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

Abstract

The role of a prominent surface-exposed loop (residues 88–97) in the α 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 β-turn formation. The mutant so formed (termed SK 88–97-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 K m 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 K d 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 α domain of SK contributes towards catalytic turn-over, even though its individual contribution towards enzyme–substrate affinity per se is minimal.