THIMET OLIGOPEPTIDASE SPECIFICITY - EVIDENCE OF PREFERENTIAL CLEAVAGENEAR THE C-TERMINUS AND PRODUCT INHIBITION FROM KINETIC-ANALYSIS OF PEPTIDE HYDROLYSIS

The substrate-size specificity of human thimet oligopeptidase (EC 3.4. 24.15) was investigated with oligomers of glycyl-prolyl-leucine (GPL)( n) where n = 2, 3, 4 and 5. These peptides were cleaved only at Leu-Gl y bonds to give GPL as the single final product. Hydrolysis was most r apid with (GPL)(3) and slowest with (GPL)(5). The more water-soluble o ligomers of Gly-Hyp-Leu showed the same trend. (Gly-Hyp-Leu)(6) was no t hydrolysed, consistent with the previous finding that substrates lar ger than 17 amino acids are not cleaved by thimet oligopeptidase. The cleavage of (GPL)(3) to GPL fitted a sequential first-order model. Fir st-order kinetics were unexpected as the initial substrate concentrati on was greater than K-m. The anomaly was also seen during the cleavage of bradykinin and neurotensin, and in these cases first-order behavio ur was due to potent competitive inhibition by the C-terminal product. The sequential mechanism for (GPL)(3) breakdown by thimet oligopeptid ase does not discriminate between initial cleavages towards the N- or C-terminus. As isoleucine is an unfavourable residue in P1, substrates were made in which selected leucine residues were replaced by isoleuc ine. GPL--GPI--GPL (where -- represents the bond between the tripeptid e units) was resistant to hydrolysis and GPI--GPL--GPL was cleaved onl y at the -Leu-Gly- bond. Experiments with isoleucine-containing analog ues of (Gly-Hyp-Leu)(4) showed that thimet oligopeptidase preferred to cleave these peptides near the C-terminus.