W. Plaga et al., Modification of Cys-418 of pyruvate formate-lyase by methacrylic acid, based on its radical mechanism, FEBS LETTER, 466(1), 2000, pp. 45-48
The recently determined crystal structure of pyruvate formate-lyase (PFL) s
uggested a new view of the mechanism of this glycyl radical enzyme, namely
that intermediary thiyl radicals of Cys-418 and Cys-419 participate in diff
erent ways [Becker, A. et al, (1999) Net. Struct, Biol, 6, 969-975]. We rep
ort here a suicide reaction of PFL that occurs with the substrate-analog me
thacrylate with retention of the protein radical (K-I=0.42 mM, k(i)=0.14 mi
n(-1)). Using [1-C-14]methacrylate (synthesized via acetone cyanhydrin), th
e reaction end-product was identified by peptide mapping and cocrystallizat
ion experiments as S-(2-carboxy-(2S)-propyl) substituted Cys-418, The stere
oselectivity of the observed Michael addition reaction is compatible with a
radical mechanism that involves Cys-418 thiyl as nucleophile and Cys-419 a
s H-atom donor, thus supporting the functional assignments of these catalyt
ic amino acid residues derived from the protein structure. (C) 2000 Federat
ion of European Biochemical Societies.