Active-site residues of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli. Bromopyruvate inactivation and labeling of glutamate 45.
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.
Vlahos
C J
CJ
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
Dekker
E E
EE
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D005971:Glutamates; D018698:Glutamic Acid; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D007652:Oxo-Acid-Lyases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010449:Peptide Mapping; D011487:Protein Conformation; D011773:Pyruvates / Q000494:pharmacology*; D014357:Trypsin
D000345:Affinity Labels; D005971:Glutamates; D007656:Ketoglutaric Acids; D011773:Pyruvates; D018698:Glutamic Acid; C017092:bromopyruvate; D014357:Trypsin; D007652:Oxo-Acid-Lyases; C021415:4-hydroxy-2-oxoglutarate aldolase