Abstract

Carboxypeptidase II from malted barley is a serine carboxypeptidase which exhibits peptidase activity with a pH optimum below 4.0 and amidase, esterase, and peptidyl amino acid amide hydrolase activities with a pH optimum above 7.5. These different pH-optima reflect variations of the pH dependence of Km. The enzyme hydrolyses effectively substrates containing basic and hydrophobic amino acid residues on the P1 and/or P 1 ′ positions. Addition of salt or phenylguanidine inhibits the activity towards substrates with basic amino acid residues in either of these positions and enhances the activity towards peptide substrates with hydrophobic amino acid residues in both positions and towards ester substrates with hydrophobic amino acid residues in the P1 position. A binding site model to account for the observations is proposed. Malt carboxypeptidase II catalyses the formation of peptide bonds using an N-blocked amino acid ester as acyl component and amino acids, amino acid methyl esters or amino acid amides as nucleophiles. Peptides may also be used as acyl components with the result that the nucleophile is incorporated in place of the C-terminal amino acid residue, i.e. a transpeptidation takes place. The enzyme is easily saturated with amino acid amides indicating the formation of a complex between nucleophile and acyl-enzyme intermediate prior to deacylation. The apparent dissociation constant of this complex (KN(app)) is approximately 1 mM with H−Lys−NH2 and H−Arg−NH2 while it is 13 mM with amino acid amides with uncharged side-chains. At saturation with these nucleophiles the fraction of aminolysis are 0.80–0.95. However, saturation cannot be achieved with amino acids and amino acid esters as nucleophiles and the fractions of aminolysis are lower than with amino acid amides. The specificity of carboxypeptidase II renders it suitable for synthesis of peptide bonds where the acyl and/or the imine portion is donated by a basic amino acid residue.

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