Diverse stability and catalytic properties of human tryptase alpha and beta isoforms are mediated by residue differences at the S1 pocket.

Recombinant human tryptases (rHTs) corresponding to alpha and beta isoforms were characterized. rHTbeta was similar to tryptase isolated from skin (HST); it was a tetramer, hydrolyzed model substrates efficiently, and was functionally unstable when incubated under physiological conditions. Activity was lost rapidly (t(1/2) approximately 1 min) by a reversible process similar to that observed for the spontaneous inactivation of HST. Circular dichroism (CD) and intrinsic fluorescence emission (IFE) spectra of active rHTbeta corresponded to those of active HST and upon spontaneous inactivation IFE decreased in parallel to activity loss. rHTalpha differed from HST in catalytic ability and stability. rHTalpha did not react with model substrates, an active site titrant, or a competitive inhibitor of HST/rHTbeta. IFE and CD spectra were similar to those of the active and not the spontaneously inactivated form of HST. Under physiological conditions, rHTalpha IFE decreased at a rate 900-fold slower than that observed for HST, and rHTalpha remained tetrameric when examined by size exclusion chromatography at physiological salt concentration. Thus, rHTalpha is a stable "inactive" form of HT. Three active site variants of rHTalpha, K192Q, D216G, and K192Q-D216G were characterized. Residues 192 and 216 (chymotrypsinogen numbers for residues 191 and 215 of rHTalpha) lie at the entrance to the primary specificity (S1) pocket, and the mutations converted them to the residues of HTbeta. While K192Q displayed the same properties as rHTalpha, the catalytic and stability characteristics of D216G and K192Q-D216G progressively approached those of HST. Thus, the contrasting stability/activity properties of rHTalpha and rHTbeta are largely related to differences at the S1 pocket. On the basis of the properties of the variants, we suggest that the side chain of Asp216 is blocking and stabilizing the S1 pocket and that this stabilization is sufficient to prevent spontaneous inactivation.