Direct spectroscopic observation of a Brewer's yeast pyruvate decarboxylase-bound enamine intermediate produced from a suicide substrate. Evidence for nonconcerted decarboxylation

  • Kuo D
  • Jordan F
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The conjugated alpha-keto acid (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid, a suicide substrate (Kuo, D. J., and Jordan, F. (1983) Biochemistry 22, 3735-3740), when reacted with brewer's yeast pyruvate decarboxylase (EC, was found to produce a new absorption band centered at 440 nm. The band was attributed to the formation of a thiamindiphosphate-bound intermediate produced upon decarboxylation and was not observed in the absence of either thiamindiphosphate or apoprotein. Simultaneously, with the appearance of the spectral band, the enzyme was inactivated irreversibly. The combined evidence suggested that the spectral band pertains to an enzyme-bound conjugated enamine and the results constitute the first direct observation of such an intermediate in any thiamindiphosphate requiring enzymatic reaction. It could be concluded that for the alpha-keto acid employed, CO2 loss and C-protonation of the enamine take place in a stepwise, rather than concerted, manner and that the steps culminating in CO2 loss are faster overall than the subsequent steps that lead to the release of the product. The observation of stoichiometric concentration of enzyme-bound enamine intermediate supports the previous suggestion that alpha-hydroxyethyl thiamindiphosphate is not the true intermediate, but rather the enamine is (Ullrich, J., and Mannshreck, A. (1967) Eur. J. Biochem 1, 110-116).

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  • ISSN: 00219258
  • PUI: 14204537
  • PMID: 6417127
  • SGR: 0021048023
  • SCOPUS: 2-s2.0-0021048023


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