Dissecting substrate recognition by thrombin using the inactive mutant S195A.
The catalytically inactive mutant S195A was used to study the interaction of thrombin with substrates under equilibrium conditions. By monitoring changes in intrinsic fluorescence, we measured dissociation constants for a variety of synthetic substrates, PAR peptides and the inhibitor PPACK. The S195A mutant retains the Na(+)-binding properties of the wild type, and substrate binding to the mutant is enhanced by the presence of Na(+). Temperature dependence studies allowed calculation of the thermodynamic parameters of substrate binding at the active site and showed a negligible deltaC(p). Titration of synthetic substrates carrying substitutions at the P1-P3 positions revealed energetics consistent with the specificity hierarchy identified in hydrolysis by the wild type. Titration with PAR peptides, which interact with both the active site and exosite I of thrombin, also showed consistency with the results obtained with the wild type at steady state. These findings demonstrate that inactive mutants of enzymes make it possible to dissect the equilibrium components linked to substrate binding and complement information on the kinetic properties of the wild type.
Krem
Maxwell M
MM
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St. Louis, MO 63110, USA.
Di Cera
Enrico
E
D000465:Algorithms; D000590:Amino Acid Chloromethyl Ketones / Q000737:chemistry; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013917:Thrombin / Q000737:chemistry* / Q000235:genetics*
D000590:Amino Acid Chloromethyl Ketones; D005456:Fluorescent Dyes; D008024:Ligands; D013917:Thrombin; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone