Reactive sulfhydryl groups in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase.
Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) is inactivated by several thiol- and vicinal dithiol-specific reagents. Titration experiments of the enzyme with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) show the presence of reactive monothiol and vicinal dithiol groups, whose modifications lead to enzyme inactivation. The enzyme is also inactivated by N-(1-pyrenyl)iodoacetamide (PyrIAM), with a binding stoichiometry of approx. 2 mol per mol of enzyme subunit. A high level of pyrene excimer fluorescence is detected on the labeled enzyme, thus implying the reaction of the reagent with two spatially close sulfhydryl groups in the protein. The carboxykinase is not completely inactivated by different vicinal dithiol-specific reagents, thus implying a catalytically non-essential character for these groups. From substrate protection experiments of the enzyme inactivation by DTNB, PyrIAM and vicinal dithiol-specific reagents, it is concluded that the loss of enzyme activity is caused by the modification of both thiol and vicinal dithiol groups in the substrate binding region.
Cardemil
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Departamento de Química, Facultad de Ciencia, Universidad de Santiago de Chile.
Encinas
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MV
Jabalquinto
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AM
D001151:Arsenic / Q000494:pharmacology; D001152:Arsenicals / Q000494:pharmacology; D018053:Arsenites; D002104:Cadmium / Q000494:pharmacology; D004112:Dimercaprol / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D007700:Kinetics; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
D001152:Arsenicals; D018053:Arsenites; D013438:Sulfhydryl Compounds; D002104:Cadmium; D004112:Dimercaprol; C029341:oxophenylarsine; D004228:Dithionitrobenzoic Acid; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D015032:Zinc; C015001:arsenite; D001151:Arsenic