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...

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Bibliographic Details
Published in:Biochimica et biophysica acta 1997-02, Vol.1337 (2), p.166-174
Main Authors: Bazaes, S, Montecinos, L, Krautwurst, H, Goldie, H, Cardemil, E, Jabalquinto, A M
Format: Article
Language:English
Subjects:
Adenosine Diphosphate - metabolism
Amino Acid Sequence
Binding Sites
Conserved Sequence
Diethyl Pyrocarbonate - pharmacology
Enzyme Inhibitors - pharmacology
Escherichia coli - enzymology
Escherichia coli - genetics
Histidine - chemistry
Molecular Sequence Data
Peptide Fragments - chemistry
Peptide Fragments - genetics
Phosphoenolpyruvate - metabolism
Phosphoenolpyruvate Carboxykinase (GTP) - antagonists & inhibitors
Phosphoenolpyruvate Carboxykinase (GTP) - chemistry
Phosphoenolpyruvate Carboxykinase (GTP) - genetics
Saccharomyces cerevisiae - enzymology
Saccharomyces cerevisiae - genetics
Sequence Homology, Amino Acid
Species Specificity
Substrate Specificity
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Summary: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.
ISSN:0006-3002
0167-4838
DOI:10.1016/S0167-4838(96)00155-0