Differential utilization of enzyme-substrate interactions for acylation but not deacylation during the catalytic cycle of Kex2 protease.
Kex2 protease from Saccharomyces cerevisiae is the prototype for a family of eukaryotic proprotein processing proteases belonging to the subtilase superfamily of serine proteases. Kex2 can be distinguished from degradative subtilisins on the basis of stringent substrate specificity and distinct pre-steady-state behavior. To better understand these mechanistic differences, we have examined the effects of substrate residues at P(1) and P(4) on individual steps in the Kex2 catalytic cycle with a systematic series of isosteric peptidyl amide and ester substrates. The results demonstrate that substrates based on known, physiological cleavage sites exhibit high acylation rates (> or =550 s(-1)) with Kex2. Substitution of Lys for the physiologically correct Arg at P(1) resulted in a > or =200-fold drop in acylation rate with almost no apparent effect on binding or deacylation. In contrast, substitution of the physiologically incorrect Ala for Nle at P(4) resulted in a much smaller defect in acylation and a modest but significant effect on binding with Lys at P(1). This substitution also had no effect on deacylation. These results demonstrate that Kex2 utilizes enzyme-substrate interactions in different ways at different steps in the catalytic cycle, with the S(1)-P(1) contact providing a key specificity determinant at the acylation step.
Rockwell
N C
NC
Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
Fuller
R S
RS
D000215:Acylation; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D005470:Fluorometry; D045683:Furin; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008956:Models, Chemical; D043484:Proprotein Convertases; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D013381:Subtilisins / Q000378:metabolism*
D029701:Saccharomyces cerevisiae Proteins; D001120:Arginine; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae; D045683:Furin; D008239:Lysine