REACTION OF NEPRILYSIN (NEUTRAL ENDOPEPTIDASE) AND THERMOLYSIN WITH CYCLIC-PEPTIDES

The reaction of neprilysin and thermolysin with a series of cyclic bet a-turn peptides, varying in length from 6 to 14 residues, has been stu died. All of the cyclic peptides bind to neprilysin with their affinit y increasing from 113 mu M for the 6-membered ring to 17 mu M for the 14-membered ring. The 6-membered cyclic peptide was not hydrolyzed. Ho wever, k(cat) increased from 1.5 min(-1) for the 8-membered cyclic pep tide to 148 min(-1) for the 14-membered cyclic peptide, With thermolys in binding of the 6- or 8-membered cyclic peptides was not detected. T he K-m values for the 10-, 12-, and 14-membered cyclic peptides were a ll in the 100 mu M range. With thermolysin, k(cat) increased from 7 mi n(-1) for the 10-membered cyclic peptide to 27,000 min(-1) for the 14- membered cyclic peptide. Cyclic peptides were all cleaved at N-termina lly directed sites. Modeling of the binding of a cyclic peptide, struc turally similar to the 12-membered cyclic beta-turn peptide described above, into the active site of thermolysin shows that only half of the substrate makes contact with the enzyme and that only residues on one side of the peptide could fit into the active site, From these studie s it is concluded that key factors which influence catalysis include n ot only peptide sequence, but the flexibility of the peptide and the o rientation of the S'1 residue in a cyclic peptide. (C) 1995 Academic P ress, Inc.