Acyl peptide hydrolase

The role of the serine residue in hydrolysis was further examined using pseudo-substrates, e.g. p-nitrophenylacetate—substrates which were only very slowly utilized by the enzyme. The p-nitrophenyl group was slowly released and the acyl group became attached to the same serine in hydrolases which had been detected by DIPF (Kilby and Youatt, 1954). Mechanisms for peptide and ester hydrolysis were therefore proposed in which the acyl group became transiently and covalently bound to serines in catalytic sites (see Hartley et al. 1969). 

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.

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. 

Some new approaches to suppress competitive reactions in protease-catalyzed peptide synthesis have been developed in our group [14], namely leaving group manipulations at the acyl donor in kinetically controlled reactions, enzymatic synthesis in organic solvent-free microaqueous systems, cryoenzymatic peptide synthesis, and biotransformations in frozen aqueous systems using the reverse hydrolysis potential of proteases and other hydrolases... 

Classical enzymes employed for peptide coupling of the serine hydrolase family are chymotrypsin, trypsin, and subtilisin. Chymotrypsin and trypsin are secreted in the mammalian gut as inactive precursors, which are activated by autoproteolysis and structural reorganization. The use of chymotrypsin for peptide synthesis has been reported since the 1930s [50]. Most early examples concern peptide s)mthesis using amides or (m)ethyl esters as acyl donors and free amino acids and their amides, short peptides, or short peptide amides as nucleophilic acyl acceptors [3]. These studies revealed that a high pH, high nucleophile concentration, and low product solubility stimulate the formation of synthetic product. Ethyl esters appeared suitable acyl donors in kinetically controlled conversions, and amino acid amides act better as nucleophilic acyl acceptors than the free amino acids [4]. Furthermore, tripeptides often performed better than dipeptides or amino acids as acyl acceptors. 

Prolyl aminopeptidases (PAP) are exopeptidases that hydrolytically cleave off an N-terminal Pro from peptides. The enzymes belong to the a/p hydrolase fold proteins. A PAP from Streptomyces thermoluteus carrying the active site nucleophile mutation S144C was used as a catalyst for the synthesis of proline-containing peptides. Dipeptide synthesis was obtained with an amino acid methyl or benzyl ester as the acyl donor and prolyl-OBz as the nucleophile [21]. Under alkaline conditions, cycli-zation and polymerization of prolyl-OBz was observed. 

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