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A Computational Study of Substrate Mechanism of Pyruvate-Formate Lyase

High-level quantum mechanical calculations have been performed on small model systems relevant to the substrate mechanism of Pyruvate Formate-Lyase (PFL), comparing the reactivity of the natural substrate pyruvate with that of the known inhibitor oxamate. For both substrates the presently accepted (consensus) mechanism, involving the addition of a cystine-based thiyl radical to the carbonyl carbon of the substrate, is compared to an alternative mechanism involving hydrogen abstraction as the primary step. This mechanism, relevant because of the known structural homology between PFL and the ribonucleotide reductase family of enzymes, is found to display similar reaction barriers to the consensus mechanism, but is much less favorable with respect to the reaction energetics. The inhibitory effect of oxamate can be traced back to an increase in reaction barrier and reaction energy along the consensus mechanism pathway. The high-level quantum results have been subsequently used as benchmarks to examine the suitability of applying a coupled quantum-mechanical/molecular mechanical (QM/MM) approach to this system. This comparison shows that while the QM/MM technique gives reaction barriers and enthalpies in good agreement with the complete quantum treatment, some caution must be exercised for properties such as complexation energies. DFT also encounters some difficulties in determining these complexation energies, possibly related to difficulties with dispersion forces.

PFL; glycyl radical enzyme; substrate mechanism; QM/MM

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