Record Details

Multiple exosites distributed across the three domains of streptokinase co-operate to generate high catalytic rates in the streptokinase-plasmin activator complex.

DIR@IMTECH: CSIR-Institute of Microbial Technology

View Archive Info
 
 
Field Value
 
Title Multiple exosites distributed across the three domains of streptokinase co-operate to generate high catalytic rates in the streptokinase-plasmin activator complex.
 
Creator Aneja, Rachna
Datt, Manish
Yadav, Suman
Sahni, Girish
 
Subject QR Microbiology
 
Description To examine the global function of the key surface-exposed loops of streptokinase, bearing substrate-specific exosites, namely, the 88-97 loop in the α domain, the 170 loop in the β domain, and the coiled-coil region (Leu321-Asn338) in the γ domain, mutagenic as well as peptide inhibition studies were carried out. Peptides corresponded to the primary structure of an exosite, either individual or stoichiometric mixtures of various disulfide-constrained synthetic peptide(s) inhibited plasminogen activation by streptokinase. Remarkably, pronounced inhibition of substrate plasminogen activation by the preformed streptokinase-plasmin activator complex was observed when complementary mixtures of different peptides were used compared to the same overall concentrations of individual peptides, suggesting co-operative interactions between the exosites. This observation was confirmed with streptokinase variants mutated at one, two, or three sites simultaneously. The single/double/triple exosite mutants of streptokinase showed a nonadditive, synergistic decline in kcat for substrate plasminogen activation in the order single > double > triple exosite mutant. Under the same conditions, zymogen activation by the various mutants remained essentially native- like in terms of nonproteolytic activation of partner plasminogen. Multisite mutants also retain affinity to form 1:1 stoichiometric activator complexes with plasmin when probed through sensitive equilibrium fluorescence studies. Thus, the present results strongly support a model of streptokinase action, wherein catalysis by the streptokinase-plasmin complex operates through a distributed network of substrate-interacting exosites resident across all three domains of the cofactor protein.
 
Publisher ACS Publications
 
Date 2013-12-10
 
Type Article
PeerReviewed
 
Relation http://pubs.acs.org/doi/abs/10.1021/bi400142s
http://crdd.osdd.net/open/1507/
 
Identifier Aneja, Rachna and Datt, Manish and Yadav, Suman and Sahni, Girish (2013) Multiple exosites distributed across the three domains of streptokinase co-operate to generate high catalytic rates in the streptokinase-plasmin activator complex. Biochemistry, 52 (49). pp. 8957-68. ISSN 1520-4995