Transfer reactions peptidases

An understanding of the molecular interactions between the acylenzyme and the attacking nucleophilic amine component allows an optimization of the acyl transfer efficiency. The efficiency of the nucleophilic attack of the amine component depends essentially on an optimal binding within the active site by S - P interactions (Fig. 12.5-11). Consequently, more information on the specificity of the S subsites of serine and cysteine peptidases are useful, which can be obtained by systematic acyl transfer studies using libraries of nucleophilic amine components. According to the definition of the p value (see above) small values of p indicate high S subsite specificity for the appropriate amine component in peptidase-catalyzed acyl transfer reactions. 

Gln-Gln-Phe-Gly9-OMe was formed by chymotrypsin-catalyzed transpeptidation in the presence of H-Phe-GIy-OMe. In a papain-catalyzed acyl transfer reaction and subsequent tryptic cleavage, the resulting dodecapeptide ester was converted into substance P. These results indicate that peptides can be readily produced by a combination of recombinant DNA technology and peptidase-catalyzed conversion with the advantage of possible incorporation of groups other than coded amino acids into the recombinant product. 

The amide transfer reactions can be distinguished from the transamidation reactions studied by Fruton et al. as follows. The enzyme is not a peptidase or protease. The amide (or hydroxylamine) transfer is restricted to the /3-carboxyl of aspartic acid or the y-carboxyl of glutamic acid the transfer is not associated with hydrolysis of the amide it does depend on ATP or ADP, arsenate or phosphate, and Mn++, but this dependence notwithstanding, it shares with the peptidase-protease type of transamidation its independence of energy or phosphate transfer. 

When one compares the inhibitor picture of amino acid transfer by peptidases and of 7-glutamyl and glycine transpeptidases on the one hand, with that of amino acid incorporation on the other, one sees at a glance that they are quite different (Table VIII). Additional examples of contrast not shown in the table are HCN, which activates the transferase activity as it does the hydrolytic action of papain and inhibits amino acid incorporation the amide transfer reactions are activated by arsenate the synthesis of serum albumin is inhibited. 

This claim is based on the following observations (1) Like that of their prokaryotic ancestors, mitochondrial translation uses fMet-tRNA (rather than the Met-tRNA used by cytoribosomes) as the initiator. (2) It can do so successfully because the transformylase which converts the Met-tRNA into its fMet derivative is mitochondrial in its localization. (3) The formation of the initiation complex can be monitored by the transfer of labeled formate (f ) to f Met to puromycin, resulting in its quantitative conversion to f Met-puro —a reaction that is restricted both in vitro and in vivo to the mitochondrial fraction and can be shown to go on with linear kinetics for extended periods. (4) Retention of f Met on nascent polypeptide chains is restricted to mitochondrial polyribosomes and can be used as a specific means for the identification and characterization of the latter. (5) Mitochondria, at least of the yeast species examined by us, appear to be deficient in both a deformylase capable of removing formate from fMet, whether free or on polypeptides, as well as in peptidases capable of removing either this component itself or small peptides from the N-terminal end. (6) Initiation by fMet is absent in p" mutants. In principle then, presence of formate as N-terminal fMet in a polypeptide provides an unambiguous means for its identification as having been synthesized on mitoribosomes. In practice, although feasible, as will be shown in the next section, this is difficult because it requires the prior... 

---