Identification of a new exosite involved in catalytic turnover by the streptokinase-plasmin activator complex during human plasminogen activation.
IR@IMTECH: CSIR-Institute of Microbial Technology, Chandigarh
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| Title |
Identification of a new exosite involved in catalytic turnover by the streptokinase-plasmin activator complex during human plasminogen activation.
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| Creator |
Aneja, Rachna
Datt, Manish Singh, Balvinder Kumar, Shekhar Sahni, Girish |
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| Subject |
QR Microbiology
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| Description |
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.
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| Publisher |
ASBMB
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| Date |
2009-11-20
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| Type |
Article
PeerReviewed |
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| Format |
application/pdf
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| Identifier |
http://crdd.osdd.net/open/546/1/sahni09.pdf
Aneja, Rachna and Datt, Manish and Singh, Balvinder and Kumar, Shekhar and Sahni, Girish (2009) Identification of a new exosite involved in catalytic turnover by the streptokinase-plasmin activator complex during human plasminogen activation. The Journal of biological chemistry, 284 (47). pp. 32642-50. ISSN 1083-351X |
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| Relation |
http://www.jbc.org/content/284/47/32642.full.pdf+html
http://crdd.osdd.net/open/546/ |
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