Peptide and protein carboxyl-terminal labeling through carboxypeptidase Y-catalyzed transpeptidation.
A survey of carboxypeptidase Y-catalyzed carboxyl-terminal modification of short peptides in the presence of various amino acids revealed that transpeptidation occurred in significant yield only with peptides containing a proline at the penultimate or antepenultimate position. For these peptides, transpeptidation was shown to occur specifically at the carboxyl side of the proline, thus suggesting a determining role of this residue for transpeptidation. Two model peptides, YPFP-GPI and YPFVEPI, were studied in detail. Initial yields of transpeptidation in the presence of various nucleophiles were compared. Among natural amino acids, the highest yield was obtained with methionine, followed by other amino acids bearing hydrophobic side chains. In order to transpose the method of transpeptidation to a protein, a variant of Escherichia coli methionyl-tRNA synthetase bearing the carboxyl-terminal Glu-Pro-Met sequence was genetically created. Under the conditions optimized for the transpeptidation of YPF-VEPI with methionine, this protein could be labeled specifically at its carboxyl-terminal end. Moreover, the parameters of the labeling reaction were in agreement with those observed in the transpeptidation of the model peptide.
Berne
P F
PF
Laboratoire de Biochimie, U.R.A. 240 Centre National de la Recherche Scientifique, Ecole Polytechnique, Palaiseau, France.
Schmitter
J M
JM
Blanquet
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D002268:Carboxypeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005934:Glucagon / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008715:Methionine; D008718:Methionine-tRNA Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D011392:Proline; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D009842:Oligopeptides; D011506:Proteins; D005934:Glucagon; D011392:Proline; D008715:Methionine; D002268:Carboxypeptidases; C022633:serine carboxypeptidase; D008718:Methionine-tRNA Ligase