Parast C V, Wong K K, Lewisch S A, Kozarich J W, Peisach J, Magliozzo R S
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Biochemistry. 1995 Feb 28;34(8):2393-9. doi: 10.1021/bi00008a001.
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.
