Protein Science, Vol 7, Issue 8 1728-1737, Copyright © 1998 by Cold Spring Harbor Laboratory Press

ARTICLE

Role of P225 and the C136-C201 disulfide bond in tissue plasminogen activator

A. VINDIGNI and E. D. CERA
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St. Louis, Missouri 63110

The protease domain of tissue plasminogen activator (tPA), a key fibrinolytic enzyme, was expressed in Escherichia coli with a yield of 1 mg per liter of media. The recombinant protein was titrated with the Erythrina caraffa trypsin inhibitor (ETI) and characterized in its interaction with plasminogen and the natural inhibitor plasminogen activator inhibitor-1 (PAI-1). Analysis of the catalytic properties of tPA using a library of chromogenic substrates carrying substitutions at P1, P2, and P3 reveals a strong preference for Arg over Lys at P1, unmatched by other serine proteases like thrombin or trypsin. In contrast to these proteases and plasmin, tPA shows little or no preference for Pro over Gly at P2. A specific inhibition of tPA by Cu(2+) was discovered. The divalent cation presumably binds to H188 near D189 in the primary specificity pocket and inhibits substrate binding in a competitive manner with a K(d) = 19 {mu}M. In an attempt to engineer Na(+) binding and enhanced catalytic activity in tPA, P225 was replaced with Tyr, the residue present in Na(+)-dependent allosteric serine proteases. The P225Y mutation did not result in cation binding, but caused a significant loss of specificity (up to 100-fold) toward chromogenic substrates and plasminogen and considerably reduced the inhibition by PAI-1 and ETI. Interestingly, the P225Y substitution enhanced the ability of Cu(2+) to inhibit the enzyme. Elimination of the C136-C201 disulfide bond, that is absent in all Na(+)-dependent allosteric serine proteases, significantly enhanced the yield (5 mg per liter of media) of expression in E. coli, but caused no changes in the properties of the enzyme whether residue 225 was Pro or Tyr. These findings point out an unanticipated crucial role for residue 225 in controlling the catalytic activity of tPA, and suggest that engineering of a Na(+)-dependent allosteric enhancement of catalytic activity in this enzyme, must involve substantial changes in the region homologous to the Na(+) binding site of allosteric serine proteases.
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