PMID:1978721
| Citation |
Vlahos, CJ and Dekker, EE (1990) Active-site residues of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli. Bromopyruvate inactivation and labeling of glutamate 45. J. Biol. Chem. 265:20384-9 |
|---|---|
| Abstract |
Treatment of pure 2-keto-4-hydroxyglutarate aldolase from Escherichia coli, a "lysine-type," Schiff-base mechanism enzyme, with the substrate analog bromopyruvate results in a time- and concentration-dependent loss of enzymatic activity. Whereas the substrates pyruvate and 2-keto-4-hydroxyglutarate provide greater than 90% protection against inactivation by bromopyruvate, no protective effect is seen with glycolaldehyde, an analog of glyoxylate. Inactivation studies with [14C] bromopyruvate show the incorporation of 1.1 mol of 14C-labeled compound/enzyme subunit; isolation of a radioactive peptide and determination of its amino acid sequence indicate that the radioactivity is associated with glutamate 45. Incubation of the enzyme with excess [14C]bromopyruvate followed by denaturation with guanidine.HCl allow for the incorporation of carbon-14 at cysteines 159 and 180 as well. Whereas the presence of pyruvate protects Glu-45 from being esterified, it does not prevent the alkylation of these 2 cysteine residues. The results indicate that Glu-45 of E. coli 2-keto-4-hydroxyglutarate aldolase is essential for catalytic activity, most likely acting as the amphoteric proton donor/acceptor that is required as a participant in the overall mechanism of the reaction catalyzed. |
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| Keywords |
Affinity Labels; Amino Acid Sequence; Binding Sites; Escherichia coli/enzymology; Glutamates; Glutamic Acid; Ketoglutaric Acids/metabolism; Kinetics; Models, Structural; Molecular Sequence Data; Oxo-Acid-Lyases/antagonists & inhibitors; Oxo-Acid-Lyases/metabolism; Peptide Mapping; Protein Conformation; Pyruvates/pharmacology; Trypsin |
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