Transferases dipeptidyl transferase

The use of cell-free extracts to produce proteins has been known for a long time. Early work in this area has been reviewed [53-55]. However, it is much less common to isolate the enzymes, sometimes to purify them, and use them for in vitro production of proteins. A successful example is a publication by Fruton et al. [56], who demonstrated the action of dipeptidyl transferase as a polymerase. Using this enzyme, they made a number of oligopeptides. 

A potentially active area is the in vitro synthesis of proteins via dipeptidyl transferase and cyanophycin synthetase. In specific cases these enzymes may perhaps be useful. Potential handicaps of this approach in the commercial context are the lack of general availability and higher costs of these two enzymes. 

Cathepsin C. Cathepsin J. Dipeptidyl aminopeptidase I. Dipeptidyl transferase. 

Cat heps in C, dipeptidyl transferase, dipeptidyl amino peptidase I. Isoln from beef spleen Tallan el al, J. Biot. Chem. 194, 793 (1952) de la Haba et al, ibid. 234, 316 0 959)-Hydrolyzes dipeptidyl amides or esters bearing a free a-amino (or a-imino) group in the N-terminal position, esp. those containing an aromatic amino acid adjacent to the free a -amino group Planta, Gruber, Biochtm. Biophys. Acta 53, 443 (1961) Wurz et al. Biochemistry 1, 19 (1962). Enhances the proteolysis of prothrombin to thrombin and thus plays an important role in blood dotting Purcell, Barnhart, flio-chim. Biophys. Acta 78, 800 (1963),... 

A different type of peptide hydrolase, dipeptidyl transferase (dipeptidylpep-tide hydrolase), catalyzed the polymerization of dipeptide amide in an aqueous solution. In the case of glycyl-L-phenylalaninamide, trimer was formed in 78% yield (69). The polymerization of glycyl-L-tyrosinamide produced the corresponding oligomer with DP up to 8 (70). 

In the second step of elongation, the a-amino group of the amino acid in the A site (AA2) acts as a nucleophile and attacks the carbonyl group of AA1 (in this case fMet). This reaction leads to the formation of a dipeptidyl-tRNA in the A site and a deacylated-tRNAfMet in the P site (Fig. 26.12). As shown by Harry Noller in 1992, this peptidyl transferase reaction is catalyzed by ribozyme activity present in the 23 S rRNA of the 50S ribosome subunit (and the 28S rRNA in the 60S ribosome subunit in eukaryotes), rather than by ribosomal proteins, as originally thought. 

A peptide bond is then formed in a reaction catalyzed by peptidyl transferase, which is a part of the SOS subunit (Step 3). The mechanism for this reaction is shown in Figure 12.13. The a-amino group of the amino acid in the A site performs a nucleophilic attack on the carbonyl group of the amino acid linked to the tRNA in the P site. There is now a dipeptidyl-tRNA at the A site and a tRNA with no amino acid attached (an uncharged tRNA ) at the P site. 

In the initiation complex, location of the charged initiator tRNA in the P site of the ribosome allows transfer of the methionine residue to the amino group of another aminoacyl-tRNA in the A site (Fig. 7b) by pep-tidyl transferase to form dipeptidyl-tRNA (see Fig. 7c). Functional insertion of Met-tRNAf directly into the P site can be demonstrated using the trinucleotide AUG as a synthetic mRNA and another trinucleotide, for example UUU, to bind an acceptor aminoacyl-tRNA (in this case Phe-tRNA). 

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