Amino acid sequence of the pyruvate and the glyoxylate active-site lysine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase.
Pure 2-keto-4-hydroxyglutarate aldolase of Escherichia coli, a "lysine-type" trimeric enzyme which has the unique properties of forming an "abortive" Schiff-base intermediate with glyoxylate (the aldehydic product/substrate) and of showing strong beta-decarboxylase activity toward oxalacetate, binds any one of its substrates (2-keto-4-hydroxyglutarate, pyruvate, or glyoxylate) in a competitive manner. To determine whether the substrates bind at the same or different (juxta-positioned) sites and what degree of homology might exist between the active-site lysine peptide of this enzyme and that of other lysine-type (Class I) aldolases or beta-decarboxylases, the azomethine formed separately by this aldolase with either [14C]pyruvate or [14C]glyoxylate was reduced with CNBH3-. After each enzyme adduct was digested with trypsin, the 14C-labeled peptide was isolated, purified, and subjected to amino acid analysis and sequence determination. In each case, the same 14-amino acid lysine-peptide was isolated and found to have the following primary sequence: Glu-Phe-*Lys-Phe-Phe-Pro-Ala-Glu-Ala-Asn-Gly-Gly-Val-Lys (where * = the active-site lysine). Hence, glyoxylate competes for, and inhibits aldolase activity by reacting with, the one active-site lysine residue/subunit. This active-site lysine peptide has a high degree (65%) of homology with that of 2-keto-3-deoxy-6-phosphogluconate aldolase of Pseudomonas putida but is not similar to that of any Class I fructose-1,6-bisphosphate aldolase or of acetoacetate beta-decarboxylase of Clostridium acetobutylicum. Furthermore, it was found that extensive reaction of glyoxylate with the N-terminal amino group of this enzyme may well be general complicating factor in sequence studies with proteins plus glyoxylate.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D001894:Borohydrides / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006038:Glyoxylates / Q000378:metabolism; D008239:Lysine; D008956:Models, Chemical; D007652:Oxo-Acid-Lyases / Q000032:analysis / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011773:Pyruvates / Q000378:metabolism*; D019289:Pyruvic Acid; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D001894:Borohydrides; D006038:Glyoxylates; D010446:Peptide Fragments; D011773:Pyruvates; D019289:Pyruvic Acid; D014357:Trypsin; D007652:Oxo-Acid-Lyases; C021415:4-hydroxy-2-oxoglutarate aldolase; C031150:glyoxylic acid; D008239:Lysine