Tissue kallikrein processes small proenkephalin peptides

Tissue kallikrein may play a role in processing precursor polypeptide hormones. We investigated whether hydrolysis of natural enkephalin precursors, peptide F and bovine adrenal medulla docosapeptide (BAM-22P), by hog pancreatic kallikrein is consistent with this concept. Incubation of peptide F with this tissue kallikrein resulted in the release of Met⁵-enkephalin and Met⁵-Lys⁶-enkephalin. Met⁵-Lys⁶-enkephalin was the main peptide released, indicating that the major cleavage site was between two lysine residues. At 37°C and pH 8.5, the K_M values for formation of Met⁵-enkephalin and Met⁵-Lys⁶-enkephalin were 129 and 191 μM, respectively. Corresponding k_cat values were 0.001 and 0.03 s^?1 and k_cat/K_M ratios were 8 and 1.6·10² M^?1 · s^?1, respectively. Cleavage of peptide F at acidic pH (5.5) was negligible. When BAM-22P was used as a substrate, Met⁵-Arg⁶-enkephalin was released, thus indicating cleavage between two arginine residues. At pH 8.5, K_M was 64 μM, k_cat was 4.5 s^?1, and the k_cat/K_M ratio was 7 · 10⁴ M^?1 · s^?1. At 5.5, the pH of the secretory granules, K_M, k_cat and k_cat/K_M were 184 μM, 1.9 s^?1 and 10⁴ M^?1 · s^?1, respectively. It is unlikely that peptide F could be a substrate for kallikrein in vivo; however, tissue kallikrein could aid in processing proenkephalin precursors such as BAM-22P by cleaving Arg-Arg peptide bonds.