Identification of a New Exosite Involved in Catalytic Turnover by the Streptokinase-Plasmin Activator Complex during Human Plasminogen Activation

Rachna Aneja,Manish Datt,Balwinder Singh,Shekhar Kumar,Girish Sahni

doi:10.1074/jbc.m109.046573

Rachna Aneja, Manish Datt + Show 3 more

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https://doi.org/10.1074/jbc.m109.046573

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Abstract

With the goal of identifying hitherto unknown surface exosites of streptokinase involved in substrate human plasminogen recognition and catalytic turnover, synthetic peptides encompassing the 170 loop (CQFTPLNPDDDFRPGLKDTKLLC) in the beta-domain were tested for selective inhibition of substrate human plasminogen activation by the streptokinase-plasmin activator complex. Although a disulfide-constrained peptide exhibited strong inhibition, a linear peptide with the same sequence, or a disulfide-constrained variant with a single lysine to alanine mutation showed significantly reduced capabilities of inhibition. Alanine-scanning mutagenesis of the 170 loop of the beta-domain of streptokinase was then performed to elucidate its importance in streptokinase-mediated plasminogen activation. Some of the 170 loop mutants showed a remarkable decline in k(cat) without any alteration in apparent substrate affinity (K(m)) as compared with wild-type streptokinase and identified the importance of Lys(180) as well as Pro(177) in the functioning of this loop. Remarkably, these mutants were able to generate amidolytic activity and non-proteolytic activation in "partner" plasminogen as wild-type streptokinase. Moreover, cofactor activities of the 170 loop mutants, pre-complexed with plasmin, against microplasminogen as the substrate showed a similar pattern of decline in k(cat) as that observed in the case of full-length plasminogen, with no concomitant change in K(m). These results strongly suggest that the 170 loop of the beta-domain of streptokinase is important for catalysis by the streptokinase-plasmin(ogen) activator complex, particularly in catalytic processing/turnover of substrate, although it does not seem to contribute significantly toward enzyme-substrate affinity per se.

Highlights

32642 JOURNAL OF BIOLOGICAL CHEMISTRY minogen by forming a high affinity stoichiometric complex with “partner” plasminogen, which, after a conformational activation step, acquires the capability to selectively cleave the Arg561-Val562 scissile peptide bond in “substrate” plasminogen, thereby converting the latter into HPN, plasmin [2, 3]
The crystal structure of SK complexed with ␮PN, with which SK forms a tight binary complex as it does with full-length HPG, reveals that the domains of SK form a “threesided crater,” with the ␮PN situated at the bottom of a valley into which the substrate’s catalytic domain, bearing the “target” scissile peptide bond to be cleaved, can potentially dock [9]
The results, Expression and Purification of wtSK/SK Loop Mutants—The presented below, reveal that this loop contributes in a major wtSK and 170 loop mutants cloned in pET 23d were expressed way toward catalytic processing of the substrate plasminogen, intracellularly in E. coli BL21 (DE3) cells as inclusion bodies because various point-mutations within this loop resulted in under the control of the T7 phage RNA polymerase promoter

Summary

Introduction

32642 JOURNAL OF BIOLOGICAL CHEMISTRY minogen by forming a high affinity stoichiometric complex with “partner” plasminogen, which, after a conformational activation step, acquires the capability to selectively cleave the Arg561-Val562 scissile peptide bond in “substrate” plasminogen, thereby converting the latter into HPN, plasmin [2, 3]. The relative slopes of the progress curves so obtained (see Fig. 3, which shows the time course of plasmin generation by some of the alanine mutants compared with that of wtSK) clearly indicate that the alanine substitution mutants of 170 loop exhibited varied extents of reduction in their HPG activation capabilities.

Results

Conclusion