Kringles of substrate plasminogen provide a “catalytic switch” in plasminogen to plasmin turnover by Streptokinase

1 To understand the role of substrate plasminogen kringles in its differential catalytic processing by 2 the streptokinase - human plasmin (SK-HPN) activator enzyme, Fluorescence Resonance Energy Transfer 3 (FRET) model was generated between the donor labeled activator enzyme and the acceptor labeled 4 substrate plasminogen (for both kringle rich Lys plasminogen – LysPG, and kringle less microplasminogen 5 - µPG as substrates). Different steps of plasminogen to plasmin catalysis i.e. substrate plasminogen docking 6 to scissile peptide bond cleavage, chemical transformation into proteolytically active product, and the 7 decoupling of the nascent product from the SK-HPN activator enzyme were segregated selectively using 8 (1) FRET signal as a proximity sensor to score the interactions between the substrate and the activator 9 during the cycle of catalysis, (2) active site titration studies and (3) kinetics of peptide bond cleavage in the 10 substrate. Remarkably, active site titration studies and the kinetics of peptide bond cleavage have shown 11 that post docking chemical transformation of the substrate into the product is independent of kringles 12 adjacent to the catalytic domain. Stopped-flow based rapid mixing experiments for kringle rich and kringle less substrate plasminogen derivatives under substrate saturating and single-cycle turn-over conditions have 14 shown that the presence of kringle domains adjacent to the catalytic domain in the macromolecular substrate 15 contributes by selectively speeding up the final step, namely the product release/expulsion step of catalysis 16 by the streptokinase-plasmin(ogen) activator enzyme. (solid squares) and 2 µM µPG (solid spheres) without NPGB and 2 µM LysPG (open squares) and 2 µM µPG (open spheres) in the presence of 10 molar excess of NPGB inhibitor. Assay curves represents molar concentration of p-nitroaniline generated following hydrolysis of Chromozym ® PL. Reaction was carried out in assay buffer (50 mM Tris-Cl pH 7.5, 100 mM NaCl) supplemented with 0.5 mM Chromozym ® PL, (see materials and methods). Data represent the mean of three independent determinations (B) Fluorescence traces for the rapid mixing of donor labeled activator enzyme with acceptor labeled kringle rich substrate plasminogen under pseudo first order conditions where a slight excess of enzyme (3.5 µM) over substrate (3 µM) is taken. The mixture was excited at 336 nm and the emission from the acceptor (520 nm) following energy transfer from the donor was monitored after passing


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Human plasminogen (HPG), a single chain multi domain glycoprotein of molecular weight ~92 residues 543-791) in SK-HPN mediated catalysis (16,17). Strikingly this 'substrate-assisted' proteolysis 48 as observed for SK has not been observed for 'direct' plasminogen activators like tPA or Urokinase which 49 do not discriminate between kringle rich and kringle less macromolecular substrates (18). Amongst (19,20). One working hypothesis is that conformational transitions which are 58 originated in the activation loop and/or other adjoining regions such as the kringle domains following 59 scissile bond cleavage in the nascent product plasmin may be 'utilized' in dis-engagement of the product 60 from proteolytic enzyme (SK-HPN) and facilitating the decoupling of macromolecular complexation 61 between the erstwhile substrate plasminogen and the activator enzyme at the end of each catalytic cycle.

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In order to get an insight into the mechanism utilized by kringle rich and kringle less plasminogen 63 derivatives as substrate, a FRET based spectroscopic approach has been utilized as an intermolecular 64 proximity sensor on a stopped-flow apparatus to get real time visualization of the catalytic events happening 65 on a millisecond time scale (21,22). For its very high sensitivity and flexibility, FRET has been used 66 extensively for studying protein-protein interactions (23)(24)(25). Experiments in this study have been designed

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Amongst all plasminogen activators (as in SAK, tPA, uPA), it has been observed that SK-HPN 458 activator complex has highest catalytic power likely due to productive utilization of long-range interactions 459 between the activator and the enzyme (16,18

