Peptidases kinetically controlled synthesis

Animal tissue is a soiuce of papain-related lysosomal cysteine peptidases that have been used in di- and tripeptide synthesis. Due to the insufficient homogenic preparations of cathepsin B, earlier reports of its synthetic activity have to be taken witii caution [16]. The availability of a recombinant production system for hiunan cathepsin B will allow experiments with pure enzyme. The endopeptidase cathepsin L from parasite Fasciola hepatica was expressed in yeast and used in the kinetically controlled synthesis of Cbz-Phe-Arg-Ser-NH starting from Cbz-Phe-Arg-OMe and H-Ser-NH in an aqueous medimn [31]. 

In contrast to the equilibrium-controlled approach the peptidase-catalyzed kinetically controlled peptide synthesis (for a review see reference1851) needs much less enzyme, the reaction time to reach maximal product yield is significantly shorter, and the product yield depends both on the properties of the enzyme used and the substrate specificity. Kinetic control means that the product appearing with the highest rate and disappearing with the lowest velocity would accumulate. Whereas the equilibrium-controlled approach ends with a true equilibrium, in the kinetic approach the concentration of the product formed goes through a maximum before the slower hydrolysis of the product becomes important. The product will be hydrolyzed if the reaction is not stopped after the acyl donor ester is consumed and true equilibrium is allowed to be reached. 

Figure 12.5-9. Comparison of the equilibrium (a) and the kinetically controlled approach (b) of peptidase-catalyzed peptide synthesis.

Serine and cysteine peptidases are not perfect acyltransferases. Therefore, it is useful to have a method for the prediction of the outcome of the kinetically controlled peptide synthesis. In order to get a simple efficiency parameter we decided to introduce the partition value p11061 analogous with the definition of the Michaelis constant according to Eq. (3), where P2 = Ac-OH, P3 = Ac-N, and N = HN. 

Many related so-called thermolysin-like proteinases (TLPs) from various Grampositive strains have been described [47], including neutral proteases from Bacillus subtilis, and some of these variants are applied in peptide synthesis. Several metal-loenzymes acting as carboxy- or aminopeptidase have also been characterized, but these variants have not been extensively used in peptide synthesis. A bovine carboxy-peptidase A [39] and orange carboxypeptidase C [68] have been applied for dipeptide synthesis in water-organic solvent mixtures, both under thermodynamic and xmder kinetic control. 

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