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 with a single lysine to alanine mutation showed significantly reduced capabilities of inhibition. Alanine-scanning mutagenesis of the 170 loop of the ␤-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 ␤-domain of streptokinase is important for catalysis by the streptokinaseplasmin(ogen) activator complex, particularly in catalytic processing/turnover of substrate, although it does not seem to contribute significantly toward enzyme-substrate affinity per se.Plasminogen activation, a key event in physiological fibrinolysis, is a widely employed therapeutic means to rapidly alleviate circulatory disorders that arise due to obstruction of blood flow by pathological thrombi (1). Streptokinase (SK), 2 a bacteria-derived thrombolytic protein, activates plasminogen by forming a high affinity stoichiometric complex with "partner" plasminogen, which, after a conformational activation step, acquires the capability to selectively cleave the Arg 561 -Val 562 scissile peptide bond in "substrate" plasminogen, thereby converting the latter into HPN, plasmin (2, 3). Elegant equilibrium binding and kinetic studies directed toward resolving the steps involved in the phenomenon of conformational activation of HPG by SK demonstrate a relatively rapid formation of a SK⅐HPG* complex, a "conformationally activated" state, due to the generation of a nonproteolytically activated active center, which is also amidolytically active (4). This conformationally activated SK⅐HPG* complex can then recruit a substrate molecule of HPG (3, 9) and proteolytically activate it, likely through inter-molecular proteolytic scission, into HPN. In addition, the HPG moiety in the SK⅐HPG* activator complex can exchange with free HPN preferentially (because the lat...

Pro42 and Val45 of staphylokinase modulate intermolecular interactions of His43–Tyr44 pair and specificity of staphylokinase–plasmin activator complex

“…The amidolysis profile of SAK or SAK mutants was measured spectrophotometrically at 405 nm in microtitre plate as described above [22].…”

Identification through Combinatorial Random and Rational Mutagenesis of a Substrate-interacting Exosite in the γ Domain of Streptokinase

“…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).…”

“…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.…”

“…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.…”

“…Mechanistic insights on the specificity switch engendered by SK upon binding onto the HPN active site are crucial in major part because of possibly enabling the engineering of altogether new target specificities into pre-existent protease active sites for biomedical applications (20,21). The binding of SK to HPN results in the expression of substrate recognition "exosites" on the SK⅐HPG/HPN activator complex, which are located physically away from the active center of the enzyme (19).…”