Spectroscopic studies and synthetic applications of thiamin diphosphate dependent enzymes

Thiamin diphosphate (ThDP) is an important cofactor in many metabolic pathways such as the oxidative and non-oxidative decarboxylation of alpha-keto acids. ThDP-dependent enzymes share a common mechanism. It starts with the deprotonation of the C2-H of the thiazolium ring of ThDP which forms the ylid that attacks the carbonyl carbon on pyruvate, yielding the first tetrahedral adduct C2alpha-lactyl-ThDP (LThDP). Decarboxylation forms the enamine or the 2-alpha-carbanion and its protonation yields C2alpha-hydroxyethyl-ThDP (HEThDP).; The 4'-aminopyrimidine group of ThDP undergoes tautomerization to the 1',4'-imino form. Appropriate compounds were synthesized to create models for the 1',4'-imino tautomer of the 4'-aminopyrimidine ring of ThDP. To help with the 15N chemical shift assignments, the model compounds were specifically labeled with 15N at the amino nitrogen atom. The 15N chemical shift for the 1,4-iminopyrimidine tautomer should serve as useful guides to the assignment of enzyme-bound signals. Michaelis complex is an intermediate resulting from a non-covalent interaction involving ThDP. It was shown earlier by our group that it gives rise to an electronic transition near 320--330 nm, readily detected by circular dichroism. Chemical models created showed that this band corresponds 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.; In addition to the decarboxylation of 2-oxo acids, thiamin diphosphate (ThDP)-dependent decarboxylases/dehydrogenases can also carry out so-called carboligation reactions, where the central ThDP-bound enamine intermediate reacts with an electrophilic substrate/product. In this thesis we report detailed analysis of the stereoselectivity for forming the carboligase products acetoin, acetolactate, and phenylacetylcarbinol by yeast pyruvate decarboxylase and pyruvate dehydrogenase complex E1 subunit (from Escherichia coli) active-center variants. The enantiomeric excess was analyzed by a combination of NMR, circular dichroism and chiral-column gas chromatographic methods. The different stereoselectivities exhibited by the two enzymes could be utilized in the chiral synthesis of important intermediates.; We have synthesized acetylphosphinate (AP) and acetyl methylphosphinate (AMP) as close substrate analogues which form the first LThDP-like tetrahedral intermediate and methyl benzoylphosphonate, which forms the phosphonomandelylthiamin diphosphate intermediate. We have studied the formation and different tautomeric forms of the 4'-aminopyrimidine ring of LThDP and phosphonomandelylthiamin diphosphate like intermediates by circular dichroism, stopped-flow CD and stopped-flow photo diode array on yeast pyruvate decarboxylase, dehydrogenase complex E1 subunit, pyruvate oxidase and benzoylformate decarboxylase.