Modification of Cys-418 of pyruvate formate-lyase by methacrylic acid, based on its radical mechanism.
The recently determined crystal structure of pyruvate formate-lyase (PFL) suggested a new view of the mechanism of this glycyl radical enzyme, namely that intermediary thiyl radicals of Cys-418 and Cys-419 participate in different ways [Becker, A. et al. (1999) Nat. Struct. Biol. 6, 969-975]. We report here a suicide reaction of PFL that occurs with the substrate-analog methacrylate with retention of the protein radical (K(I)=0.42 mM, k(i)=0.14 min(-1)). Using [1-(14)C]methacrylate (synthesized via acetone cyanhydrin), the reaction end-product was identified by peptide mapping and cocrystallization experiments as S-(2-carboxy-(2S)-propyl) substituted Cys-418. The stereoselectivity of the observed Michael addition reaction is compatible with a radical mechanism that involves Cys-418 thiyl as nucleophile and Cys-419 as H-atom donor, thus supporting the functional assignments of these catalytic amino acid residues derived from the protein structure.
Plaga
W
W
Biochemie-Zentrum Heidelberg (BZH), Ruprecht-Karls-Universität, Im Neuenheimer Feld 501, 69120, Heidelberg, Germany.
Vielhaber
G
G
Wallach
J
J
Knappe
J
J
D000123:Acetyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D005609:Free Radicals / Q000737:chemistry; D007700:Kinetics; D008689:Methacrylates / Q000494:pharmacology; D008956:Models, Chemical; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism
D004791:Enzyme Inhibitors; D005609:Free Radicals; D008689:Methacrylates; D011994:Recombinant Proteins; C008384:methacrylic acid; D000123:Acetyltransferases; C022560:formate C-acetyltransferase; D003545:Cysteine