Reaction Performance Medium Engineering and Kinetics

As pointed out before, the mechanism of aldolase-catalyzed aldol addition is different for class I and class II enzymes. 

For class II DHAP-dependent aldolases, an ordered two-substrate mechanism has been derived from structural studies (Dreyer and Shulz 1996). DHAP binds the zinc ion with its hydroxyl and keto oxygen atoms, conducting to an enediolate intermediate, before the entrance of the acceptor aldehyde. For instance, FucA and RhuA are homotetramers with a Zn + atom in each subunit and the formed enediolate is linked to the carbonyl group of the acceptor aldehyde by either the si face (in the case of FucA) or the re face (in the case of RhuA). Thus, aldol addition products with 3R, 4R configuration are obtained in the case of FucA, and 3R, 4S configuration in the case of RhuA (Fig. 6.5.3). 

As mentioned before, these enzymes are strictly dependent of DHAP as donor substrate, and DHAP is usually unstable at the reaction conditions (Fessner and Walter 1997). In aqueous medium, DHAP suffers decomposition and isomerization reactions as shown in Fig. 6.5.4. 

The reactions proceed through the formation of an enediolate intermediate and it has been demonstrated that there are only two final reaction products inorganic phosphate and a small amount of methylglyoxal due to its polymerization (Richard 1993). The irreversible DHAP decomposition has been shown to be of first order in a pH range between 7 and 9 (Phillips and Thornalley 1993), and becomes almost negligible for temperatures lower than 4 °C. On the other hand, as the decomposition 

for an aldol addition reaction, the significance of non-synthetic DHAP decomposition has to be taken into account. In order to improve the yield and selectivity, different approaches have been employed a) temperature reduction, exploiting the different temperature dependence of the reaction rates as a consequence of the different magnitudes of their energies of activation b) use of a high aldehyde excess to drive the process toward synthesis. 

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