The Single-Chain Form of Tissue-Type Plasminogen Activator Has Catalytic Actvity: Studies with a Mutant Enzyme That Lacks the Cleavage Site

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 V_max and K_m 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 V_max of the cleaved enzyme, rather than to any change in the K_m 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).