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

Abstract

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 k(cat)/K(m) 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 k(cat)/K(m) value for PC1, whereas furin activity is seriously impaired by the Glu substitution. Elongating the peptidyl sequence up to the P8 position decreases the k(cat)/K(m) value for furin but not for PC1. In both the P3 or P5 Glu substitution, the decrease of k(cat)/K(m) was due primarily to lower k(cat) rather than higher K(m), 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 furin. The 811-fold difference in the apparent K(app)/[I] (1.63 x 106 s-1 M-1), and k(cat)/K(m) determined with the corresponding peptidyl MCA substrate (2.01 x 103 s-1 M-1), supports the proposal that cleavage of the acylenzyme represents the rate-limiting step for PC1 and furin.