Reaction kinetics identification for the analysis of reaction mechanisms of ThDP-dependent enzymes

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The elucidation of reaction mechanisms and kinetics for thiamine diphosphat (ThDP)- dependent enzymes in synthetic applications is by far not closed. Although mechanistic postulates exist for a range of enzymes and their respective reactions, there is yet no conclusive information available to deduce and predict the catalytic abilities of different enzymes and correlate them with structural information. The analysis of steady-statereaction kinetics allows suggesting suitable reaction kinetic expressions and the related kinetic parameters and thus the determination of the rate-limiting step. An important feature to achieve kinetic identification is the planning of optimal experiments for model discrimination and parameter estimation. Reaction kinetic identification becomes increasingly complex with increasing number of reactants. In case of 2-hydroxy ketones, two different substrates result in the formation of up to four distinct products, each in two enantiomeric forms. For the identification of reactions of that complexity, suitable experimental and identification strategies will be implemented and verified. The suggested reaction mechanisms are verified using NMR methods and molecular modeling. Objective of this project is to support the differentiation and classification of ThDPdependent enzymes in terms of their ability to transfer the acyl functionalities to different acceptors and thus contribute to the development of an integrated understanding of ThDP reaction range and its application in biocatalytic synthesis.