The determination of pyruvate formate lyase crystallographic structure brought new insights to its mechanism of reaction and presented the possibility of a direct attack to the substrate from cysteine 418 as opposed to the previously expected cysteine 419. An inhibition study performed by Knappe and co-workers, using substrate-analogue methacrylate, confirms that cysteine 418 is most likely to add directly to pyruvate, since an inhibition product has been found as a substituent in this residue. The work presented here consists of a study of the inhibition mechanism of pyruvate formate lyase by methacrylate, using density functional theory with the hybrid B3LYP functional. We were able to determine all pertinent structures, confirm the proposed experimental mechanism, and add important detail to the energy profile associated with the mechanism of inhibition. Additionally, the obtained results provide the energy values for both the chemical reaction and the stereochemical reorganization necessary in order for the thiol-methacrylate adduct to come within reactional reach of Cys419.