Characterization of Three Distinct Catalytic Forms of Human Tryptase-β: Their Interrelationships and Relevance

Abstract

Human tryptase-beta (HTbeta) is a serine protease that is isolated as a tetramer of four identical, catalytically active subunits (HTbeta-AT). Tetramer activity is not affected by protein-based physiological inhibitors but instead may be regulated by an autoinactivation process we have called spontaneous inactivation. Unless stabilized by heparin or high salt, the active tetramer converts to an inactive state consisting of an inactive-destabilized tetramer that reversibly dissociates to inactive monomers upon dilution. We refer to this mixture of inactive species as siHTbeta and show in this study that previous reports of monomeric catalytic forms are derived from this mixture. siHTbeta itself did not hydrolyze model substrates but unlike the tetramer did react slowly with the serpin alpha2-antiplasmin (alpha2-AP), suggesting a highly limited catalytic potential. In the presence of heparin (or other highly charged polysaccharides), we demonstrate that siHTbeta formed a well-defined complex with the heparin (siHTbeta-HC) that reacted 70-fold faster with alpha2-AP than siHTbeta and also hydrolyzed model substrates and fibrinogen. Formation of siHTbeta-HC was limited to dilute subunit solutions since high subunit concentrations resulted in the reformation of the active tetramer. By compensating for changes in the strength of heparin binding, siHTbeta-HC could be formed over the pH range of 6.0-8.5. The activity dependence on pH was bell-shaped with highest activity between pH 6.8 and pH 7.5. In contrast, HTbeta-AT activity showed no dependence upon heparin, increased over the pH range of 6.0-8.5, and was much higher than that of siHTbeta-HC especially above pH 6.8. HTbeta-AT incubated with excess heparin of different size (3-15 kDa) was functionally stable at 25 degrees C but lost activity regardless of heparin size at 37 degrees C above pH 6.8. The change in stability, which is likely due to weakened heparin binding, did not result in the formation of a stable catalytic monomer. These results confirm that siHTbeta is for the most part an inactive species and that any active monomer is a consequence of heparin binding to siHTbeta under dilute conditions where unfavorable thermodynamics and/or kinetics restrict formation of active tetramer. Heparin binding under these conditions drives a limited reorganization of the active site to a conformation that is catalytic but not the equivalent of a subunit within the active tetramer.