Quantitative characterization of furin specificity. Energetics of substrate discrimination using an internally consistent set of hexapeptidyl methylcoumarinamides.
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.
Krysan
D J
DJ
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Rockwell
N C
NC
Fuller
R S
RS
D000577:Amides; D000595:Amino Acid Sequence; D003374:Coumarins / Q000378:metabolism*; D004734:Energy Metabolism; D045683:Furin; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D043484:Proprotein Convertases; D029701:Saccharomyces cerevisiae Proteins; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000577:Amides; D003374:Coumarins; D029701:Saccharomyces cerevisiae Proteins; D015842:Serine Proteinase Inhibitors; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae; D045683:Furin