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) Net. 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-C-14]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. (C) 2000 Federation of European Biochemical Societies.