Definition of the Extended Substrate Specificity Determinants for β-Tryptases I and II

• Published in: The Journal of biological chemistry 2001-09, Vol.276 (37), p.34941-34947
• Main Authors: Harris, Jennifer L., Niles, Andrew, Burdick, Keith, Maffitt, Mark, Backes, Bradley J., Ellman, Jonathan A., Kuntz, Irwin, Haak-Frendscho, Mary, Craik, Charles S.
• Format: Article
• Language: English
• Subjects: Humans; Isoenzymes - metabolism; Pichia - genetics; Recombinant Proteins - metabolism; Serine Endopeptidases - chemistry; Serine Endopeptidases - metabolism; Serine Proteinase Inhibitors - pharmacology; Substrate Specificity; Tryptase beta I; Tryptase beta II; Tryptases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M102997200

Tryptases βI and βII were heterologously expressed and purified in yeast to functionally characterize the substrate specificity of each enzyme. Three positional scanning combinatorial tetrapeptide substrate libraries were used to determine the primary and extended substrate specificity of the proteases. Both enzymes have a strict primary preference for cleavage after the basic amino acids, lysine and arginine, with only a slight preference for lysine over arginine. βI and βII tryptase share similar extended 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 β-tryptases reveal that selectivity is highly dependent on ground state substrate binding. Coupled with the functional determinants, structural determinants of tryptase substrate specificity were identified. Molecular docking of the preferred substrate sequence to the three-dimensional tetrameric tryptase structure reveals a novel extended substrate binding mode that involves interactions from two adjacent protomers, including P4 Thr-96′, P3 Asp-60B′ and Glu-217, and P1 Asp-189. Based on the determined substrate information, a mechanism-based tetrapeptide-chloromethylketone inhibitor was designed and shown to be a potent tryptase inhibitor. Finally, the cleavage sites of several physiologically relevant substrates of β-tryptases show consistency with the specificity data presented here.