Transpeptidation transfer

Transpeptidation experiments. The pepsin-catalyzed hydrolysis of Leu-Tyr-Leu gives the product Leu-Leu, which can be formed from the acyl transfer shown in equation 16.31.166,167... 

One of the essential questions relating to the mechanism is whether the catalytic process involves any covalent intermediates between the substrate moieties and the enzyme. In the case of pepsin two possible intermediates could be formed the amino intermediate which involves the transfer of the amino moiety of the substrate to one of the carboxyl groups on the enzyme and the acyl intermediate which involves the transfer of the acyl moiety as shown on page 165. The examples used are substrates involved in transpeptidation reactions in which the respective intermediate has been demonstrated see below). 

However, since the time these mechanisms were proposed, we have obtained more direct evidence for an acyl intermediate in pepsin- and penicillopepsin-catalyzed reactions from transpeptidation reactions which only proceed via an acyl transfer 129). Some of the experimental evi-... 

This ambiguity was resolved in the experiment shown in Table XL The peptide in this case represents the C-terminal sequence of the B-chain of insulin. The major products were the penta-peptide lacking the N-terminal phenylalanine and free phenylalanine indicating that hydrolysis was the major reaction. In addition, however, there was a significant amount of Phe-Phe. This dipeptide could only come from a transpeptidation involving an acyl transfer. The acyl transfer presumably proceeds via a covalent acyl intermediate. The evidence for this comes from the experiment shown in Table XII where Leu-Tyr-Leu was incubated with both porcine pepsin and penicillopepsin in the presence of a lO-fold excess of C-leucine over Leu-Tyr-Leu. The products, leucine and leucylleucine, were separated by high voltage electrophoresis and analyzed for their specific radioactivity. At most, only traces of radio-... 

Gln-Gln-Phe-Gly9-OMe was formed by chymotrypsin-catalyzed transpeptidation in the presence of H-Phe-GIy-OMe. In a papain-catalyzed acyl transfer reaction and subsequent tryptic cleavage, the resulting dodecapeptide ester was converted into substance P. These results indicate that peptides can be readily produced by a combination of recombinant DNA technology and peptidase-catalyzed conversion with the advantage of possible incorporation of groups other than coded amino acids into the recombinant product. 

The soluble DD-Cbases catalyze transfer reactions between donor and acceptor peptides. These enzymes also hydrolyze donor peptides at the same time as transpeptidation is occurring. 

Not only transpeptidation, but also a certain amount of peptide-peptide condensation, is possibly involved in the plastein reaction. With some of the proteases used for plastein formation, especially a-chymotrypsin (26, 52, 53, 54), the acyl-enzyme intermediate can be formed at pHs 5 from the reversal of the degradative reaction (Equation 1 E-OH + HOOC-CHR-NH E-O-OCCHR-NH- + H20). Once the acyl-enzyme intermediate is formed, the acyl group can be transferred to a nucleophile resulting in peptide bond synthesis. 

During hydrolysis catalyzed by serine proteases an acyl-enzyme complex transfers the acyl group to water. However, in enzymatic synthesis, the acyl group is not transferred to water but to a nucleophile, that is, to an amino group or amino acids/peptides. Thus a transpeptidation reaction takes place during the enzymatic modification [46,57]. 

Transpeptidation, transamidation, a reaction involving the transfer of one or more amino acids from one peptide chain to another. This term was first coined by Fruton, in 1950, by analogy with transglycosidation for the papain-catalyzed displacement reaction between Bz-Gly-NH2 and aniline forming Bz-Gly-NHPh. Of special importance in relation to protease-catalyzed transpeptidation reactions in a preparative scale is the one-step tryptic conversion of porcine insulin into human insulin, despite the controversial interpretation of the mechanism involved. A bacterial transpeptidase, serim-type u-Ala-u-Ala carhoxypeptidase (EC... 

Transpeptidation In transpeptidation (peptidyl transfer), the peptide bond is formed via nucleophilic displacement of the P-site tRNA of the peptidyl-tRNA by the amino group of the aa-tRNA in the A-site. The nascent polypeptide chain is thereby lengthened at its C-terminus by one residue and transferred to the A-site tRNA (Figure 13.16). The peptidyl transfer reaction is catalyzed by the peptidyl transferase ribozyme of 23S rRNA of the 50S ribosome subunit. 

Studies by Mirelman, Bracha and Sharon (1972, 1974a, b) have done much to clarify this. They used Micrococcus luteus as a source of broken cell walls and showed that penicillin inhibited the incorporation of GlcNAcMur-NAc-pentapeptide, or A-acetylglucosamine, with an efficiency approaching that with which it blocked the release of D-alanine. Hence the glycosyl transfer was, in some way, probably connected with transpeptidation. Studies... 

Transpeptidation, i.e., transfer of the y-glutamyl residue from one peptide to a different peptide... 

The enzyme works optimally at pH > 9 with N-protected amino acid esters as acyl donors, and amino acids or amino acid amides as nucleophiles (acceptors). At that pH value it has high esterase activity an amino acyl-enzyme complex will be formed rapidly which transfers the acyl residue to the acceptor molecule (transpeptidation), and since at alkaline pH the rate of peptide cleaving is minimal, the product formed will be released in excellent yield. As an example the last coupling step in a synthesis of the opioide peptide Met-enkephalin that started with benzoylarginine ethylester may be shown (Fig. 8). 

The enzymatic synthesis of peptides (Scheme 6.24) from which proteins can be constructed is not so limited, and chemical synthesis has an even wider application, but these are not yet suitable techniques for manufacture. Moreover, there are no general methods for building the peptides into full protein structures. Nevertheless, enzymes do have a role in the manufacture of peptides themselves. In a mixture of butan-l,4-diol and water, trypsin will catalyse the exchange of the carboxy-terminal alanine of porcine insulin with threonine t-butyl ester (Scheme 6.25). The reaction is essentially a transpeptidation in which the acyl group of lysine is transferred from one amino group on alanine to another on the threonine. This converts porcine insulin into the ester of the human hormone, and a simple deprotection yields one of the commercial products. 

All the known transpeptidation and transamidation reactions are of the carboxyl-transfer type and so far no example has been reported of the alternative type which might be envisaged, namely transfer of the amino moiety of a donor peptide to new linkage ivith the carboxyl group of an acceptor. 

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