Transpeptidation, enzymatic

Peptide bond formation occurs immediately following the dissociation of the binding factor from the ribosome. This reaction is known as transpeptidation, and the enzymatic center that catalyzes it is known as peptidyl transferase, although it promotes the conversion of an ester to a... 

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]. 

Polyethylene glycol is used to make the enzymes soluble in organic solvents [88], and high reaction yields have been obtained with polyethylene-glycol-modified chymotrypsin [89], and papain in benzene [90], Enzymatic modification reactions with deacylation, via aminolysis, of an intermediate covalent acyl-enzyme also support the mechanism of transpeptidation in kinetically controlled peptide syn-... 

Methionine-enriched protein was produced also from an enzymatically prehydrolyzed milk protein using an enzymatic peptide modification method with a-chymotrypsin as catalyst. Amino acid incorporation leading to methionine enrichment of the product proceeded via formation of covalent bonds. The concentration of the substrate was 25% (w/v). Methionine was added to the reaction mixture in the form of methionine methyl ester hydrochloride. An ester/substrate ratio of 1 5 was used in the enzymatic peptide modification reaction. The methionine content of the product was twice as high as that of the substrate. The slight change in the degree of hydrolysis revealed that part of the amino acids were bound to the peptide chains and that transpeptidation was the main force during this enzyme-catalyzed reaction. The newly incorporated Met was located in C- and N-termi-nals in a ratio of 3 1 [82],... 

Methionine-enriched protein was produced also from an enzymatically prehydrolyzed milk protein (SR) by the EPM process, and the methionine contents of SR and the EPM product were determined by a microbiological method. The methionine content in the EPM product was more than twice as high as in the substrate. These protein fractions were separated by thin-layer chromatography [83]. The separated peptides and peptide mixtures were eluted and their molar amino acid contents were determined. The ratio of polar and apolar amino acids in the peptides was found to be influenced basically by transpeptidation taking place in the EPM reaction. The analysis of the peptides of these products showed that methionine was incorporated mainly in peptides with a relatively high ratio of apolar amino acids. 

The functional properties of proteins depend also on their structure and interactions with the environment. The functional properties of surfactants depend on their hydrophilic-hydrophobic balance, too. Protein chains modified by proteolysis, amino acid incorporation, and transpeptidation may display different functional properties. As milk proteins possess good surface activities [131], the question of the changes in the functional properties of the enzymatically modified protein products is of especial interest. 

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. 

Other hand, formation of y-glutamyl peptides by enzymatic transpeptid-ation reactions, also in fermentation broths, is possible (cf. Section VII. 3). 

Transpeptidase activity was further studied in extracts of root and sprouting bulbs of Allium cepa. The pH optimum for the hydrolytic activity was 9.0, and it was again concluded that hydrolytic activity was adequate to explain the hydrolysis of y-glutamyl peptides during sprouting of onions. It was confirmed that no enzymatic activity was present in dormant bulbs 8). Subsequently a partially purified enzyme was obtained from sprouted onion bulbs and was shown to have both hydrolytic activity and transpeptidase activity with an optimum pH of 9, although the transpeptidation action was presumably the essential one. The possibility that the crude onion extract also contained a genuine... 

A simple case of the general transpeptidation reaction was the trans-amidation resulting in the formation of hippuric anilide from aniline and hippuric amide in the presence of papain. Since this reaction proceeded much faster than the enzymatic synthesis of hippuric anilide from hippuric acid and aniline, it seems reasonable to infer that exchange, in the former reaction, took place between the aniline and ammonia. Waley and Watson subjected L-lysyl-L-tyrosyl-L-lysine and L-lysyl-L-tyrosyl-L-leucine to treatment with chymotrypsin and trypsin at pH 7.8. In the hydrolysis mixture of either of these substrates they were able to identify lysyllysine which could have arisen only by rearrangement of the amino acids in peptide bond. The peptide may have reacted with the lysine liberated by hydrolysis ... 

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