Question

Why are the peptide fragments generated by trypsin digestion incompatible with C-terminal sequencing using carboxypeptidase A?

Answer 1

• Carboxypeptidases are proteolytic enzymes which only cleave the C-terminal peptide bond in polypeptides. Those characterized until now can, dependent on their catalytic mechanism, be classified as either metallocarbopeptidases or as series carbopeptidases.
• Enzymes from the latter group are found in the vacuoles of higher plant and fungi and in the lysosomes of animal cells.
• It is probable that the well known catalytic mechanism of the series endopeptidases is also employed by the serine carboxypeptidases but presumably with the difference that the pKa of the catalytically essential histidyl residue is somewhat lower in the carboxypeptidases than in the endopeptidases.
• The leaving group specificity of these two groups of enzymes differ since the carboxypeptidases only release C-terminal amino acids from peptides and not longer peptide fragement.
• Other proteolytic enzymes the serine carboxypeptidases contain binding sites which secure the interaction between enzyme and substrate.
• All hydrolytic reaction catalyzed by carboxypeptidase Y proceed via an acyl-enzyme intermediate, and used as acyl components in transacylation reaction to other nucleophiles than water.
• Carboxypeptidase Y catalyzed transpeptidation reaction using N-blocked dipeptides as acyl components and amino acid amides as amine components normally result in formation of only two products, a transpeptidation product where the C-terminal amino acid residue has been replaced and a hydrolysis product.