SUBSTRATE-SPECIFICITY OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR - CHARACTERIZATION OF THE FIBRIN INDEPENDENT SPECIFICITY OF T-PA FOR PLASMINOGEN

Tissue-type plasminogen activator (t-PA) is a remarkably specific prot ease: the only known substrate of this enzyme in vivo is a single pept ide bond (Arg(560)-Va1(561)) within the proenzyme plasminogen. Part of the substrate specificity of t-PA is due to a ternary interaction bet ween fibrin, BPA, and plasminogen which reduces the K-m of t-PA for pl asminogen 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 spe cificity of t-PA for plasminogen by assaying t-PA and trypsin toward s ubstrates modeled after the peptide sequence in plasminogen surroundin g Ar-960-Val(561). Surprisingly, t-PA hydrolyzes these substrates with kappa(cat)/K-m 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 pr otease domain. This suggests that the protease domain, in spite of its high homology to the nonspecific enzyme trypsin, is inherently specif ic for recognition of one or more structural features displayed by nat ive plasminogen.