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Modification of Cys‐418 of pyruvate formate‐lyase by methacrylic acid, based on its radical mechanism
Author(s) -
Plaga Wulf,
Vielhaber Gabriele,
Wallach Jochen,
Knappe Joachim
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)01752-4
Subject(s) - chemistry , nucleophile , lyase , radical , stereochemistry , substrate (aquarium) , catalysis , reaction mechanism , formate , enzyme , organic chemistry , oceanography , geology
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‐(2 S )‐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.