Enhanced carboxypeptidase efficacies and differentiation of peptide epimers

Carboxypeptidases enzymatically cleave the peptide bond of C-terminal amino acids. In humans, it is involved in enzymatic synthesis and maturation of proteins and peptides. Carboxypeptidases A and Y have difficulty hy-drolyzing the peptide bond of dipeptides and some other amino acid sequences. Early investigations into different N-blocking groups concluded that larger moieties increased substrate susceptibility to peptide bond hydrolysis with carboxypeptidases. This study conclusively demonstrates that 6-aminoquinoline-N-hydroxysuc-cimidyl carbamate (AQC) as an N-blocking group greatly enhances substrate hydrolysis with carboxypepti-dase. AQC addition to the N-terminus of amino acids and peptides also improves chromatographic peak shapes and sensitivities via mass spectrometry detection. These enzymes have been used for amino acid sequence determination prior to the advent of modern proteomics. However, most modern proteomic methods assume that all peptides are comprised of L-amino acids and therefore cannot distinguish L-from D-amino acids within the peptide sequence. The majority of existing methods that allow for chiral differentiation either require synthetic standards or incur racemization in the process. This study highlights the resistance of D-amino acids within peptides to enzymatic hydrolysis by Carboxypeptidase Y. This stereoselectivity may be advantageous when screening for low abundance peptide stereoisomers.

Automated summary

Carboxypeptidases are enzymes that cleave the peptide bond of C-terminal amino acids. This study demonstrates that the addition of 6-aminoquinoline-N-hydroxysuccinimidyl carbamate (AQC) as an N-blocking group greatly enhances substrate hydrolysis with carboxypeptidase. The study also highlights the resistance of D-amino acids within peptides to enzymatic hydrolysis by Carboxypeptidase Y, which could be advantageous for screening low abundance peptide stereoisomers.