Identification of reactive conserved histidines in phosphoenolpyruvate carboxykinases from Escherichia coli and Saccharomyces cerevisiae.
Escherichia coli and Saccharomyces cerevisiae phospho enol pyruvate (PEP) carboxykinases are inactivated by diethylpyrocarbonate (DEP). Inactivation follows pseudo-first-order kinetics and exhibits a second order rate constant of 0.8 M-1 s-1 for the bacterial enzyme and of 3.3 M-1 s-1 for the yeast carboxykinase. A mixture of ADP + PEP + MnCl2 protects against inactivation by DEP, suggesting that residues within the active site are being modified. After digestion of the modified proteins with trypsin, the labeled peptides were isolated by reverse-phase high-performance liquid chromatography and sequenced by Edman degradation. His-271 of E. coli carboxykinase and His-273 of the yeast enzyme were identified as the reactive amino-acid residues. The modified histidine residues occupy equivalent positions in these enzymes, and they are located in a highly conserved region of all ATP-dependent phospho enol pyruvate carboxykinases described so far.
Bazaes
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Departamento de Química, Universidad Metropolitana de Ciencias de la Educación, Santiago, Chile.
Montecinos
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Krautwurst
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Goldie
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Cardemil
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Jabalquinto
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D006639:Histidine; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D004047:Diethyl Pyrocarbonate