Abstract
Pyruvate formate-lyase (PPL) catalyzes the reversible conversion of CoA and pyruvate into acetyl-CoA and formate. Active enzyme contains a glycyl radical whose α-hydrogen undergoes rapid exchange with solvent (t1/2 ~ 5 min at 0 °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 -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.
Original language | English |
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Pages (from-to) | 2393-2399 |
Number of pages | 7 |
Journal | Biochemistry |
Volume | 34 |
Issue number | 8 |
DOIs | |
State | Published - Feb 1995 |