Polypeptide sequential synthesis

Some overlap between categories can occur. For example, the sequential synthesis of a polypeptide on a polymer support is equivalent to grafting the polypeptide onto the original polymer support, whereas each step of the solid-phase peptide synthesis can be regarded as a polymer-analogous transformation. 

The techniques for automated solid phase synthesis were first highly developed for polypeptides and the method is abbreviated as SPPS. Polypeptide synthesis requires the sequential coupling of the individual amino acids. After each unit is added, it must be deprotected for use in the next coupling step. 

From a chemical point of view, the El/ubiquitin thiol ester should be competent to donate ubiquitin to a substrate amino group. In fact, aminoacyl-errzyme thiol esters are used in exactly this way in non-ribosomal polypeptide synthesis, a process that was discovered around the same time as ubiquitin-protein conjugation [5]. In spite of the attractive simplicity of this model, however, biochemical reconstitution studies showed that besides El two additional fractions were required to conjugate ubiquitin to a model substrate. They were called ubiquitin carrier protein (E2) and ubiquitin-protein ligase (E3), respectively, since the respective factors seemed to act sequentially [6]. Interestingly, the E2 factor apparently formed a thiol ester with ubiquitin. Based on these results, Hershko and co-workers proposed the ubiquitin conjugation cascade (Figure 5.1). 

The synthesis of active centers is not a small problem. The enzyme car-boxypeptidase A is a pancreatic exopeptidase that catalyzes the sequential release of amino acids from the C terminus of polypeptide chains as shown in reaction (1). Much work has been done on the structure 31). Although the... 

The techniques for automated solid-phase synthesis were first highly developed for polypeptides. Polypeptide synthesis requires the sequential coupling of the individual amino acids. 

The first successful synthesis of a biologically active cyclic peptide, gramicidin S, was accomplished by Schwyzer and Sieber[6,7l via the 4-nitrophenyl ester. The fact that -protected peptide esters can be deprotected to give the peptide active ester salts has made this approach popular not only in the synthesis of sequential polypeptides but also of cyclic peptides. Among the various active esters examined for this purpose, the pentafluorophenyl esters have emerged as the most reactive ones, although a high risk of epimerization is encountered when C-terminal chiral amino acids are involved. 

The animal fatty acid synthase (FAS EC 2.3.1.85) is one of the most complex multifunctional enzymes that have been characterized, as this single polypeptide contains all the catalytic components required for a series of 37 sequential transactions (Smith, 1994). The animal FAS consists of two identical polypeptides of approximately 2500 amino acid residues (MW, ca. 270 kDa), each containing seven catalytic subunits (1) ketoacylsynthase, (2) malonyl/acetyl transferase, (3) dehydrase, (4) enoyl reductase, (5) (3-kcto reductase, (6) acyl carrier protein (ACP), and (7) thioesterase. Although some components of the complex are able to carry out their respective catalytic steps in the monomeric form, only in the FAS dimer do the subunits attain conformations that facilitate coupling of the individual reactions of fatty acid synthesis to occur (Smith et al., 2003). 

The most widely nsed method to construct polypeptides is the use of a polymeric substrate to achieve multiple reactions. The Merrifield polypeptide synthesis on a polymeric substrate (crosslinked polystyrene) as the solid phase, revolutionized the synthesis of sequential polypeptides. The initial attachment of a protected amino acid to chloromethyl group containing polystyrene is achieved with triethylamine followed by hydrolysis to give 679 (Scheme 2.338, Step A). After removal of the protecting group, coupling of... 

Once the first amino acid is bound to the polymer, additional amino acids can be added sequentially. The steps of the solid phase peptide synthesis technique are illustrated in the accompanying scheme. In the last step, HF cleaves the polypeptide chain from the polymer. 

Figure 2.8. Linking the RNA and Protein Worlds. Polypeptide synthesis is directed by an RNA template. Adaptor RNA molecules, with amino acids attached, sequentially bind to the template RNA to facilitate the formation of a peptide bond between two amino acids. The growing polypeptide chain remains attached to an adaptor RNA until the completion of synthesis.

Polypeptide synthesis proceeds at peptidyl iP) and aminoacyl (.4) sites in the ribosome (Fig. 21-11). The synthesis in Fig. 21-11 shows initiation followed by sequential addition of Gly and lie. The 5 end of mRNA, located in the smaller subunit of the ribosome, is prepared to receive the tRNAs for Met and Gly (by matching of codon of mRNA and anticodon of tRNA). The tRNAs for Met and Gly pick up Met and Gly from the cytosol, enter the ribosome, and line up at the P and A sites, respectively, by base pairing of codons of mRNA and anticodons of tRNA (Fig. 21-1 la). The enzyme peptidyl transferase, contained in the large subunit of the ribosome, catalyzes the transfer and... 

For the production purpose, abolishment of feedback control and fusion of sequential enzymes may be desirable. The recombinant technology is the method of choice when the redesigning involves substimtions between coded amino acids. However, for substitutions involving artificial, noncoded amino acids or their analogues/derivatives (e.g. post-translationally modified amino acids, isotopic label of specific residue), chemical methods in particular semi-chemical synthesis becomes necessary (Chaiken, 1981). The frag-ments/chains of modified, synthetic or expressed polypeptides are enzymatically or chemically ligated (Muir, 2003). 

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