REGULATION OF HUMAN MAST-CELL P-TRYPTASE - CONVERSION OF INACTIVE MONOMER TO ACTIVE TETRAMER AT ACID PH

At neutral pH, human mast cell beta-tryptase is stabilized in its enzy matically active, tetrameric form by heparin, and resists inhibition b y biologic protease inhibitors. After dissociation of beta-tryptase fr om heparin, active tetramers rapidly convert to inactive monomers in a n isotonic, neutral pH environment. Although reversible transition sta tes probably exist during this conversion, once inactive monomers form , addition of heparin fails to reconstitute active tetramer at neutral pH. The current study shows that complete reactivation of inactive mo nomers can occur at acidic pH in a heparin-independent manner. The res pective rate-determining steps for formation of tetramer and active en zyme from inactive monomers exhibit second and first order kinetics ba sed on an analysis of initial reaction rates. The optimal pH for tetra mer formation and reactivation is about 6, suggesting His residues pla y a critical role. The optimal ionic strength equivalent is 160 mM NaC l; and the optimal temperature range is 22 degrees C to 37 degrees C. We propose a sequential three-step reactivation process at acidic pH, dimerization of monomers (rate-determining second order step), rapid f ormation of inactive tetramers, and slow formation of active tetramers (overall rate-determining first order step). Whether reactivation of human beta-tryptase occurs at extracellular or intracellular sites, wh ere the pH is acidic in vivo, should be considered.