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In the present study, therefore, we have segregated this system into different steps of catalysis. (rate determining) step is approximately 100-fold lower for kringle less substrate than that of kringle rich 507 substrate plasminogen, and thus elegantly correlates with the respective steady state parameters for catalytic 508 turnover. Interestingly, from the stopped-flow data it is observed that it is the complex dissociation or 509 product decoupling step which is the rate determining step in the catalytic scheme. This data has clearly 510 shown that substrate kringles provide the critical "catalytic switch" by enhancing the rate of product release 511 by nearly two orders of magnitude compared to kringle less substrate.

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One plausible explanation for this mechanism can be that following scissile bond cleavage, strain plasminogen activation (36,37). The role of various surface exposed exosites in SK cofactor in modulating 533 the catalytic turnover of the substrate plasminogen has been well explored (11,13,15,17,38 contrast to, say, the ATP driven conformational-catalytic enzyme systems in the genetic machinery of 541 eukaryotic cells) and provide a tantalizing peep into the catalytic mechanism whereby the SK attaches itself 542 to human plasmin (a non-specific protease) and converts into an extremely specific enzyme (39,40).

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In the present study, thus, for the first time,     Preformed equimolar SK-HPN (7.5 µM) activator enzyme was added to assay buffer containing 10 mM Na-phosphate buffer, pH 7.2. Slightly more than equimolar substrate plasminogen derivative/s (kringle rich (HPG) or kringle less (µPG)) at 9.5 µM final conc. was added to reaction buffer followed by immediate addition of 100 µM NPGB. The "burst" of p-nitrophenol release was monitored spectrophotometrically at 405 nm (see materials and methods section for details). The curves shown represent molar concentration of p-nitrophenol generated following NPGB hydrolysis by SK-HPN alone (solid triangles), SK-HPN with HPG as substrate (solid squares) and SK-HPN with µPG as substrate (open spheres).    . Following substrate docking, there is a catalytic transformation of substrate to product plasmin through scissile peptide bond cleavage and finally product release, resulting in the decoupling of newly formed product HPN (or µPN with µPG as substrate) from the complex and consequent dissociation of FRET pairs. (B) In the presence of active site inhibitor NPGB, there is a formation of ternary complex but the catalytic steps following complex formation (i.e. peptide bond cleavage and the product release) are blocked. Color code: green -SK; orange-HPN as a binary partner; purple-kringle rich Plasminogen as a substrate molecule; black-NPGB; red flag-donor fluorophore IAEDANS, blue flag-acceptor fluorophore F5M. Assay curves represents molar concentration of p-nitroaniline generated following hydrolysis of Chromozym ® PL. Reaction was carried out in assay buffer (50 mM Tris-Cl pH 7.5, 100 mM NaCl) supplemented with 0.5 mM Chromozym ® PL, (see materials and methods). Data represent the mean of three independent determinations (B) Fluorescence traces for the rapid mixing of donor labeled activator enzyme with acceptor labeled kringle rich substrate plasminogen under pseudo first order conditions where a slight excess of enzyme (3.5 µM) over substrate (3 µM) is taken. The mixture was excited at 336 nm and the emission from the acceptor (520 nm) following energy transfer from the donor was monitored after passing through an emission long pass filter 495 nm (C) Fluorescence traces for the rapid mixing of donor labeled activator enzyme in the presence of 10 molar excess of NPGB with acceptor labeled kringle rich substrate plasminogen under similar experimental conditions. (D) and (E) represents fluorescence traces for the rapid mixing of donor labeled activator enzyme with acceptor labeled kringle less substrate plasminogen under similar reaction conditions in the absence and presence of NPGB, respectively. Fluorescence traces thus obtained were then fitted to a bi-exponential model using Bio-kine32 V4.66 software (shown as a thin red line superimposed on each relaxation curve) and the kinetic constants were determined.