Peptide transpeptidation reaction

The peptidoglycan layer confers mechanical stability to the cell wall of the bacteria. An important intermediate of the peptidoglycan biosynthesis is the GlcNAc- MurNAc-L-Ala-D-y-Gln-L-Lys-D-Ala-D-Ala peptide (muramyl-pentapeptide), which is in its lipid-carrrier bound form transglycosylated to a linear polysaccharide. The linear polysaccharide is then cross-linked to peptidoglycan by transpeptidation reactions. Perkins observed that vancomycin binds to the Lys-D-Ala-D-Ala peptide motif of bacterial cell wall intermediates. This observation was later investigated on a mole-cular level by NMR and by x-ray crystallographic studies. ... 

Figure 8.30. Conformations of Penicillin and a Normal Substrate. The conformation of penicillin in the vicinity of its reactive peptide bond (A) resembles the postulated conformation of the transition state of R-d-Ala-d-Ala (B) in the transpeptidation reaction. [After B. Lee. J. Mol. Biol. 61(1971) 464.]...

R -NH2 peptide chain, amino acid or another peptide of the bacterial cell wall involved in the transpeptidation reaction... 

The final steps in cell wall synthesis involve cross-linkage of the peptidoglycan chains. This converts water-soluble polymeric substances (due to many polar functions) with mobility into a tough, insoluble, and inflexible material.9 In simplest terms this is a transpeptidation reaction where the end amine function of the pentaglycine side chain forms a new peptide bond at the expense of the terminal D-alanyl-D-alanine linkage of 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]. 

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],... 

The antibiotic penicillin is a transition state analog that binds very tightly to gly-copeptidyl transferase, an enzyme required by bacteria for synthesis of the cell wall (Fig. 8.18). Glycopeptidyl transferase catalyzes a partial reaction with penicillin that covalently attaches penicillin to its own active site serine. The reaction is favored by the strong resemblance between the peptide bond in the p-lactam ring of penicillin and the transition state complex of the natural transpeptidation reaction. Active site inhibitors such as penicillin that undergo partial reaction to form irreversible inhibitors in the active site are sometimes termed suicide inhibitors. ... 

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... 

My ne. t comments are concerned with the function of the enzymes responsible for the hydrolysis of GSH. We have felt from the start of our work in 1946 that the hydrolysis of GSH is concerned with absorption mechanisms and onlj" indirectly with the synthesis of protein. Thus, we would explain the transpeptidation reactions f rom GSH as a mechanism of transfer of amino acids across cellular membranes. The formation of 7-glutamyl peptides would provide the concentration differential necessary for the absorption of amino acids, for e.xample, from the tubular filtrate or from the intestine. Thus, we would explain the high concentration of the enzymes responsible for transpeptidation in kidney and intestine on the basis that these tissues are the ones most concerned with absorption phenomena. 

This conversion was designated as a transpeptidation reaction, since the exchange occurred at the peptide bond. 

So far transpeptidation reactions have been carried out with approximately 0.02 M amino acid and peptide, which is far greater than the concentration of free amino acids or peptides in the cell. The question arises to what extent the degree of transpeptidation depends on the concentra tions of reactants. In all cases so far studied, even with the above concentrations of reactants, the degree of hydrolysis exceeded that of transpeptidation, and the reaction was always stopped at an arbitrarily chosen stage of incomplete hydrolysis of the parent peptide otherwise the newly formed peptide would have been completely hydrolyzed also. If the ratio of transpeptidation to hydrolysis decreases significantly with decreasing concentration of reactants, the importance of transpeptidation reactions in the economy of the cell would, then, appear to be as significantly lessened. There is a need, clearly, for kinetic data. 

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 ... 

If this should be the case, either apparent acyl transfer or amine transfer would be possible by a direct condensation of the preferentially retained product and an acceptor that can readily displace the product that leaves more easily. That such condensation reactions are catalyzed by pepsin in the case of oligopeptides was demonstrated many years ago (54), and is consistent with the neglible free energy decrease in the hydrolysis of interior peptide bonds (55). For transpeptidation reactions in which an apparent E-Tyr amino-enzyme has been postulated, the free energy change in the condensation of an acceptor such as Ac-Phe with tyrosine would be more unfavorable in free solution, but the possibility must be considered that the ammonium pKa of the tyrosine retained at the active site may be lower than that of tyrosine in free solution, perhaps by virtue of the interaction of the carboxylate group of the amino acid with a complementary cationic group of the active site. 

---