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 transacyl ation reactions and that carboxypeptidase Y (CPD-Y) is suitable for th is due to its broad specificity and stability in the presence of denat urants. Furthermore, CPD-Y is characterized by a S-1' binding site tha t is open to solvent and, thus, capable of catalyzing a transpeptidati on 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 doe s not exceed 15%. In the present publication it is demonstrated that m utants of CPD-Y, designed for low leaving group dependence, efficientl y incorporate biocytin amide as well as a new fluorescent nucleophile, N-E-Abz-Lysine amide (ablysin amide), into peptides and proteins. (C) 1997 Academic Press.