title: Subunit level crosslinking of rabbit muscle pyruvate kinase by o-phthaldialdehyde.
abstract: o-Phthaldialdehyde caused irreversible inhibition of rabbit muscle pyruvate kinase following preliminary formation of an enzyme-reagent complex. At pH 7.5, 35 degrees C, the dissociation constant for the complex and the maximal pseudo-first-order rate constant for covalent modification were 0.32 +/- 0.08 mM and 2.54 +/- 0.23 min-1, respectively. The inactivation was accompanied by uv-spectral changes pointing to isoindole formation, with a limiting stoichiometry of 1 isoindole linkage per enzyme subunit. Phosphoenolpyruvate, ADP, and ATP effectively protected the enzyme against inactivation, suggesting that the active site is the target of o-phthaldialdehyde action. As native and modified enzymes were indistinguishable with respect to mobility of the major band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was concluded that the crosslinkage was intrasubunit in character, and that the amino acid residues involved must be closely positioned in the polypeptide backbone. Lysine 366, previously shown to be selectively reactive toward 2',3'-dialdehyde ADP (Bezares et al., 1987, Arch, Biochem. Biophys. 253, 133-137), and cysteine 325 or 357 are implicated.
authors.affiliation: Department of Biochemistry, School of Pharmacy, Hacettepe University, Ankara, Turkey.
authors.forename: S
authors.initials: S
authors.lastname: Yilmaz
authors.forename: I
authors.initials: I
authors.lastname: Ozer
mesh_terms: D000447:Aldehydes / Q000493:pharmacokinetics*; D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000493:pharmacokinetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D007527:Isoenzymes / Q000378:metabolism*; D009132:Muscles / Q000187:drug effects / Q000201:enzymology*; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity; D009764:o-Phthalaldehyde / Q000493:pharmacokinetics*
chemical_list: D000447:Aldehydes; D003432:Cross-Linking Reagents; D007527:Isoenzymes; D009764:o-Phthalaldehyde; D011770:Pyruvate Kinase