title: Multiple exosites distributed across the three domains of streptokinase co-operate to generate high catalytic rates in the streptokinase-plasmin activator complex.
abstract: 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.
authors.affiliation: The Institute of Microbial Technology (CSIR) , Sector 39-A, Chandigarh-160036, India.
authors.forename: Rachna
authors.initials: R
authors.lastname: Aneja
authors.forename: Manish
authors.initials: M
authors.lastname: Datt
authors.forename: Suman
authors.initials: S
authors.lastname: Yadav
authors.forename: Girish
authors.initials: G
authors.lastname: Sahni
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D055162:Biocatalysis; D020134:Catalytic Domain; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D010958:Plasminogen / Q000737:chemistry*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D013291:Streptococcus / Q000201:enzymology*; D013300:Streptokinase / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D010958:Plasminogen; D013300:Streptokinase