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 displayed k(cat)/K(m) values greater than 10(6) M(-1) s(-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 P(1), P(2), and P(4) demonstrates that whereas interactions at P(1) make comparable contributions to catalysis by the two enzymes, furin exhibited a approximately 10-fold lesser dependence on P(2) recognition but a 10-100-fold greater dependence on P(4) recognition. Furin has recently been shown to exhibit P(6) recognition and we found that this interaction contributes approximately 1.4 kcal/mol toward catalysis independent of the nature of the P(4) residue. We have also shown that favorable residues at P(2) and P(6) will compensate for less than optimal residues at either P(1) or P(4). 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.
Highlights
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
The 83-kDa anthrax protective antigen was hydrolyzed by furin with a kcat/Km 5000-fold higher than that for cleavage of a tetrapeptidyl amino(methyl)coumarinamide corresponding to the P1-P4 residues of the cleavage site in anthrax protective antigen [13, 17]
Summary
ENERGETICS OF SUBSTRATE DISCRIMINATION USING AN INTERNALLY CONSISTENT SET OF HEXAPEPTIDYL METHYLCOUMARINAMIDES*. A well defined physiological substrate has yet to be identified conclusively, coexpression studies, studies of furin-deficient cell lines, and studies using purified secreted, soluble furin have implicated furin in processing a wide range of proproteins including precursors of growth factors [5], receptors [6, 7], coagulation factors [8], metalloproteases [9], viral envelope glycoproteins [10, 11], and bacterial toxins [12] These studies, along with a limited number of studies using model peptide substrates, indicate that furin cleavage occurs C-terminal to an Arg that is preceded by one or more basic residues at P2, P4, or P6 [13, 14].1. Such substrates reacted much faster than related peptidyl-MCA2 substrates (MCA, methylcoumarinamide) and, based on these data as well as data for the cleavage of anthrax protective antigen, it has been suggested that furin may require specific residues C-terminal to the cleavage site for efficient catalysis, that simple chain extension N-terminal to the P4 site may enhance reactivity or that the bulky coumarin moiety of the peptidyl-MCA substrates may interfere with binding through interactions with the S1Ј position [17]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
