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  • Title: Multiple exosites distributed across the three domains of streptokinase co-operate to generate high catalytic rates in the streptokinase-plasmin activator complex.
    Author: Aneja R, Datt M, Yadav S, Sahni G.
    Journal: Biochemistry; 2013 Dec 10; 52(49):8957-68. PubMed ID: 23919427.
    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.
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