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

Thiamine diphosphate (ThDP)-dependent enzymes are a group of biocatalysts taking part in numerous biosynthetic pathways and catalysing a broad range of reactions. They are able to form and cleave bonds between carbons and other atoms like hydrogen, oxygen, sulphur and nitrogen. Of special interest are carboligation reactions between two carbon atoms yielding enantiopure 2-hydroxyketones (figure 1), which are important building blocks of drugs and natural products. Aliphatic and aromatic aldehydes can be used as substrates for the enzymatic production of 2-hydroxyketones. From two different substrates up to four different hydroxyketones can be formed (figure 2). The underlying individual reactions share reaction intermediates. Therefore, all individual reaction steps can be illustrated as a complex reaction network.

To optimize such an enzyme process, the complex reaction network needs to be fully characterized. For this purpose, the identification of reaction kinetics is an important tool. Therefore, the reaction is investigated by progress curve experiments and the derived kinetic information is used for the formulation of a mechanistically motivated kinetic model and the accurate estimation of kinetic parameters (figure 3). Based on the estimated kinetic parameters rate-limiting steps of the reaction network are identified and predictions about the catalytic abilities of different ThDP-dependent enzymes are enabled.

Figure 1: Suggested general reaction schemes for carboligations using ThDP-dependent enzymes

 

 

Figure 2: Competing single reactions of the carboligation using ThDP-enzymes. A, B = different aldehydes (substrate); AA, AB, BA, BB = 2-hydroxyketones (products)

 

 

Figure 3: Reaction kinetic identification workflow