2.7.3. Products in General Base-Catalyzed Reactions of HBnT 2.7.4. Fragmentation Products of the C2R Conjugate Base of HBnT 2.7.5. Avoiding Fragmentation on the Enzyme 2.8. The Reactivity of R-Mandelyl-thiamin (MT) 2.8.1. Synthesis of MT 2.8.2. Decarboxylation of MT 2.8.3. Pyridinium Catalysis of Decarboxylation of MT 2.8.4. Internal Return of CO 2 2.8.5. Overcoming the Return of CO 2 2.8.6. Competing with the Internal Return of Carbon Dioxide 3. Spectroscopic Detection of Covalent Intermediates in TDP Enzymes 3.1. Chemical Quench and Analysis of Covalent TDP Intermediates by 1 H NMR 3.1.1. General Considerations in the Use of Quenching 3.1.2. Kinetic Analysis of Steady-State Intermediate Distributions 3.1.3. Acid-Quench Analysis of Covalent TDP Intermediates Deriving from Pyruvate 3.1.4. Acid Quench-NMR Studies in Transketolase 3.2. Direct Spectroscopic Observation of the Enamine Intermediate 3.3. Spectroscopic Detection, Electronic and X-ray Structures of Radical Intermediates in TDP Enzymes 3.3.1. Pyruvate:Ferredoxin Oxidoreductase 3.3.2. Pyruvate Oxidases 3.3.3. Pyruvate Dehydrogenase Complex 3.3.4. Acetohydroxyacid Synthase and Glyoxylate Carboligase 3.4. Different Protonation States of the TDP Aminopyrimidine in the Course of Catalysis and Spectroscopic Signatures 4. Structural Studies on Covalent TDP Intermediates on Enzymes 4.1. Structures of Covalent TDP Intermediates in Pyruvate Oxidase 4.2. Structural Studies on Covalent TDP Intermediate Analogues 4.3. Structural Studies on Transketolase 4.3.1. Relations between Transketolases and Transaldolases/Aldolases 4.4. Pyramidal Carbanion or Planar Enamine?

Enzymic catalysis proceeds via intermediates formed in the course of substrate conversion. Here, we directly detect key intermediates in thiamin diphosphate (ThDP)-dependent enzymes during catalysis using (1)H NMR spectroscopy. The quantitative analysis of the relative intermediate concentrations allows the determination of the microscopic rate constants of individual catalytic steps. As demonstrated for pyruvate decarboxylase (PDC), this method, in combination with site-directed mutagenesis, enables the assignment of individual side chains to single steps in catalysis. In PDC, two independent proton relay systems and the stereochemical control of the enzymic environment account for proficient catalysis proceeding via intermediates at carbon 2 of the enzyme-bound cofactor. The application of this method to other ThDP-dependent enzymes provides insight into their specific chemical pathways.