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J Biol Chem, Vol. 274, Issue 28, 19670-19676, July 9, 1999

Human Tryptases  and /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

Chifu Huang^§, Lixin Li^¶, Steven A. Krilis^¶, Kara Chanasyk^§, Yinzi Tang^§, Zhiqin Li^§, John E. Hunt, and Richard L. Stevens^§

From the  Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, the ^§ Department of Medicine, Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Boston, Massachusetts 02115, the ^¶ Department of Medicine, the University of New South Wales and Department of Immunology, Allergy, and Infectious Disease, St. George Hospital, Kogarah, New South Wales 2217, Australia, and the  School Pathology, the University of New South Wales, Randwick, New South Wales 2052, Australia

Tryptases  and /II were expressed in insect cells to try to ascertain why human mast cells express these two nearly identical granule proteases. In contrast to that proposed by others, residue 3 in the propeptide did not appear to be essential for the three-dimensional folding, post-translational modification, and/or activation of this family of serine proteases. Both recombinant tryptases were functional and bound the active-site inhibitor diisopropyl fluorophosphate. However, they differed in their ability to cleave varied trypsin-susceptible chromogenic substrates. Structural modeling analyses revealed that tryptase  differs from tryptase /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 therefore carried out to determine the importance of this residue. Because the D215G derivative of tryptase  exhibited potent enzymatic activity against fibrinogen and other tryptase /II-susceptible substrates, Asp²¹⁵ dominantly restricts the substrate specificity of tryptase . These data indicate for the first time that tryptases  and /II are functionally different human proteases. Moreover, the variation of just a single amino acid in the substrate-binding cleft of a tryptase can have profound consequences in the regulation of its enzymatic activity and/or substrate preference.

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