Nardilysin cleaves peptides at monobasic sites.
Nardilysin (N-arginine dibasic convertase, EC 3.4.24.61) was first identified on the basis of its ability to cleave peptides containing an arginine dibasic pair, i.e., Arg-Arg or Arg-Lys. However, it was observed that an aromatic residue adjacent to the dibasic pair (i.e., Phe-Arg-Lys) could alter the cleavage site. In this study we determined whether nardilysin can cleave peptides at a single basic residue. Nardilysin cleaves beta-endorphin at the monobasic site, Phe(17)-Lys(18), with a k(cat)/K(m) of 2 x 10(8) M(-)(1) min(-)(1). This can be compared to a k(cat)/K(m) of 8.5 x 10(8) M(-)(1) min(-)(1) for cleavage between a dibasic pair in dynorphin B-13. Nardilysin also cleaves calcitonin at His-Arg and somatostatin-14 at Cys-Lys. We examined the hydrolysis of fluorogenic peptides based on the beta-endorphin 12-24 sequence, Abz-T-P-L-V-T-L-X(1)-X(2)-N-A-I-I-K-Q-EDDnp. Nardilysin hydrolyzes the peptides when X(1)-X(2) = F-K, F-R, W-K, M-K, Y-K, and L-K. The kinetics of cleavage at F-K and F-R are similar; however, K-F is not hydrolyzed. Nardilysin cleaves at two monobasic sites M-K and F-R of the kallidin model peptide Abz-MISLMKRPPGFSPFRSSRI-NH(2), releasing desArg(10) kallidin (KRPPGFSPF). However, nardilysin does not release desArg(10) kallidin from the physiological precursor low molecular weight kininogen. These studies extend the range of potential substrates for nardilysin and further substantiate that nardilysin is a true peptidase.
Chow
K Martin
KM
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536-0298, USA.
Oakley
Oliver
O
Goodman
Jack
J
Ma
Zhangliang
Z
Juliano
Maria Aparecida
MA
Juliano
Luiz
L
Hersh
Louis B
LB
D000595:Amino Acid Sequence; D024322:Amino Acids, Aromatic / Q000737:chemistry; D024361:Amino Acids, Basic / Q000737:chemistry*; D000599:Amino Acids, Diamino / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D007704:Kininogens / Q000737:chemistry; D008666:Metalloendopeptidases / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013004:Somatostatin / Q000737:chemistry; D013552:Swine; D001615:beta-Endorphin / Q000737:chemistry
D024322:Amino Acids, Aromatic; D024361:Amino Acids, Basic; D000599:Amino Acids, Diamino; D007704:Kininogens; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D013004:Somatostatin; D001615:beta-Endorphin; D008666:Metalloendopeptidases; C087976:nardilysin