1995
DOI: 10.1021/bi00008a001 View full text |Buy / Rent full text
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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… Show more

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“…H/D exchange at Gly-734 requires that this residue be in close proximity to Cys-419, which mediates the exchange via reversible H atom abstraction (supplemental Scheme S3). When PFL is activated with catalytic amounts of PFL-AE, the observed H/D exchange is consistent with that previously reported (13). However, as the ratio of PFL-AE to PFL is increased to 0.1 and 1.0, the extent of H/D exchange at short incubation times decreases, as indicated by the decreased collapse of the Gly-734 radical doublet signal to a singlet.…”
Section: Discussionsupporting
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“…H/D exchange at Gly-734 requires that this residue be in close proximity to Cys-419, which mediates the exchange via reversible H atom abstraction (supplemental Scheme S3). When PFL is activated with catalytic amounts of PFL-AE, the observed H/D exchange is consistent with that previously reported (13). However, as the ratio of PFL-AE to PFL is increased to 0.1 and 1.0, the extent of H/D exchange at short incubation times decreases, as indicated by the decreased collapse of the Gly-734 radical doublet signal to a singlet.…”
Section: Discussionsupporting
“…(2, 3, 13). The active site residue Cys-419 has been demonstrated to be essential for the H/D exchange at Gly-734 because the mutant C419S-PFL does not undergo H/D exchange (13). These results provide evidence that the Gly-734 radical in activated PFL abstracts H ⅐ from Cys-419 to generate a thiyl radical, which can subsequently abstract a hydrogen from Gly-734 to regenerate the glycyl radical; this reversible H ⅐ abstraction involving a species with an exchangeable hydrogen (Cys-419) results in deuterium exchange into the Gly-734 (supplemental Scheme S3).…”
Section: Stability Of the Pfl Glycyl Radical Is Affected By The Presementioning
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“…The L. lactis sequence ISCCVSP (residues 414 to 420 [ Fig. 4]) is highly conserved and includes two adjacent Cys residues that have been implicated in the cleavage of the C-C bond of pyruvate and hydrogen exchange of the glycyl radical of the enzyme (25).…”
Section: Resultsmentioning
“…In its active form, this enzyme contains a stable glycyl radical (Gly-734) required for catalysis (2). Studies of mutants and substrate analogs propose that on substrate binding, the spin is transferred from Gly-734 to the reaction center (Cys-418/Cys-419), where a thiyl radical initiates the homolytic cleavage of the pyruvate C-C bond (3,4).…”
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