The Interaction of Human Tryptase-β with Small Molecule Inhibitors Provides New Insights into the Unusual Functional Instability and Quaternary Structure of the Protease

• Published in: Biochemistry (Easton) Vol. 44; no. 9; pp. 3580 - 3590
• Main Authors: Selwood, Trevor, Smolensky, Holly, McCaslin, Darrell R, Schechter, Norman M
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 08.03.2005
• Subjects: Aprotinin - chemistry; Aprotinin - metabolism; Binding, Competitive; Chromatography, Gel; Enzyme Reactivators - chemistry; Enzyme Reactivators - metabolism; Enzyme Stability; Humans; Hydrogen-Ion Concentration; Hydrolysis; Leupeptins - chemistry; Leupeptins - metabolism; Protein Structure, Quaternary; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Serine Endopeptidases - chemistry; Serine Endopeptidases - metabolism; Serine Proteinase Inhibitors - chemistry; Serine Proteinase Inhibitors - metabolism; Sulfones - chemistry; Sulfones - metabolism; Tryptases
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi047765u

Human tryptase-β (HTβ) is a serine protease with an atypical tetrameric structure and an unusual dependence on heparin binding or high salt for functional and structural stability. In the absence of heparin and at physiological salt, pH, and temperature, HTβ rapidly loses activity by a reversible process that we have called spontaneous inactivation. The role of tetramer dissociation in this process is controversial. Using small irreversible or competitive inhibitors of HTβ as stabilizing ligands, we were able to examine tetramer stability under inactivating (decay) conditions in the absence of heparin and to define further the process of spontaneous inactivation. Size exclusion chromatography showed that interaction with inhibitors stabilized the tetramer. Using sedimentation equilibrium, spontaneously inactivated HTβ (si-HTβ) was shown to be a destabilized tetramer that dissociates upon dilution and which in the presence of a competitive inhibitor re-formed a stable tetramer. Addition of inhibitors to si-HTβ rescued catalytic activity as was shown after inhibitor displacement. At high concentrations of si-HTβ (4−5 μM), the binding of inhibitor alone provided sufficient free energy for complete reactivation and tetramer stabilization, whereas at low si-HTβ concentration (0.1 μM) where the destabilized tetramer would be mostly dissociated, reactivation required more free energy which was provided by the binding of both an inhibitor and heparin. The results demonstrate that HTβ is a tetramer in the absence of heparin and that tetramer dissociation is a consequence of and not a prerequisite for inactivation. Heparin binding likely stabilizes the tetramer by favoring a functionally active conformation with stable intersubunit contacts, rather than by simply cross-linking active monomers.