Mapping Toe Active Site of Carboxypeptidase‐A: A Proposed Scheme for Substrate Binding at the Active Site

The aim of the work presented here was to characterize the active site of carboxypeptidase A. The kinetic constants \documentclass{article}\pagestyle{empty}\begin{document}$$ {\rm \bar K}_{\rm m} $$\end{document}, kcatand \documentclass{article}\pagestyle{empty}\begin{document}$$ {\rm C ( = \bar K}_{\rm m} \times {\rm k}_{{\rm cat}} ) $$\end{document}for the enzymatic hydrolysis of the C‐terminal bond in a number of homologous series of peptide substrates were measured. Changes in the kinetic constants due to systematic structural changes in a particular amino acid residue of the substrate are discussed in terms of the interaction of this residue with the corresponding subsite of the enzyme active site. The structural changes included are: (a) elongation of the peptide substrate by one amino acid residue, (b) blocking of the positively‐charged N‐terminal residue by an N‐blocking group, (c) substitution of an L‐amino acid residue by its D‐antipode, and (d) substitution of a particular residue of the substrate from glycyl to alanyl, leucyl, phenylalanyl, cyclohexalanyl or one of the following N‐blocking groups: N‐acetyl, N‐methyloxycarbonyl, N‐phenylpropionyl and N‐benzyloxycarbonyl. The effect of these structural changes in the substrate on the kinetic constants for the hydrolysis of the C‐terminal bond is given by the ratio of the kinetic constants of pairs of homologous substrates differing by a single structural change. Changes in free energy of binding of the substrate‐active site complexes were derived from the ratio of \documentclass{article}\pagestyle{empty}\begin{document}$$ {\rm \bar K}_{\rm m} $$\end{document}constants of homologous pairs of substrate. Based on the kinetic data presented, the active site of carboxypeptidase A was characterized in terms of (a) length, (b) stereospecificity of the various subsites, and (c). the nature of the interactions between each subsite and the corresponding residue of the substrate, as well as the cooperativity effect of these interactions. Evidence obtained from fitting of model peptide substrate to an atomic model of the active site support the conclusions derived from the kinetic data. In conclusion, we suggest a scheme for binding of peptide substrate to the active site of carboxypeptidase A.