Equilibrium-controlled synthesis

As discussed above, proteases are peptide bond hydrolases and act as catalysts in this reaction. Consequently, as catalysts they also have the potential to catalyze the reverse reaction, the formation of a peptide bond. Peptide synthesis with proteases can occur via one of two routes either in an equilibrium controlled or a kinetically controlled manner 60). In the kinetically controlled process, the enzyme acts as a transferase. The protease catalyzes the transfer of an acyl group to a nucleophile. This requires an activated substrate preferably in the form of an ester and a protected P carboxyl group. This process occurs through an acyl covalent intermediate. Hence, for kineticmly controlled reactions the eii me must go through an acyl intermediate in its mechanism and thus only serine and cysteine proteases are of use. In equilibrium controlled synthesis, the enzyme serves omy to expedite the rate at which the equilibrium is reached, however, the position of the equilibrium is unaffected by the protease. 

The equilibrium-controlled synthesis (thermodynamic approach) is the direct reversal of proteolysis. For this reason, independent of the individual mechanism, aU proteases can be used. The thermodynamic barrier to peptide bond synthesis is predonoinantly due to the energy required for the transfer of a proton from the positively charged a-annino group to the negatively charged a-carboxy group. The equilibrium constant for tho conversion of the un-ionized reactants into the product is determined by the concentration of the unionized forms in (Scheme 1). 

The two main approaches to glycosidase-catalyzed synthesis of glycosidic linkages involve direct reversal of hydrolysis (equilibrium-controlled synthesis) and trapping of a glycosyl-enzyme intermediate (kinetically-controlled process) [217]. 

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