Synthetic model and bioactive peptides as potential substrates for enteropeptidase

Enteropeptidase (enterokinase EC 3.4.21.9), catalyzing trypsinogen activation, exhibits unique properties for high efficiency hydrolysis of the polypeptide chain after the N-terminal tetraaspartyl-lysyl sequence. This makes it a convenient tool for the processing of fusion proteins containing this sequence. We found the enteropeptidase-catalysing degradation of some bioactive peptides: cattle hemoglobin beta-chain fragments Hb (2–8) (LTAEEKA) and Hb (1–9) (MLTAEEKAA), human angiotensin II (DRVYIHPF) (AT). Model peptideswith truncated linker WDDRG and WDDKG also were shown to be susceptible to enteropeptidase action. Kinetic parameters ofenteropeptidase hydrolysis for these substrates were determined.Km values for all substrates with truncated linker (∼10-3 M) are an order of magnitude higher thancorresponding values for typical enteropeptidase artificial peptide or fusion protein substrates with full enteropeptidase linker –DDDDK– (Km ∼ 10-4 M). kcat values for AT, Hb (2–8), WDDRG and WDDKG are ∼30–40 min-1. But one additional amino acid residue at both N- and C-terminus of Hb (2–8) results in a drastic increase of hydrolysis efficiency: kcat value for Hb (1–9) is 1510 min-1. Recent study demonstrates the possibility of undesirable cleavage of target peptides or proteins containing the above-mentioned truncated linker sequences; further, the ability of enteropeptidase to hydrolyse specifically several biologically active peptides in vitro along with its unique natural substrate trypsinogen was demonstrated.