Spectroscopic evidence for participation of the 1',4'-imino tautomer of thiamin diphosphate in catalysis by yeast pyruvate decarboxylase

The 1′,4′-iminopyrimidine tautomeric form of the coenzyme thiamin diphosphate (ThDP), implicated in catalysis on the basis of the conformation of enzyme-bound ThDP, has been observed by both ultraviolet absorption and circular dichroism spectroscopy. On yeast pyruvate decarboxylase, the unusual tautomer is observed in an active center variant in which catalysis in the post-decarboxylation regime of the reaction is compromised. In a model system consisting of N1-methyl-4-aminopyrimidinium or N1-methyl-N4- n -butylpyrimidinium salts, on treatment with either NaOH in water, or DBU in DMSO there is an intermediate formed with λ max near 310 nm, and this intermediate reverts back to the starting salt on acidification. Proton NMR chemical shifts are consistent with the intermediate representing the 1-methyl-4-imino tautomer. On the enzyme, the intermediate could be observed by rapid-scan stopped flow with UV detection when reacting holoenzyme of the E477Q active center variant with pyruvate, and by circular dichroism even in the absence of pyruvate. This represents the first direct observation of the imino tautomeric form of ThDP both on the enzyme and in models, although some years ago, this laboratory had already reported some pertinent acid-base properties for its formation [Jordan, F., and Mariam, Y. H. (1978) J. Am. Chem. Soc. 100 , 2534–2541]. The work also represents the first instance in which a rare tautomer implicated in catalysis is identified and suggests that such tautomeric catalysis may be more common in biology than hitherto recognized.