Mechanism and Catalytic Site Atlas

Step 1. The TTP cofactor adopts the V configuration which brings the 4 imino group of the aminopyrimidine ring close to the C2 carbon of the thiazolium ring [PMID:16680160]. Hydrogen bonding of the carboxylate group from Glu59' to N1' of the aminopyrimidine ring increases the basic nature of the 4' position by stabilising the 4'-imino tautomeric form, allowing the group to deprotonate the C1 position of the thiamine ring. As a result, the cofactor is activated towards electrophilic attack.

Step 2. The carbanion of thiamine diphosphate initiates a nucleophilic attack on the carbonyl carbon of pyruvate in an addition reaction. The conjugated double bond system of the cofactor undergoes rearrangement which results in the deprotonation of Glu59'. The 4' enamine tautomer is favoured by the formation of a strong hydrogen bond between the side chain carbonyl group of Glu59' and the N1 hydrogen.

Step 3. The covalently bound pyruvate undergoes decarboxylation.

Step 4. A single electron is transferred from the high energy thamine diphosphate enamine intermediate to the FAD, resulting in bond order rearrangement and deprotonation of the alcohol group present on the intermediate.

Step 5. Tautomerisation of the resulting radical intermediate.

Step 6. The thiamine ring nitrogen acts as an electron sink in the formation of the radical tautomer.

Step 7. Phosphate attacks the kinetically stable anion radical adduct. Kinetics experiments have shown the presence of phosphate to enhance the rate of electron transfer [PMID:19476487]. The negative charge resulting from the nucleophilic attack by phosphate is thought to reduce the potential of the intermediate and therefore increase the driving force of the following electron transfer to FAD.

Step 8. The high energy phosphate radical delivers a second reducing equivalent to the FAD semiquinone.

Step 9. Prior to this step there is a rearrangement of electrons in the TPP-substrate adduct which results in a negative charge on the oxygen atom. The tetrahedral anion intermediate collapses. This forms the high energy metabolite acetyl-phosphate.

Step 10. The FAD diradical transfers one of its electrons to dioxygen with subsequent loss of a single proton.

Step 11. The FADH transfers the second radical and proton to the dioxygen to regenerate the FAD cofactor and hydrogen peroxide.

Step 12. The C2 atom of the thamine ring is exposed to the solvent. Reprotonation of the anion regenerates the TPP cofactor.

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