Tetrahedral Intermediates in Thiamin Diphosphate-Dependent Decarboxylations Exist as a 1'4'-Imino Tautomeric Form of the Coenzyme,Unlike the Michaelis Complex or the Free Coenzyme

摘要

Two circular dichroism signals observed on thiamin diphosphate (ThDP)-dependent enzymes,a positive band in the 300-305 nm range and a negative one in the 320-330 nm range,were investigated on yeast pyruvate decarboxylase (YPDC) and on the El subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-El).Addition of the tetrahedral ThDP-acetaldehyde adduct,2-a-hydroxyethylThDP,to PDHc-El generates the positive band at 300 nm,consistent with the formation of the 1',4'-iminopyrimidine tautomer,as also demonstrated for phosphonolactylthiamin diphosphate,a stable analogue of the tetrahedral ThDP-pyruvate adduct 2-alpha-lactylThDP (Jordan,F.et ak (2003) J.Am.Chem.Soc.725,12732-12738).Therefore,we suggest that all tetrahedral ThDP-bound covalent complexes will also prefer this tautomer,and that the 4'-aminopyrimidine of ThDP participates in multiple steps of acid-base catalysis on ThDP enzymes.Studies with YPDC and PDHc-El,and their active center variants,in conjunction with chemical models,enabled assignment of the negative band at 330 nm to a charge-transfer transition between the 4'-aminopyrimidine tautomer (presumed electron donor) and the thiazolium ring (presumed electron acceptor) of ThDP,with no significant contributions from any amino acid side chain of the proteins.However,in both YPDC and PDHc-El,the presence of substrate or substrate surrogate was required to enable detection,suggesting that the band at 320-330 nm be used as a reporter for the Michaelis complex,involving the amino tautomer,on both enzymes.As the positive band near 300 nm reports on the 1',4'-imino tautomer of ThDP,methods are now available for kinetic monitoring of both tautomeric forms.