title: Hydrogen exchange of the glycyl radical of pyruvate formate-lyase is catalyzed by cysteine 419.
abstract: Pyruvate formate-lyase (PFL) catalyzes the reversible conversion of CoA and pyruvate into acetyl-CoA and formate. Active enzyme contains a glycyl radical whose alpha-hydrogen undergoes rapid exchange with solvent (t1/2 approximately 5 min at 0 degree C). We have investigated this exchange using site-directed mutagenesis and mechanism-based inactivation. Mutation of the active-site cysteine 419 into a serine, which renders the enzyme catalytically inactive, abolishes alpha-hydrogen exchange in the radical. This suggests that the exchange process is not an intrinsic property of the glycyl radical but is a consequence of its interaction with cysteine 419. This residue is also demonstrated to be involved in the transfer of the radical to acetylphosphinate, a mechanism-based inactivator of the enzyme. In contrast, mutation of the other essential cysteine 418 to a serine has no effect on the hydrogen exchange or the transfer of the radical to acetylphosphinate. A mechanism for the hydrogen exchange catalyzed by cysteine 419 consistent with a redox role for this residue in the normal catalytic reaction is proposed.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
authors.forename: C V
authors.initials: CV
authors.lastname: Parast
authors.forename: K K
authors.initials: KK
authors.lastname: Wong
authors.forename: S A
authors.initials: SA
authors.lastname: Lewisch
authors.forename: J W
authors.initials: JW
authors.lastname: Kozarich
authors.forename: J
authors.initials: J
authors.lastname: Peisach
authors.forename: R S
authors.initials: RS
authors.lastname: Magliozzo
mesh_terms: D000123:Acetyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D003903:Deuterium; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005609:Free Radicals; D005998:Glycine / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D007474:Ion Exchange; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction
chemical_list: D005609:Free Radicals; D006859:Hydrogen; D003903:Deuterium; D000123:Acetyltransferases; C022560:formate C-acetyltransferase; D003545:Cysteine; D005998:Glycine