Human tryptases alpha and beta/II are functionally distinct due, in part, to a single amino acid difference in one of the surface loops that forms the substrate-binding cleft

Tryptases alpha and beta/II were expressed in insect cells to try to ascert ain why human mast cells express these two nearly identical granule proteas es, In contrast to that proposed by others, residue -3 in the propeptide di d not appear to be essential for the three-dimensional folding, post-transl ational modification, and/or activation of this family of serine proteases, Both recombinant tryptases were functional and bound the active-site inhib itor diisopropyl fluorophosphate, However, they differed in their ability t o cleave varied trypsin-susceptible chromogenic substrates, Structural mode ling analyses revealed that tryptase alpha differs from tryptase beta/II in that it possesses an Asp, rather than a Gly, in one of the loops that form its substrate-binding cleft. A site-directed mutagenesis approach was ther efore carried out to determine the importance of this residue. Because the D215G derivative of tryptase alpha exhibited potent enzymatic activity agai nst fibrinogen and other tryptase beta/II-susceptible substrates, Asp(215) dominantly restricts the substrate specificity of tryptase alpha. These dat a indicate for the first time that tryptases alpha and beta/II are function ally different human proteases, Moreover, the variation of just a single am ino acid in the substrate-binding cleft of a tryptase can have profound con sequences in the regulation of its enzymatic activity and/or substrate pref erence.