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-fluoresce nce (qf) opioid- and bradykinin-related peptides was performed to dete rmine the main features leading the oligopeptides to hydrolysis by the recombinant rat testis thimet oligopeptidase (EC 3.4.24.15). The resu lts indicate that a minimum substrate length of six amino acids is req uired and that among the oligopeptides six to thirteen amino acid resi dues long, their susceptibility as substrates is highly variable. Thim et oligopeptidase was able to hydrolyse, with similar catalytic effici ency, peptide bonds having hydrophobic or hydrophilic amino acids as w ell 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-Ar g bond in qf dynorphin (2-8) [qf-Dyn(2-8); Abz-GGFLRRV-EDDnp, where Ab z stands for o-aminobenzoyl and EDDnp for N-(2,4-dinitrophenyl) ethyle nediamine], to Arg-Arg in qf-Dyn(2-8)Q, in which Gln was substituted f or Val at its C-terminus. Similarly, a cleavage site displacement was also observed with the hydrolysis of the internally quenched-fluoresce nce bradykinin analogues containing Gin at the C-terminal position, na mely Abz-RPPGFSPFR-EDDnp and Abz-GFSPFR-EDDnp are cleaved at the Phe-S er bond, but Abz-RPPGFSPFRQ-EDDnp and Abz-GFSPFRQ-EDDnp are cleaved at the Pro-Phe bond.