Multiple Exosites Distributed across the Three Domains of Streptokinase Co-Operate to Generate High Catalytic Rates in the Streptokinase鈥揚lasmin Activator Complex

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• 作者：Rachna Aneja ; Manish Datt ; Suman Yadav ; Girish Sahni
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：December 10, 2013
• 年：2013
• 卷：52
• 期：49
• 页码：8957-8968
• 全文大小：456K
• 年卷期：v.52,no.49(December 10, 2013)
• ISSN：1520-4995

文摘

To examine the global function of the key surface-exposed loops of streptokinase, bearing substrate-specific exosites, namely, the 88鈥?7 loop in the 伪 domain, the 170 loop in the 尾 domain, and the coiled-coil region (Leu³²¹-Asn³³⁸) in the 纬 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鈥損lasmin 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 k_cat 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鈥損lasmin complex operates through a distributed network of substrate-interacting exosites resident across all three domains of the cofactor protein.