2009
DOI: 10.1074/jbc.m109.046573
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Identification of a New Exosite Involved in Catalytic Turnover by the Streptokinase-Plasmin Activator Complex during Human Plasminogen Activation

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 (CQFTPLNPDDDFRPGLK-DTKLLC) in the ␤-domain were tested for selective inhibition of substrate human plasminogen activation by the streptokinaseplasmin activator complex. Although a disulfide-constrained peptide exhibited strong inhibition, a linear peptide with the same sequence, or a disulfide-constrained variant… Show more

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Cited by 19 publications
(38 citation statements)
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“…The amidolysis profile of SAK or SAK mutants was measured spectrophotometrically at 405 nm in microtitre plate as described above [22].…”
Section: Amidolysis Profile Of Sak-pm Bimolecular Complexmentioning
confidence: 99%
“…The amidolysis profile of SAK or SAK mutants was measured spectrophotometrically at 405 nm in microtitre plate as described above [22].…”
Section: Amidolysis Profile Of Sak-pm Bimolecular Complexmentioning
confidence: 99%
“…Other studies carried out with truncated SK derivatives suggest that the region constituting both ␤ and ␥ domains are strongly involved in 1:1 binding with the partner and also in generating an activator complex (37)(38)(39). Although studies in the recent past have implicated specific lysine residues in both ␤ and ␥ domains in enzyme-substrate interactions followed by identification of discrete structural epitopes/exosites in both the ␣ and ␤ domains (20,21,33,35), any clear-cut involvement of an exosite in the ␥ domain solely with this function has not been demonstrated so far. Bi-domain construct of SK composed of ␣ and ␤ domains exhibits only ϳ3% substrate activation ability after complexation with HPN compared with wtSK, which suggests the importance of ␥ domain in engendering the full-blown substrate activation characteristic of wt SK (40).…”
mentioning
confidence: 99%
“…Moreover, the observation that the ␥ domain binds closely to the catalytic active center of the SK⅐HPN complex suggests that it might indeed be a preferred area of substrate interaction also (17), and there is, therefore, a need to explore intensively whether this domain contains any site(s) playing a role specifically in substrate interaction under conditions that unambiguously rule out the involvement of any 1:1 interactions. Previously, alanine mutagenesis of the extensive charged side chains of the residues present in the coiled coil region of the ␥ domain of SK has revealed the overall functional relevance of this region in HPG activation in general (29), but to look for the possible presence of a discrete substrate-interacting exosite/epitope of the type demonstrated in the ␣ and ␤ domains (20,21,35,41), in the present study we took recourse to a random mutagenesis approach.The results, presented below, examine a series of randommutant clones of the C-terminal region of SK (residues 210 -414) for their functional importance in substrate HPG activation. Because this approach initially yielded several scores of functionally compromised mutants that carried multiple-site mutations, an analysis of their "deconvolved" forms, that is, all or most single-site mutants derived from the mutant-clusters with compromised activity, was then carried out to decipher the functional importance of residues present in the ␥ domain in substrate recognition and/or turnover.…”
mentioning
confidence: 99%
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