Tissue-type plasminogen activator (t-PA), the serine protease responsible for catalyzing the production of plasmin from plasminogen at the site of mood clots, is synthesized as a single-chain polypeptide precursor. Proteolytic cleavage at the C-terminal side of Arg275 generates a two-chain form of the enzyme whose subunits are held together by a single disulfide bond. We have measured the activities of both forms of the wild-type enzyme, as well as that of a mutant enzyme (Arg275 → Gly), created by oligonucleo-tide-directed mutagenesis, that cannot be cleaved into a two-chain form. Both types of single-chain t-PAs are enzymatically active and exhibit identical Vmax and Km values when assayed with synthetic peptide substrates, indicating that the single amino acid change had no effect on the amidolytic activity of the enzyme. However, cleavage of wild-type t-PA into the two-chain form results in increased activity both on a peptide substrate and on the natural substrates Lys- and Glu-plasminogen in the absence or presence of stimulation by soluble fibrin. The enhanced activity is due to a 3-5-fold increase in the Vmax of the cleaved enzyme, rather than to any change in the Km values for the various substrates. During incubation with plasminogen, the single-chain form of wild-type t-PA is converted to the two-chain form by plasmin generated during the reaction. This conversion, from the less active to the more active form of the enzyme, results in a reaction that displays biphasic kinetics. Both the single-chain, cleavage-minus t-PA and the two-chain, wild-type t-PA are stimulated by soluble fibrin, although the single-chain form requires higher levels of fibrin to achieve maximum activity. Finally, both forms of the enzyme can be inhibited to the same extent by the serpin plasminogen activator inhibitor 1 (PAI-1).
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