Aneja R, Datt M, Singh B, Kumar S, Sahni G
Identification of a new exosite involved in catalytic turnover by the streptokinase-plasmin activator complex during human plasminogen activation. [JOURNAL ARTICLE]
J Biol Chem 2009 Sep 30.
With a view to identify 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. While 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 kcat without any alteration in apparent substrate affinity (Km) as compared to wild type streptokinase, and identified the importance of Lys180 as well as Pro177 in the functioning of this loop. Remarkably, these mutants were able to generate levels of amidolytic activity and nonproteolytic activation in partner plasminogen as did 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 kcat as was observed in case of full-length plasminogen, with no concomitant change in Km. 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 towards enzyme-substrate affinity per se.
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