated by a number of sulfhydryl-directed reagents [5,5'-dithiobis(2-nitrobenzoate), iodoacetate, N-ethylmaleimide, N-(1-pyrenyl)maleimide or N-(iodoacetyl)-N'-(5-sulfo-1-naphthylethylenediamine)], unlike phosphoenolpyruvate carboxykinase from other organisms. On the other hand, the enzyme is rapidly inactivated by the arginyl-directed reagents 2,3-butanedione and 1-pyrenylglyoxal. The substrates, ADP plus PEP in the presence of Mn2+, protect the enzyme against inactivation by the diones. Quantitation of pyrenylglyoxal incorporation indicates that complete inactivation correlates with the binding of one inactivator molecule per mole of enzyme. Chemical modification by pyridoxal 5'-phosphate also produces inactivation of the enzyme, and the labeled protein shows a difference spectrum with a peak at 325 nm, characteristic of a pyridoxyl derivative of lysine. The inactivation by this reagent is also prevented by the substrates. Binding stoichiometries of 1.25 and 0.30 mol of reagent incorporated per mole of enzyme were found in the absence and presence of substrates, respectively. The results suggest the presence of functional arginyl and lysyl residues in or near the active site of the enzyme, and indicate lack of reactive functional sulfhydryl groups.
Bazaes
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Departamento de Química, Universidad Metropolitana de Ciencias de la Educación, Santiago, Chile.
Silva
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Goldie
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Cardemil
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Jabalquinto
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AM
D001120:Arginine; D001665:Binding Sites; D003545:Cysteine; D003931:Diacetyl / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006037:Glyoxal / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011721:Pyrenes / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000378:metabolism / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D011721:Pyrenes; D013439:Sulfhydryl Reagents; C066961:pyrene glyoxal; D006037:Glyoxal; D011732:Pyridoxal Phosphate; D001120:Arginine; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D003931:Diacetyl; D008239:Lysine; D003545:Cysteine