Transpeptidization

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

D,D-endopeptidase acting on maturing peptidoglycan D, D - c arb o xyp ep tidase transpeptidation is absent none apparent... 

Fig. 8.2 Interaction of transpeptidase (Enz) with its natural substrate, acyl-D-alanyl-D-alanine in the first stage of the transpeptidation reaction to form an acyl-enzyme intermediate. A similar reaction with a penicillin results in the formation of an inactive penicilloyl-enyme complex.

Fig. 8.3 A, comparison of the stmcture of the nucleus of the penicillin molecule with B, the D-alanyl-D-alanine end group of the precursor of bacterial peptidoglycan. The broken lines show the correspondence in position between the labile bond of penicillin and the bond broken during the transpeptidation reaction associated with the crosslinking in peptidoglycan. 

Mode of action Interferes with bacterial cell wall synthesis during active multiplication, causing cell wall death and resultant bactericidal activity Inhibits bacterial cell wall synthesis by binding to one or more of the penicillin-binding proteins, which in turn inhibit the final transpeptidation step of peptidoglycan synthesis in bacterial cell walls bacteria usually lyse from ongoing autolytic enzyme activity... 

Borsook s review article (1952) is a good starting point for a consideration of changes in thought which have occurred with regard to biosynthesis of protein. .. the possibility that synthesis could occur by reverse of hydrolysis of proteins. .. is discussed (and disagreed with) and so is protein synthesis by transpeptidation. Both theories were current at the time. .. Borsook needs only half a page to discuss the role of nucleic units in protein synthesis. 

This enzyme [EC 3.4.16.4], also known as serine-type D-alanyl-D-alanine carboxypeptidase, catalyzes the hydrolysis of D-alanyl-D-alanine to yield two D-alanine. This enzyme comprises a group of membrane-bound, bacterial enzymes of the peptidase family Sll. They are distinct from the zinc D-alanyl-D-alanine carboxypeptidase [EC 3.4.17.14]. The enzyme also hydrolyzes the D-alanyl-D-alanine peptide bond in the polypeptide of the cell wall. In addition, the enzyme will also catalyze the transpeptidation of peptidyl-alanyl moieties that are A-acetyl-substituents of D-alanine. The protein is inhibited by j8-lactam antibiotics, which acylate the active-site seryl residue. 

C) Inhibition of transpeptidation reaction in bacterial cell wall synthesis. 

B. Humans cannot synthesize folic acid (A) diet is their main source. Sulfonamides selectively inhibit microbially synthesized folic acid. Incorporation (B) of PABA into microbial folic acid is competitively inhibited by sulfonamides. The TMP-SMX combination is synergistic because it acts at different steps in microbial folic acid synthesis. All sulfonamides are bacteriostatic. Inhibition of the transpeptidation reaction (C) involved in the synthesis of the bacterial cell wall is the basic mechanism of action of (3-lac-tam antibiotics Changes in DNA gyrases (D) and active efflux transport system are mechanisms for resistance to quinolones. Structural changes (E) in dihydropteroate synthetase and overproduction of PABA are mechanisms of resistance to the sulfonamides. 

Formation of an amide bond (peptide bond) will take place if an amine and not an alcohol attacks the acyl enzyme. If an amino acid (acid protected) is used, reactions can be continued to form oligo peptides. If an ester is used the process will be a kinetically controlled aminolysis. If an amino acid (amino protected) is used it will be reversed hydrolysis and if it is a protected amide or peptide it will be transpeptidation. Both of the latter methods are thermodynamically controlled. However, synthesis of peptides using biocatalytic methods (esterase, lipase or protease) is only of limited importance for two reasons. Synthesis by either of the above mentioned biocatalytic methods will take place in low water media and low solubility of peptides with more than 2-3 amino acids limits their value. Secondly, there are well developed non-biocatalytic methods for peptide synthesis. For small quantities the automated Merrifield method works well. 

A characteristic feature of catalysis by the aspartic proteases is a tendency, with certain substrates, to catalyze transpeptidation reactions of the following type. 

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... 

Other well-known zinc proteases are collagenase angiotensin-converting enzyme (important in regulating blood pressure) thermolysin, a bacterial endopep-tidase of Mr 34 600 containing 316 residues in its single polypeptide chain 144 and the Zn2+ G protease from Streptomyces albus, a D-alanyl-D-alanine car-boxypeptidase that catalyzes. carboxypeptidation and transpeptidation in cell wall metabolism. 

The 1 sO-incorporation experiment was repeated for a reaction in which transpeptidation and secondary cleavage are not observed the HIV protease-catalyzed cleavage of Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 at the Tyr-Pro bond.174 The substrate was subjected to hydrolysis in 78% enriched H2180 and the remaining substrate isolated after > 80% had converted to products. 180 was found in the Tyr-Pro carbonyl group. The incorporation had to have arisen from the direct attack of H2lsO on the substrate to generate a tetrahedral adduct. 

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