Substrate specificity, acyl transfer, ester

In contrast to the equilibrium-controlled approach which ends with a true equUibrium, in the protease-catalyzed kinetically controlled synthesisf l the product appearing with the highest rate and disappearing with the lowest velocity would accumulate. This approach requires the use of acyl donor esters as carboxy components (Ac-X) and is limited to proteases which rapidly form an acyl-enzyme intermediate (Ac-E). Serine and cysteine proteases are known to catalyze acyl transfer from specific substrates to various nucleophihc amino components via an acyl-enzyme intermediate. In reactions of this type, the protease reacts rapidly with an amino acid or peptide ester, Ac-X, to form a covalent acyl-enzyme intermediate, Ac-E, that reacts, in competition with water, with the amino acid or peptide-derived nucleophile HN to form a new peptide bond (Scheme 3). The partitioning of the acyl-enzyme intermediate between water and the added nucleophile is the rate-limiting step. Under kinetic control, and if k4[HN] k3[H20], the peptide product Ac-N should accumulate. However, the soluble peptide product will be degraded if the reaction is not terminated after the acyl donor ester is consumed. 

It is known in the literature that some enzymes can catalyze the acylation reaction of polysaccharides (9,10). Not surprisingly, lipases can serve this function (9) because lipases are known to mediate ester hydrolysis or synthesis. Yet, some proteases can also catalyze this reaction (10). For example, Alcalase protease immobilized on Celite can transfer the acrylic group onto hydroxy-ethylcellulose (HEC) to form an ester(9a) (Figure 2). Thus, in this case Alcalase appears to have broad substrate specificity and can work well with esters. 

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