Sh. Ke et al., IDENTIFICATION OF A HYDROPHOBIC EXOSITE ON TISSUE-TYPE PLASMINOGEN-ACTIVATOR THAT MODULATES SPECIFICITY FOR PLASMINOGEN, The Journal of biological chemistry, 272(3), 1997, pp. 1811-1816
A wide variety of important biological processes, in eluding both the
formation and dissolution of blood clots, depend on specific cleavage
of individual target proteins by serine proteases. For example, tissue
type plasminogen activator (t-PA), a trypsin like enzyme that catalyz
es the rate limiting step of the endogenous fibrinolytic cascade, has
only one known substrate in vivo, a single peptide bond (Arg(561)-Val(
562)) in the proenzyme plasminogen. We have previously suggested that
the specificity of t-PA for plasminogen is mediated in part by direct
protein-protein interactions between the protease domain of t-PA and p
lasminogen that are distinct from those occurring within t-PA's active
site. We demonstrate in this study that residues 420-423 of t-PA, whi
ch form a fully solvent exposed, hydrophobic region of a surface loop
mapping near one edge of the active site of t-PA, form, or are essenti
al for the integrity of, an important, secondary site of interaction b
etween t-PA and plasminogen that significantly modulates the rate of p
lasminogen activation in the absence, but not the presence, of fibrin.
Identification of this secondary site of interaction between t-PA and
plasminogen provides new insight into molecular details of the evolut
ion of stringent substrate specificity by t-PA and suggests a novel st
rategy to enhance the fibrin dependence of plasminogen activation by t
-PA. While the activity of wild type t-PA is stimulated by fibrin by a
factor of approximately 650, the activity of two variants characteriz
ed in this study, t-PA/R275E,P422G and t-PA/R275E,P422E, is stimulated
by a factor of approximately 39,000 or 61,000, respectively. It is th
erefore possible that, compared with wild type t-PA, the two variants
would display enhanced ''clot selectivity'' in vivo due to reduced act
ivity in the circulation but full activity at a site of fibrin deposit
ion.