Tissue-type plasminogen activator (t-PA) is a remarkably specific protease: the only known substrate of this enzyme in vivo is a single peptide bond (Arg560-Val561) within the proenzyme plasminogen. Part of the substrate specificity of t-PA is due to a ternary interaction between fibrin, t-PA, and plasminogen which reduces the Km of t-PA for plasminogen by a factor of 440. However, even in the absence of fibrin, t-PA continues to hydrolyze plasminogen more rapidly than does trypsin, a homologous serine protease. We have measured the extent of the specificity of t-PA for plasminogen by assaying t-PA and trypsin toward substrates modeled after the peptide sequence in plasminogen surrounding Arg560-Val561. Surprisingly, t-PA hydrolyzes these substrates with kcat/Km values which are 28,000-210,000-fold lower than those obtained using trypsin. Both the high activity toward plasminogen and the low activity toward peptides are also exhibited by the isolated protease domain. This suggests that the protease domain, in spite of its high homology to the nonspecific enzyme trypsin, is inherently specific for recognition of one or more structural features displayed by native plasminogen.
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