Fluorescent peptidyl substrates as an aid in studying the substrate specificity of human prohormone convertase PC1 and human furin and designing a potent irreversible inhibitor.
The substrate specificities of two human prohormone convertases, furin and PC1, were examined with a series of 7-amino-4-methylcoumarinamide (MCA) containing peptidyl substrates. Using acetyl-Arg-Ser-Lys-Arg-MCA as model, P4 Arg substitution by Lys or Orn resulted for furin in a 538- and a 280-fold lower kcat/Km value, but only in a 14- and 18-fold decrease for PC1. Substitution of P3 Ser by either Pro, Glu, or Lys does not modify significantly the kcat/Km value for PC1, whereas furin activity is seriously impaired by the Glu substitution. Elongating the peptidyl sequence up to the P8 position decreases the kcat/Km value for furin but not for PC1. In both the P3 or P5 Glu substitution, the decrease of kcat/Km was due primarily to lower kcat rather than higher Km, possibly because of the presence of a negatively charged side chain. Finally, an octapeptidyl chloromethane derivative proved to be a potent irreversible inhibitor of either PC1 and ruin. The 811-fold difference in the apparent Kapp/[I] (1.63 x 10(6) s-1 m-1), and kcat/Km determined with the corresponding peptidyl MCA substrate (2.01 x 10(3) s-1 m-1), supports the proposal that cleavage of the acylenzyme represents the rate-limiting step for PC1 and furin.
Jean
F
F
Neuropeptides Structure and Metabolism Laboratory, Clinical Research Institute of Montréal (affiliated with the University of Montréal), Québec, Canada.
Boudreault
A
A
Basak
A
A
Seidah
N G
NG
Lazure
C
C
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002384:Catalysis; D003374:Coumarins / Q000138:chemical synthesis / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000378:metabolism*; D045683:Furin; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D043484:Proprotein Convertases; D013379:Substrate Specificity; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D003374:Coumarins; D005456:Fluorescent Dyes; D009842:Oligopeptides; D043484:Proprotein Convertases; D013381:Subtilisins; D045683:Furin; D016282:Aspartic Acid Endopeptidases; C028743:7-amino-4-methylcoumarin