C-terminal incorporation of fluorogenic and affinity labels using wild-type and mutagenized carboxypeptidase Y.
The ability to carry out specific C-terminal modification or labeling of peptides and proteins has a broad range of applications. It is well established that this may be achieved by protease-catalyzed transacylation reactions and that carboxypeptidase Y (CPD-Y) is suitable for this due to its broad specificity and stability in the presence of denaturants. Furthermore, CPD-Y is characterized by a S'1 binding site that is open to solvent and, thus, capable of catalyzing a transpeptidation reaction with nucleophiles that extend beyond the perimeter of the active site. However, one major drawback with CPD-Y is that the yield of the reaction is highly dependent on the nature of the leaving group; e.g., with large apolar leaving groups the yield of the reaction does not exceed 15%. In the present publication it is demonstrated that mutants of CPD-Y, designed for low leaving group dependence, efficiently incorporate biocytin amide as well as a new fluorescent nucleophile, N'-Abz-Lysine amide (ablysin amide), into peptides and proteins.
Stennicke
H R
HR
Carlsberg Laboratory, Department of Chemistry, Copenhagen, Valby, Denmark.
Olesen
K
K
Sørensen
S B
SB
Breddam
K
K
D000345:Affinity Labels / Q000737:chemistry*; D000588:Amines / Q000737:chemistry; D002268:Carboxypeptidases / Q000737:chemistry*; D043402:Cathepsin A; D006868:Hydrolysis; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry; D015394:Molecular Structure; D009154:Mutation; D012016:Reference Values; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D062367:ortho-Aminobenzoates / Q000737:chemistry*
D000345:Affinity Labels; D000588:Amines; C106695:N(epsilon)-anthraniloyl-lysine amide; D062367:ortho-Aminobenzoates; C065209:biocytinamide; D012259:Ribonuclease, Pancreatic; D002268:Carboxypeptidases; D043402:Cathepsin A; D008239:Lysine