Definition of the extended substrate specificity determinants for beta-tryptases I and II

Tryptases betaI and beta II were heterologously expressed and purified in y east to functionally characterize the substrate specificity of each enzyme. Three positional scanning combinatorial tetrapeptide substrate libraries w ere used to determine the primary and extended substrate specificity of the proteases. Both enzymes have a strict primary preference for cleavage afte r the basic amino acids, lysine and arginine, with only a slight preference for lysine over arginine. betaI and beta II tryptase share similar extende d substrate specificity, with preference for proline at P4, preference for arginine or lysine at P3, and P2 showing a slight preference for asparagine . Measurement of kinetic constants with multiple substrates designed for be ta -tryptases reveal that selectivity is highly dependent on ground state s ubstrate binding. Coupled with the functional determinants, structural dete rminants of tryptase substrate specificity were identified. Molecular docki ng of the preferred substrate sequence to the three-dimensional tetrameric tryptase structure reveals a novel extended substrate binding mode that inv olves interactions from two adjacent protomers, including P4 Thr-96', P3 As p-60B' and Glu-217, and P1 Asp-189. Based on the determined substrate infor mation, a mechanism-based tetrapeptide-chloromethylketone inhibitor was des igned and shown to be a potent tryptase inhibitor. Finally, the cleavage si tes of several physiologically relevant substrates of beta -tryptases show consistency with the specificity data presented here.