title: The complete amino acid sequence and identification of the active-site arginine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase.
abstract: The complete amino acid sequence of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli has been established in the following manner. After being reduced with dithiothreitol, the purified aldolase was alkylated with iodoacetamide and subsequently digested with trypsin. The resulting 19 peptide peaks observed by high performance liquid chromatography, which compared with 21 expected tryptic cleavage products, were all isolated, purified, and individually sequenced. Overlap peptides were obtained by a combination of sequencing the N-terminal region of the intact aldolase and by cleaving the intact enzyme with cyanogen bromide followed by subdigestion of the three major cyanogen bromide peptides with either Staphylococcus aureus V8 endoproteinase, endoproteinase Lys C, or trypsin after citraconylation of lysine residues. The primary structure of the molecule was determined to be as follows. (formula; see text) 2-Keto-4-hydroxyglutarate aldolase from E. coli consists of 213 amino acids with a subunit and a trimer molecular weight of 22,286 and 66,858, respectively. No microheterogeneity is observed among the three subunits. The peptide containing the active-site arginine residue (Vlahos, C. J., Ghalambor, M. A., and Dekker, E. E. (1985) J. Biol. Chem. 260, 5480-5485) was also isolated and sequenced; this arginine residue occupies position 49. The Schiff base-forming lysine residue (Vlahos, C. J., and Dekker, E. E. (1986) J. Biol. Chem. 261, 11049-11055) is located at position 133. Whereas the active-site lysine peptide of this aldolase shows 65% homology with the same peptide of 2-keto-3-deoxy-6-phosphogluconate aldolase from Pseudomonas putida, these two proteins in toto show 49% homology.
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D004229:Dithiothreitol; D010450:Endopeptidases; D004926:Escherichia coli / Q000201:enzymology*; D007460:Iodoacetamide; D008239:Lysine; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D007652:Oxo-Acid-Lyases / Q000032:analysis; D010446:Peptide Fragments / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases; D014357:Trypsin
chemical_list: D010446:Peptide Fragments; D001120:Arginine; D002268:Carboxypeptidases; D010450:Endopeptidases; D043422:Carboxypeptidases A; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D007652:Oxo-Acid-Lyases; C021415:4-hydroxy-2-oxoglutarate aldolase; D008239:Lysine; D003488:Cyanogen Bromide; D004229:Dithiothreitol; D007460:Iodoacetamide