VARIANTS OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR WITH SUBSTANTIALLY ENHANCED RESPONSE AND SELECTIVITY TOWARD FIBRIN COFACTORS

Unlike most proteases, tissue-type plasminogen activator (t-PA) is not synthesized as an inactive precursor or zymogen, Instead, the single- chain ''proenzyme'' form of t-PA possesses very significant catalytic activity, Recent investigations of the molecular basis of the unusuall y high enzymatic activity of single-chain t-PA have focused attention upon Asp-194, a residue that is invariant among chymotrypsin-like enzy mes, The critical role of this residue in securing the active conforma tion of mature chymotrypsin-like enzymes has been discussed extensivel y. Subsequent work, however, has indicated that this conserved residue can also form interactions that dramatically influence the catalytic activity of serine protease zymogens, While Asp-194 forms interactions that suppress the activity of the zymogen chymotrypsinogen, it may, b y contrast, directly promote the catalytically active conformation of single chain t-PA. To test the hypothesis that Asp-194 promotes the ac tivity of both single- and two-chain t-PA and therefore plays opposing roles in single-chain t-PA and chymotrypsinogen, and also to examine whether this invariant residue plays an essential role in the stimulat ion of t-PA by fibrin, we used site directed mutagenesis to construct the following variants of t-PA: t-PA/D194E, t-PA/D194N, t-PA/R15E,D194 E, and t-PA/R15E,D194N. In the absence of fibrin, the activity of enzy mes carrying a mutation at position 194 was reduced by factors of 1000 -2000 compared to wild type t-PA, Similar reductions of activity were observed for both single- and two chain variants, suggesting an import ant role for Asp-194 in both forms of the enzyme. The mutated enzymes, however, displayed a dramatically enhanced response to fibrin monomer s. While the activity of wild type t-PA was stimulated by fibrin monom ers by a factor of 960, the corresponding stimulation factor for the m utated enzymes varied from 498,000-1,050,000.