Role of residue Y99 in tissue plasminogen activator

Citation
A. Vindigni et al., Role of residue Y99 in tissue plasminogen activator, PROTEIN SCI, 9(3), 2000, pp. 619-622
Citations number
21
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
PROTEIN SCIENCE
ISSN journal
0961-8368 → ACNP
Volume
9
Issue
3
Year of publication
2000
Pages
619 - 622
Database
ISI
SICI code
0961-8368(200003)9:3<619:RORYIT>2.0.ZU;2-R
Abstract
The crystal structure of the fibrinolytic enzyme tissue plasminogen activat or (tPA) shows that the bulky side chain of Y99 hinders access to the activ e site by partially occluding the S2 site and may be responsible for the lo w catalytic activity of tPA toward plasminogen. We have tested the role of Y99 by replacing it with Leu, the residue found in more proficient protease s like trypsin and thrombin. The Y99L replacement results in an increase in the k(cat)/K-m for chromogenic substrates due to enhanced diffusion into t he 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, tha t 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 conc lude that the steric hindrance provided by Y99 in the crystal structure aff ects mostly nonphysiological substrates with bulky residues at P2. In addit ion, residue Y99 plays an active role in the recognition of PAI-1, but not plasminogen. Mutations of Y99 could therefore afford a resistance to inhibi tion by PAI-1 without compromising the fibrinolytic potency of tPA, a resul t of potential therapeutic relevance.