The Mechanism of Inhibition of Pyruvate Formate Lyase by Methacrylate

Pyruvate Formate Lyase (PFL) catalyzes acetyl transfer from pyruvate to coenzyme a by a mechanism involving multiple amino acid radicals. A post-translationally installed glycyl radical (G₇₃₄· in Escherichia coli) is essential for enzyme activity and two cysteines (C₄₁₈ and C₄₁₉) are proposed to form thiyl radicals during turnover, yet their unique roles in catalysis have not been directly demonstrated with both structural and electronic resolution. Methacrylate is an isostructural analog of pyruvate and an informative irreversible inhibitor of pfl. Here we demonstrate the mechanism of inhibition of pfl by methacrylate. Treatment of activated pfl with methacrylate results in the conversion of the G₇₃₄· to a new radical species, concomitant with enzyme inhibition, centered at g = 2.0033. Spectral simulations, reactions with methacrylate isotopologues, and Density Functional Theory (DFT) calculations support our assignment of the radical to a C2 tertiary methacryl radical. The reaction is specific for C₄₁₈, as evidenced by mass spectrometry. The methacryl radical decays over time, reforming G₇₃₄·, and the decay exhibits a H/D solvent isotope effect of 3.4, consistent with H-atom transfer from an ionizable donor, presumably the C₄₁₉ sulfhydryl group. Acrylate also inhibits PFL irreversibly, and alkylates C₄₁₈, but we did not observe an acryl secondary radical in H₂O or in D₂O within 10 s, consistent with our DFT calculations and the expected reactivity of a secondary versus tertiary carbon-centered radical. Together, the results support unique roles of the two active site cysteines of PFL and a C₄₁₉ S-H bond dissociation energy between that of a secondary and tertiary C-H bond.