The crystal structure of the fibrinolytic enzyme
tissue plasminogen activator (tPA) shows that the bulky
side chain of Y99 hinders access to the active site by
partially occluding the S2 site and may be responsible
for the low catalytic activity of tPA toward plasminogen.
We have tested the role of Y99 by replacing it with Leu,
the residue found in more proficient proteases like trypsin
and thrombin. The Y99L replacement results in an increase
in the kcat/Km
for chromogenic substrates due to enhanced diffusion into
the active site. The increase is modest (threefold) for
substrates specific for tPA that carry Pro or Gly at P2,
but reaches 80-fold for less specific substrates carrying
Arg at P2. On the other hand, the Y99L mutation has no
effect on the activity of tPA toward the natural substrate
plasminogen, that carries Gly at P2, and reduces more than
10-fold the inhibition of tPA by plasminogen activator
inhibitor-1 (PAI-1), that carries Ala at P2. We conclude
that the steric hindrance provided by Y99 in the crystal
structure affects mostly nonphysiological substrates with
bulky residues at P2. In addition, residue Y99 plays an
active role in the recognition of PAI-1, but not plasminogen.
Mutations of Y99 could therefore afford a resistance to
inhibition by PAI-1 without compromising the fibrinolytic
potency of tPA, a result of potential therapeutic relevance.