Insulin transpeptidation

S5mthesis ot bioactive peptides in water and water-cosolvent mixtures [6], insulin transpeptidation [9], substrate-mimetic approach [10]... 

J. Marknssen, Human Insulin by Tryptic Transpeptidation of Porcine Insulin andBiosynthetic Precursors, MTP Press, Lancaster, U.K., 1987. 

Preparation of Human Insulin. Porcine insulin can be converted to the human insulin sequence by an enzyme-catalyzed transpeptidation reaction (10,11). Under appropriate conditions trypsin acts preferentially at LysB29 rather than ArgB22 to yield a covalent des[B30]insulin/trypsin complex (acyl—enzyme intermediate). In the presence of high concentrations of organic co-solvents and the /-butyl ester of threonine, transpeptidation predominates over hydrolysis to yield the /-butyl ester of human insulin. Following appropriate purification steps and acidolytic removal of the ester, human insulin suitable for treating patients is obtained. 

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-... 

Further commercial interest was found by the transpeptidation reaction of porcine insulin to human insulin, the latter only differing in the last amino acid of Ae B-chain (Ala-B30 to Thr-B30). This reaction proceeds under kinetic control with trypsin, carboxypeptidase Y or achromobacter lyticus protease I. ... 

With the rapid increase in the incidence of diabetes among the population, it is no longer possible to satisfy the pharmaceutical requirement (estimated to be 15-20 tonnes per year in 2005) from animal sources. Furthermore, the animal-extracted insulins are slightly different from human insulin, which might cause formation of insulin-binding antibodies and allergic reactions. Porcine insulin, which differs from human insulin only by a single amino acid in position B30, can be converted to human insulin in a transpeptidation reaction, in which an alanine is replaced with a threonine [1]. 

Fig. 13.4 Conversion of insulin precursor to recombinant human insulin using transpeptidation. The C-peptide is removed from the partially purified insulin precursor using a serine protease (e.g., trypsin). By inclusion of a threonine ester (T ) and using appropriate reaction conditions, it is possible to couple threonine to Lys using...

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 reactions with proteolytic enzymes have been frequently observed, too the first example of Fraenkel-Conrat mentioned at the beginning of this section belongs to this type of reactions. A remarkable instance for a transpeptidation reaction is the transformation of pork insulin into human insulin by carboxypeptidase A [35]. This enzyme splits a carboxyl-terminal amino acid from the end of a polypeptide chain and also catalyzes the transpeptidation at the same linkage. Thus, in the B-chain of porcine insulin the terminal alanin is exchanged by threonine on incubation with the enzyme and a large excess of this amino acid. 

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. 

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