SPONTANEOUS INACTIVATION OF HUMAN TRYPTASE INVOLVES CONFORMATIONAL-CHANGES CONSISTENT WITH CONVERSION OF THE ACTIVE-SITE TO A ZYMOGEN-LIKE STRUCTURE

The conformational changes accompanying spontaneous inactivation and d extran sulfate (DS) mediated reactivation of the serine protease human tryptase were investigated by analysis of (i) intrinsic fluorescence, (ii) inhibitor binding, and (iii) catalytic efficiency. Spontaneous i nactivation produced a marked decrease in fluorescence emission intens ity that was reversed by the addition of DS. Fluorescence decreases at high (4.0 mu M) and low (0.1 mu M) tryptase concentrations were simil ar at early times and coincided with loss of enzymatic activity but de viated significantly from activity loss at later times by showing a di fference in the extent of change. The fluorescence losses were best de scribed by a two-step kinetic model in which the major decrease correl ated to activity loss (t(1/2) of 4.3 min in 0.2 M NaCl, pH 6.8, 30 deg rees C) and was followed by a further decrease (t(1/2) approximate to 60 min) whose extent differed with tryptase concentration. The ability to bind the competitive inhibitor p-aminobenzamidine was reversibly l ost upon spontaneous inactivation, providing evidence for conformation al changes affecting the major substrate binding site (S1-pocket). Est imation of catalytic efficiency using an active site titrant showed th at the specific activity of tryptase remained unchanged upon inactivat ion and reactivation. Return of enzymatic activity, intrinsic fluoresc ence, and the S1 pocket appeared to occur in the same time frame (t(1/ 2) approximate to 3 min). These studies indicate that spontaneous inac tivation involves reversible changes which convert the active site to a nonfunctional state. The association of activity loss with an intrin sic fluorescence decrease and loss of the S1-pocket is consistent with the disruption of a critical ionic bond at the active site. Formation of this ionic bond is the basis of zymogen activation for the chymotr ypsin family of serine proteases.