Development of Substrate for Carboxypeptidase-B by Employing Thiaarginine Peptides

Carboxypeptidase-B (CPB) is involved in the biosynthesis of numerous peptide hormones and neurotransmitters. CPB catalyzes hydrolysis of the basic amino acids from the C-terminal position in polypeptides during posttranslational prohormonal processing. Various peptides containing thiaarginine residue at C-terminal position were synthesized and tested for their hydrolysis by CPB. A colorimetric assay, employing Ellman's reagent to detect the thioguanidine released upon hydrolysis of the dipeptide substrates, showed that thiaarginine is a suitable mimetic for arginine. Kinetic studies on the four substrates, Z-L-Ala-DL-thia-Lys, Z-L-Ala-DL-thia-Arg, Z-L-Lys-DL-thia-Arg, and Z-L-Lys(Boc)-DL-thia-Arg, gave Km (mM) of 0.66, 5.08, 0.024, and 0.006 and kcat (min-1) of 340, 5200, 151 and 335, respectively.