Peptidases catalyzed peptide synthesis

It is interesting to note that serine peptidases can, under special conditions in vitro, catalyze the reverse reaction, namely the formation of a peptide bond (Fig. 3.4). The overall mechanism of peptide-bond synthesis by peptidases is represented by the reverse sequence f-a in Fig. 3.3. The nucleophilic amino group of an amino acid residue competes with H20 and reacts with the acyl-enzyme intermediate to form a new peptide bond (Steps d-c in Fig. 3.3). This mechanism is not relevant to the in vivo biosynthesis of proteins but has proved useful for preparative peptide synthesis in vitro [17]. An interesting application of the peptidase-catalyzed peptide synthesis is the enzymatic conversion of porcine insulin to human insulin [18][19]. 

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

In peptidase-catalyzed peptide synthesis the solubility of the starting components dramatically influences the course of the synthesis. From the ideal medium, water, the spectrum of solvents ranges from water-miscible organic solvents and aqueous-organic biphasic systems to monophasic organic solvents with trace amounts of... 

Figure 12.5-13. Influence of the reaction medium on peptidase-catalyzed peptide synthesis.

Enzymes of the pepsin family rarely catalyze the hydrolysis of esters, with the exceptions of, for example, esters of L-/3-penicillactic acid and some sulfinic acid esters. Under suitable conditions, i. e., low pH, high enzyme concentration, and formation of an insoluble peptide, aspartic peptidases are able to catalyze the synthesis of peptides [71] [72],... 

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. 

As a rule, peptidases can only make a meaningful contribution to a synthesis strategy if the full advantagee of the enzymatic reactions can be utilized. An a priori completely unrealistic position is the comparison of a stepwise peptidase-catalyzed assembley of a peptide chain with the automatic solid-phase technique. 

Fig. 3.3. Major steps in the hydrolase-catalyzed hydrolysis of peptide bonds, taking chymo-trypsin, a serine hydrolase, as the example. Asp102, His57, and Ser195 represent the catalytic triad the NH groups of Ser195 and Gly193 form the oxyanion hole . Steps a-c acylation Steps d-f deacylation. A possible mechanism for peptide bond synthesis by peptidases is represented by the reverse sequence Steps f-a.

Notwithstanding that the free energy of formation of peptide bonds from free amino acids corresponds to an equilibrium position in dilute solution beyond 99% hydrolysis, a number of authors have inclined to the view that peptide and protein synthesis may be catalyzed by proteases and peptidases. The free energy data indicate that, whether or not this is the case, the synthesis of small peptides from amino acids cannot, under physiological conditions, be a simple mass action reversal of hydrolysis. On the other hand, the condensation of large peptides may be promoted by proteases alone. 

Proteinases catalyze the hydrolysis of peptide linkages in proteins. Those proteinases which have been purified have also been shown to have peptidase activity, that is, they catalyze the hydrolysis of synthetic polypeptides of known structure. Because the proteinases split proteins to yield lower molecular weight peptones, proteoses, and peptides they have been termed endopeptidases they are capable of hydrolyzing peptide bonds that are not terminal. Exopeptidases can hydrolyze only peptide bonds that involve terminal amino acids. The synthesis of peptide substrates has been authoritatively reviewed by Fruton. ... 

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