Favourable side-chain orientation of cleavage site dibasic residues of prohormone in proteolytic processing by prohormone convertase 1/3.
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.
Brakch
N
N
Division of Hypertension and Vascular Medicine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland. Noureddine.Brakch@chuv.hopvd.ch
Rholam
M
M
Simonetti
M
M
Cohen
P
P
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D002384:Catalysis; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010121:Oxytocin / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D043484:Proprotein Convertases; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D010121:Oxytocin; C046278:oxytocin, Gly-Lys-Arg-; D010450:Endopeptidases; D043484:Proprotein Convertases; D016282:Aspartic Acid Endopeptidases; C056535:pro-ocytocin-neurophysin convertase; D004121:Dimethyl Sulfoxide