Favourable Side-Chain Orientation of Cleavage Site Dibasic Residues of Prohormone in Proteolytic Processing by Prohormone Convertase 1/3

Eur J Biochem. 2000 Mar;267(6):1626-33. doi: 10.1046/j.1432-1327.2000.01154.x.

Abstract

Previous studies using selectively modified pro-ocytocin/neurophysin substrate analogues and the purified metalloprotease, pro-ocytocin/neurophysin convertase (magnolysin; EC 3.4 24.62), have shown that dibasic cleavage site processing is associated with a prohormone sequence organized in a beta-turn structure. We have used various peptide analogues of the pro-ocytocin-neurophysin processing domain, and recombinant prohormone convertase 1/3, to test the validity of this property towards this member of the family of prohormone convertases (PCs). The enzymatic cleavage analysis and kinetics showed that: (a) with methyl amide (N-Met) modification, a secondary structure beta-turn breaker, the enzyme substrate interaction was abolished; (b) cleavage was favoured when the dibasic substrate side-chains were oriented in opposite directions; (c) the amino acid present at the P'1 position is important in the enzyme-substrate interaction; (d) the flexibility of the peptide substrate is necessary for the interaction; (e) Addition of dimethylsulfoxide to the cleavage assay favoured the cleavage of the pro-ocytocin/neurophysin large substrate over that of the smaller one pGlu-Arg-Thr-Lys-Arg-methyl coumarin amide. These data allowed us to conclude that proteolytic processing of pro-ocytocin-related peptide substrates by PC1/3 as well as by the metalloenzyme, magnolysin, involves selective recognition of precise cleavage site local secondary structure by the processing enzyme. It is hypothesized that this may represent a general property of peptide precursor proteolytic processing systems.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Aspartic Acid Endopeptidases / metabolism*
  • Catalysis
  • Dimethyl Sulfoxide / pharmacology
  • Endopeptidases / metabolism*
  • Kinetics
  • Molecular Sequence Data
  • Oxytocin / analogs & derivatives*
  • Oxytocin / biosynthesis*
  • Oxytocin / chemistry
  • Oxytocin / metabolism
  • Peptide Fragments / metabolism
  • Proprotein Convertases
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / metabolism
  • Structure-Activity Relationship
  • Substrate Specificity

Substances

  • Peptide Fragments
  • Recombinant Fusion Proteins
  • Oxytocin
  • oxytocin, Gly-Lys-Arg-
  • Endopeptidases
  • Proprotein Convertases
  • Aspartic Acid Endopeptidases
  • pro-ocytocin-neurophysin convertase
  • Dimethyl Sulfoxide