Identification of cysteine as the reactive group in pyruvate kinase alkylated by 5-chloro-4-oxopentanoic acid.
4-Hydroxypentanoic acid alanine thioether was synthesized and characterized by n.m.r. spectroscopy. This derivative corresponded to the modified amino acid obtained by allowing 5-chloro-4-oxo[3,5-3H]pentanoic acid to react with rabbit muscle pyruvate kinase. Performic acid oxidation of 4-oxo[3,5-3H]pentanoic acid alanine thioether in pyruvate kinase gave [3H]succinate (67%) and [3H]carboxymethylcysteine (33%) as expected. Evidence is presented to show that NaBH4 reduction followed by periodate oxidation and analysis of radioactive formaldehyde production may provide a convenient method for distinguishing between thiol and amino alkylation by halogenomethyl ketone compounds. Peptide 'mapping' confirms that the modification by 5-chloro-4-oxopentanoic acid occurs primarily at one region of pyruvate kinase.
D000478:Alkylation; D000600:Amino Acids, Dicarboxylic / Q000032:analysis / Q000138:chemical synthesis; D000603:Amino Acids, Sulfur / Q000032:analysis / Q000138:chemical synthesis; D000818:Animals; D001894:Borohydrides; D003545:Cysteine / Q000032:analysis*; D005561:Formates; D007651:Keto Acids; D007982:Levulinic Acids; D010084:Oxidation-Reduction; D010421:Pentanoic Acids; D010455:Peptides / Q000032:analysis; D010504:Periodic Acid; D011770:Pyruvate Kinase; D011817:Rabbits; D014631:Valerates
D000600:Amino Acids, Dicarboxylic; D000603:Amino Acids, Sulfur; D001894:Borohydrides; D005561:Formates; D007651:Keto Acids; D007982:Levulinic Acids; D010421:Pentanoic Acids; D010455:Peptides; D014631:Valerates; D010504:Periodic Acid; C013439:5-chloro-4-oxopentanoic acid; D011770:Pyruvate Kinase; D003545:Cysteine