title: 4-Chloroacetylpyridine adenine dinucleotide. A highly reactive and chromophoric affinity label of glyceraldehyde-3-phosphate dehydrogenase from sturgeon.
abstract: The analogue of NAD+, 4-chloroacetylpyridine-adenine dinucleotide (clac4PdAD+), inactivated the glyceraldehyde-3-phosphate dehydrogenase from sturgeon at a high rate. An affinity labeling was shown to occur with clac4PdAD+. The mononucleotide 4-chloroacetylpyridine 1-beta-D-ribose 5'-phosphate (clac4PdMN+) reacted with the enzyme in a second-order reaction whose rate was much smaller than that calculated for clac4PdAD+ taken as a second-order rate reagent. The rate of the reaction of clac4PdAD+ with the enzyme was determined by stopped flow, using as a probe the long-wavelength absorption maximum (430 nm) formed concomitantly with inactivation of the enzyme. Computer-assisted graphic simulation showed that the clac4PdAD+ analogue could bind to the active site of the enzyme from Bacillus stearothermophilus in a similar manner to that of NAD+, and that the reactive carbon and the reactive thiolate of Cys-149 were within bonding distance. The absorption at 430 nm was linearly proportional to the substoichiometric concentration of clac4PdAD+/mole subunit. Thiol titration suggested the modification of one thiol residue per subunit. The modified thiol was identified by degradation as Cys-149. In contrast to the absorption band generated during the reaction of the 3-chloroacetylpyridine-adenine dinucleotide (clac3PdAD+) with the same enzyme [Eur. J. Biochem. (1982) 127, 519-524; 129, 437-446], enzyme inactivated with clac4PdAD+ and clac4PdMN+ exhibited an absorption maximum at long wavelength which was still present after denaturation. The chromophore is proposed to be the enol form of the alpha-thioether ketone produced by alkylation of the thiolate of Cys-149 by the chloroacetyl group.
authors.affiliation: Laboratoire de Chimie Organique Biologique, UA-CNRS, Institut de Chimie, Université Louis Pasteur, Strasbourg, France.
authors.forename: D
authors.initials: D
authors.lastname: Tritsch
authors.forename: B
authors.initials: B
authors.lastname: Eiler-Samama
authors.forename: J
authors.initials: J
authors.lastname: Svircevic
authors.forename: A M
authors.initials: AM
authors.lastname: Albrecht
authors.forename: G
authors.initials: G
authors.lastname: Branlant
authors.forename: J F
authors.initials: JF
authors.lastname: Biellmann
mesh_terms: D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D005399:Fishes; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D009132:Muscles / Q000201:enzymology; D009243:NAD / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D010449:Peptide Mapping; D011487:Protein Conformation; D013053:Spectrophotometry
chemical_list: D000345:Affinity Labels; D009243:NAD; C032564:3-chloroacetylpyridine-adenine dinucleotide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases