Quantitative Characterization of Furin Specificity

Abstract

Furin, an essential mammalian proprotein processing enzyme of the kexin/furin family of subtilisin-related eukaryotic processing proteases, is implicated in maturation of substrates involved in development, signaling, coagulation, and pathogenesis. We examined the energetics of furin specificity using a series of peptidyl methylcoumarinamide substrates. In contrast to previous reports, we found that furin can cleave such substrates with kinetics comparable to those observed with extended peptides and physiological substrates. With the best of these hexapeptidyl methylcoumarinamides, furin displayedkcat/Kmvalues greater than 106 m−1s−1. Furin exhibited striking substrate inhibition with hexapeptide but not tetrapeptide substrates, an observation of significance to the evaluation of peptide-based furin inhibitors. Quantitative comparison of furin and Kex2 recognition at P1, P2, and P4 demonstrates that whereas interactions at P1 make comparable contributions to catalysis by the two enzymes, furin exhibited a ∼10-fold lesser dependence on P2 recognition but a 10–100-fold greater dependence on P4 recognition. Furin has recently been shown to exhibit P6 recognition and we found that this interaction contributes ∼1.4 kcal/mol toward catalysis independent of the nature of the P4 residue. We have also shown that favorable residues at P2 and P6 will compensate for less than optimal residues at either P1 or P4. The quantitative analysis of furin and Kex2 specificity sharply distinguish the nature of substrate recognition by the processing and degradative members of subtilisin-related proteases.