Kinetic investigation on d-amino acid containing peptides and carboxypeptidase Y

Carboxypeptidases catalyze the hydrolysis of the peptide bond of C terminal amino acid residues. Carboxypeptidase Y can hydrolyze all 20 naturally-occurring amino acids at varying rates under optimal conditions. Previous data suggests that carboxypeptidase Y has more difficulty hydrolyzing d-amino acids from the C terminus of peptides than l-amino acids. This enzyme was used for amino acid sequence determination prior to modern proteomics. However, most modern proteomic methods assume that all peptides are comprised of l-amino acids and do not distinguish L-from d-amino acids within the peptide sequence. Most existing methods that allow for chiral differentiation require synthetic standards or incur racemization. Steady-state kinetic analysis was performed to elucidate differences in hydrolytic rates between peptides composed solely of l-amino acids at the carboxy-terminal and those incorporating d-amino acids. Our data suggests that the l-amino acid exclusive peptides are hydrolyzed at a rate two-to-five orders of magnitude higher than d-amino acid containing peptides. Such differences are necessary to completely digest and eliminate l-amino acid exclusive peptides while leaving the carboxy-terminal d-amino acid peptides intact. The d-amino acid containing peptides can then be preconcentrated to enhance detection limits to facilitate scouting for the desired d-amino acid containing peptides. Interestingly, the peptide epimers bind equally well to carboxypeptidase Y on a low μM level, indicating high substrate affinity.