Peptide enzyme-catalyzed

Knowing how the protein chain is folded is a key ingredient m understanding the mechanism by which an enzyme catalyzes a reaction Take carboxypeptidase A for exam pie This enzyme catalyzes the hydrolysis of the peptide bond at the C terminus It is... 

There are two basic strategies for enzyme-catalyzed peptide synthesis equiUbrium- and kineticaHy controlled synthesis. The former is the direct reversal of proteolysis and involves the condensation of an amino component with unactivated carboxyl component. The latter proceeds by the aminolysis of an activated peptide ester. 

For the equiUbrium-controUed enzyme-catalyzed peptide synthesis the equiUbrium position Hes far over in the direction of the hydrolysis, and under physiological conditions, the product yield is negligible. The equiUbrium position is deterrnined exclusively by thermodynamic factors and like any other catalysts the enzymes only accelerate the attainment of the equiUbrium. 

The years that have passed since Sanger determined the structure of insulin have seen refinements in technique while retaining the same overall strategy. Enzyme-catalyzed hydrolysis to convert a large peptide to smaller fragments remains an important... 

Protease inhibitor (Section 28.13) A substance that interferes with enzyme-catalyzed hydrolysis of peptide bonds. 

An example for proteases are the (3-lactamases that hydrolyse a peptide bond in the essential (3-lactam ring of penicillins, cephalosporins, carbapenems and monobac-tams and, thereby, iireversibly inactivate the diug. 13-lactamases share this mechanism with the penicillin binding proteins (PBPs), which are essential enzymes catalyzing the biosynthesis of the bacterial cell wall. In contrast to the PBPs which irreversibly bind (3-lactams to the active site serine, the analogous complex of the diug with (3-lactamases is rapidly hydrolyzed regenerating the enzyme for inactivation of additional (3-lactam molecules. 

Since the beginning of the 20th century, organic solvents have been used in enzymatic reaction media [30]. Biocatalytic reactions in water-organic biphasic media were first carried out by Cremonesi et al. [31] and by Buckland et al. [32] less than 30 years ago. Their work aimed at the conversion of high concentrations of poorly water soluble components, particularly steroids. Later, biphasic systems were used for enzyme-catalyzed synthesis reactions that were unfavored in water, changing the reaction equilibrium towards the higher yield of the product, such as esters or peptides. 

In another study, the carrier protein was replaced by an enzyme compatible solid-phase resin (PEGA), and enzyme-catalyzed cyclization was used to probe substrate specificity. This study demonstrated also that oxo-esters are tolerated as substrates for TE domains, and then-preparation in library format served as an excellent tool for substrate specificity studies, as well as for preparation of cyclized peptides. Figure 13.11 shows how the TycA TE showed selectivity for only residues 1 and 9 (colored in red), and changes at all other residues were tolerated [42]. Hydrogen bonding interactions are shown in green. Several compounds made from this series were shown to demonstrate improved therapeutic indices (with respect to hemolysis) while retaining antimicrobial activity. 

TE-catalyzed cyclization is not limited to the synthesis of macrocyclic peptides by catalyzing the formation of aC N bond. These enzymes are also responsible for the cyclization of NRP depsipeptide and PK lactone. Indeed, a di-domain excised from fengycin synthase was... 

The suitability of the Aloe group for the construction of lipidated peptides is emphasized by the synthesis of the maleimidocaproyl-modified, S-palmi-toylated and farnesylated heptapeptide 16 which corresponds to the N-Ras C-terminus (Scheme 10).1211 In contrast to classical urethane-type protecting groups, the Aloe group can be removed in the presence of additional functional groups and under neutral conditions. It is therefore a very convenient protecting group for the synthesis of very hydro-phobic lipid-modified peptides, which are not soluble in the aqueous media required for enzyme catalyzed transformations. 

Virtually all biological reactions are stereospecific. This generalization applies not only to the enzyme-catalyzed reactions of intermediary metabolism, but also to the processes of nucleic acid synthesis and to the process of translation, in which the amino acids are linked in specific sequence to form the peptide chains of the enzymes. This review will be restricted mainly to some of the more elementary aspects of the stereospecificity of enzyme reactions, particularly to those features of chirality which have been worked out with the help of isotopes. 

This enzyme catalyzes the covalent insertion of a tyrosine at the C-terminal glutamate of the tubulin a subunit to effect the posttranslational synthesis of a peptide bond (Flavin et al., 1982 Thompson, 1982). The role of this modification reaction remains to be established... 

The hydrolysis of peptide bonds catalyzed by the serine proteases has been the reaction most extensively studied by low-temperature trapping experiments. The reasons for this preference are the ease of availability of substrates and purified enzymes, the stability of the proteins to extremes of pH, temperature, and organic solvent, and the existence of a well-characterized covalent acyl-enzyme intermediate. Both amides and esters are substrates for the serine proteases, and a number of chromo-phoric substrates have been synthesized to simplify assay by spectrophotometric techniques. 

Stehle, P, Bahsitta, H. P., and Piirst, P. (1986). Analytical control of enzyme-catalyzed peptide-synthesis using capillary isotachophoresis.. Chromatogr. 370, 131—138. 

Once assembly of a mature peptide has been completed by the NRPS, the product remains covalently linked to the PCP domain of the last module as a thioester. Release into solution from the assembly line is accomplished by a variety of enzyme-catalyzed reactions as described below (Figure 8). 

Frequently, after assembly of the peptide natural product by the NRPS, enzyme-catalyzed reactions impart additional functionality and structural elements. The reactions are very diverse and a thorough survey of the known enzymology is beyond the scope of this chapter, however, several recent review articles describe the various chemistries that are known. °... 

Use of Proteases in Peptide Synthesis. Typically peptides are synthesized the standard solid or liquid phase methodologies (56, 57). However, both of these techniques require harsh chemical reactions which are detrimental to certain amino acids. Furthermore, in practical terms most peptide syntheses are limited to the range of 30 to 50 amino acid residues. Hence, peptide synthesis is still somewhat problematic in many cases. In certain situations, the alternative method of peptide synthesis using proteases is an attractive choice. With this form of synthesis, one can avoid the use of the noxious and hazardous chemicals used in solid or liquid phase peptide synthesis. Since the reactions are enzyme catalyzed, racemization of the peptide bond does not occur. This technique has been used with success in the synthesis and semisynthesis of several important peptides including human insulin (55,59). 

This enzyme [EC 3.4.22.17] is an intracellular, nonlyso-somal member of the peptidase family C2. The enzyme catalyzes the calcium ion-dependent hydrolysis of peptide bonds with preference for Tyr-Xaa, Met-Xaa, or Arg-Xaa with a leucyl or valyl residue at the P2 position. There are two main types of calpain. One has a high calcium sensitivity in the micromolar range and is called (,-calpain or calpain I. The other calpain has a low calcium sensitivity in the millimolar range and is called m-calpain or calpain II. Forms of calpain exhibiting intermediate calcium sensitivity also exist. 

This proteolytic enzyme catalyzes the removal of N-ter-minal signal peptide extensions of secretory proteins and certain intracellular proteins. 

This enzyme [EC 3.4.21.62], a serine endopeptidase that evolved independently of chymotrypsin, contains no cys-teinyl residues. This enzyme catalyzes the hydrolysis of peptide bonds in proteins and has a broad specificity, with a preference for a large uncharged aminoacyl residue in the PI subsite. 

---