Role of the 88-97 loop in plasminogen activation by streptokinase probed through site-specific mutagenesis.

Yadav, Suman and Datt, Manish and Singh, Balvinder and Sahni, Girish (2008) Role of the 88-97 loop in plasminogen activation by streptokinase probed through site-specific mutagenesis. Biochimica et biophysica acta, 1784 (9). pp. 1310-8. ISSN 0006-3002

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The role of a prominent surface-exposed loop (residues 88-97) in the alpha domain of streptokinase (SK), in human plasminogen (HPG) activation was explored through its selective mutagenesis and deletion studies. We first made a conformationally constrained derivative of the loop by the substitution of sequences known to possess a strong propensity for beta-turn formation. The mutant so formed (termed SK88-97-Beta Turn), when tested for co-factor activity against substrate HPG, after first forming a 1:1 molar complex with human plasmin (HPN), showed a nearly 6-fold decreased co-factor activity compared to the wild-type, native SK. The major catalytic change was observed to be at the k(cat) level, with relatively minor changes in Km values against HPG. Real-time binary interaction (i.e. the 1:1 complexation between SK, or its mutant/s, with HPG), and ternary complexation studies (i.e. the docking of a substrate HPG molecule into the preformed SK-HPG complex) using Surface Plasmon Resonance were done. These studies revealed minor alterations in binary complex formation but the ternary interactions of the substitution and/or deletion mutants were found to be decreased for full-length HPG compared to that for native SK.HPG. In contrast, their ternary interactions with the isolated five-kringle domain unit of plasminogen (K1-5) showed Kd values comparable to that seen with the native SK.HPG complex. Taking into consideration the overall alterations observed in catalytic levels after site-specific mutagenesis and complete loop deletion of the 88-97 loop, on the one hand, and its known position at the SK-HPG interface in the binary complex, suggests the importance of this loop. The present results suggest that the 88-97 loop of the alpha domain of SK contributes towards catalytic turn-over, even though its individual contribution towards enzyme-substrate affinity per se is minimal.

Item Type: Article
Additional Information: Copyright of this article belongs to Elsevier Science.
Uncontrolled Keywords: Streptokinase; Human plasminogen activation; Plasminogen activator; Protein–protein interaction; Enzyme–substrate interaction
Subjects: Q Science > QR Microbiology
Depositing User: Dr. K.P.S.Sengar
Date Deposited: 21 Feb 2012 18:07
Last Modified: 21 Feb 2012 18:07

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