Cysteinyl peptides labeled by dibromobutanedione in reaction with rabbit muscle pyruvate kinase.
The bifunctional reagent 1,4-dibromobutanedione (DBBD) reacts covalently with pyruvate kinase from rabbit muscle to cause inactivation of the enzyme at a rate that is linearly dependent on the reagent concentration, giving a second order rate constant of 444 min-1 M-1. The individual substrates phosphoenolpyruvate (with KCl), ADP, or ATP in the presence of divalent metal cation provide marked protection against inactivation suggesting that reaction occurs in the region of the active site. The limited incorporation of DBBD into pyruvate kinase was measured by reduction of the carbonyl groups of the enzyme-bound reagent using [3H]NaBH4. When pyruvate kinase was reacted with 120 microM DBBD at pH 7.0 for 50 min in the absence of protectants, 1.8 mol of tritium/mol of subunit was incorporated, whereas in the presence of phosphoenolpyruvate with KCl, only 1.0 mol of tritium was incorporated per mole of subunit. Modified peptides were isolated from tryptic digests of pyruvate kinase. Reaction of enzyme in the presence of substrate (showing no activity loss) yielded a single peptide, Asn-Ile-X1-Lys, where X1 corresponds to Cys164 of the known amino acid sequence of muscle pyruvate kinase. In the absence of protectants, reaction for 10 min (when the enzyme retained substantial activity) yielded Asn-Ile-X1-Lys as the major labeled peptide, whereas reaction for 50 min (when the enzyme was 88% inactivated) yielded predominantly Asn-Ile-X1-Lys cross-linked to X2-Asp-Glu-Asn-Ile-Leu-Trp-Leu-Asp-Tyr-Lys, where X2 corresponds to Cys151. Because activity loss correlates with the appearance of the cross-linked peptides but not with formation of Asn-Ile-X1-Lys, inactivation is likely caused by the reaction leading to the cross-link between Cys151 and Cys164. The distance between the alpha-carbons of these residues in the crystal structure is 15.5 A, whereas only 12.0 A can be spanned by the two side chains linked by a dioxobutyl group, suggesting either that pyruvate kinase undergoes a conformational change in forming the cross-link or that local rapid fluctuations in structure occur in solution to the extent of 3.5 A in this region of pyruvate kinase.
Vollmer
S H
SH
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Colman
R F
RF
D000595:Amino Acid Sequence; D000818:Animals; D002074:Butanones / Q000737:chemistry* / Q000494:pharmacology; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry*; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011817:Rabbits; D013439:Sulfhydryl Reagents
D002074:Butanones; D003432:Cross-Linking Reagents; D008024:Ligands; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; C081487:1,4-dibromobutanedione; D011189:Potassium Chloride; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase; D003545:Cysteine