Structural features that make oligopeptides susceptible substrates for hydrolysis by recombinant thimet oligopeptidase.

A systematic analysis of the peptide sequences and lengths of several homologues of bioactive peptides and of a number of quenched-fluorescence (qf) opioid- and bradykinin-related peptides was performed to determine the main features leading the oligopeptides to hydrolysis by the recombinant rat testis thimet oligopeptidase (EC 3.4.24.15). The results indicate that a minimum substrate length of six amino acids is required and that among the oligopeptides six to thirteen amino acid residues long, their susceptibility as substrates is highly variable. Thimet oligopeptidase was able to hydrolyse, with similar catalytic efficiency, peptide bonds having hydrophobic or hydrophilic amino acids as well as proline in the P1 position of peptides, ranging from a minimum of six to a maximum of approximately thirteen amino acid residues. An intriguing observation was the shift of the cleavage site, at a Leu-Arg bond in qf dynorphin-(2-8) [qf-Dyn2-8; Abz-GGFLRRV-EDDnp, where Abz stands for o-aminobenzoyl and EDDnp for N-(2,4-dinitrophenyl)ethylenediamine], to Arg-Arg in qf-Dyn2-8Q, in which Gln was substituted for Val at its C-terminus. Similarly, a cleavage site displacement was also observed with the hydrolysis of the internally quenched-fluorescence bradykinin analogues containing Gln at the C-terminal position, namely Abz-RPPGFSPFR-EDDnp and Abz-GFSPFR-EDDnp are cleaved at the Phe-Ser bond, but Abz-RPPGFSPFRQ-EDDnp and Abz-GFSPFRQ-EDDnp are cleaved at the Pro-Phe bond.