Pyruvate kinase: studies on affinity labeling and active-site structure using the rabbit muscle enzyme.
Important advances have been made in recent years in the study of the structure of pyruvate kinase: the amino acid sequence of the enzymes from chicken muscle and yeast have been established and the three-dimensional structure of the cat muscle enzyme has been determined at 0.26 nm resolution. Work in our laboratory has shown that dialdehyde-ADP (oADP) can be used as an affinity label of rabbit muscle pyruvate kinase: if the enzyme is incubated with cold oADP in the presence of high ADP concentrations, dialyzed and then incubated with 14C-oADP, the enzyme inactivates and one mole of radioactive oADP incorporates per mole of enzyme subunit. A labeled peptide with a molecular weight of about 5900 has been purified from a tryptic digest of the modified enzyme. The first 26 residues of the peptide have been sequenced and this sequence is identical to a region in the chicken muscle enzyme and a peptide isolated from the bovine muscle enzyme specifically labeled with trinitrobenzenesulfonate. High homology is also found with a region of the yeast enzyme. All this suggests that the isolated peptide is part of the active site; the modified amino acid, probably a lysine, seems to be located in one of the alfa helices of domain A of the enzyme, according to the x-ray data.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D010455:Peptides; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits
D000345:Affinity Labels; D010455:Peptides; D000244:Adenosine Diphosphate; D011770:Pyruvate Kinase