Reaction of neprilysin (neutral endopeptidase) and thermolysin with cyclic peptides.
The reaction of neprilysin and thermolysin with a series of cyclic beta-turn peptides, varying in length from 6 to 14 residues, has been studied. All of the cyclic peptides bind to neprilysin with their affinity increasing from 113 microM for the 6-membered ring to 17 microM for the 14-membered ring. The 6-membered cyclic peptide was not hydrolyzed. However, kcat increased from 1.5 min-1 for the 8-membered cyclic peptide to 148 min-1 for the 14-membered cyclic peptide. With thermolysin binding of the 6- or 8-membered cyclic peptides was not detected. The Km values for the 10-, 12-, and 14-membered cyclic peptides were all in the 100 microM range. With thermolysin, kcat increased from 7 min-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-terminally directed sites. Modeling of the binding of a cyclic peptide, structurally 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 studies it is concluded that key factors which influence catalysis include not only peptide sequence, but the flexibility of the peptide and the orientation of the S'1 residue in a cyclic peptide.
Vijayaragahaven
J
J
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235, USA.
Tucker
M
M
Fehrentz
J A
JA
Isbell
D
D
Hersh
L B
LB
D000595:Amino Acid Sequence; D000818:Animals; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015260:Neprilysin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051381:Rats; D013379:Substrate Specificity; D013820:Thermolysin / Q000378:metabolism*
D010456:Peptides, Cyclic; D015260:Neprilysin; D013820:Thermolysin