Substrate specificity characterization of recombinant metallo oligo-peptidases thimet oligopeptidase and neurolysin

We report a systematic and detailed analysis of recombinant neurolysin (EC 3.4.24.16) specificity in parallel with thimet oligopeptidase (TOP, EC 3.4. 24.15) using Bk sequence and its C- and N-terminal extensions as in human k ininogen as motif for synthesis of internally quenched fluorescent substrat es, The influence of the substrate size was investigated, and the longest p eptide susceptible to TOP and neurolysin contains 17 amino acids. The speci ficities of both oligopeptidases to substrate sites P-4 to P-3' were also c haracterized in great detail using seven series of peptides based on Abz-GF SPFRQ-EDDnp taken as reference substrate. Most of the peptides were hydroly zed at the bond corresponding to P-4-F-5 in the reference substrate and som e of them were hydrolyzed at this bond or at F-2-S-3 bond. No restricted sp ecificity was found for P-1' as found in thermolysin as well for P-1 substr ate position, however the modifications at this position (P-1) showed to ha ve large influence on the catalytic constant and the best substrates for TO P contained at P-1, Phe, Ala, or Arg and for neurolysin Asn or Arg. Some am ino acid residues have large influence on the K-m constants independently o f its position. On the basis of these results, we are hypothesizing that so me amino acids of the substrates can bind to different sub-sites of the enz yme fitting P-F or F-S bond, which requires rapid interchange for the diffe rent forms of interaction and convenient conformations of the substrate in order to expose and fit the cleavage bonds in correct position for an effic ient hydrolysis. Finally, this plasticity of interaction with the substrate s can be an essential property for a class of cytosolic oligopeptidases tha t are candidates to participate in the selection of the peptides to be pres ented by the MHC class I.