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• W2021471987 abstract "Binding of the fibrinolytic proteinase plasmin (Pm) to streptokinase (SK) in a tight stoichiometric complex transforms Pm into a potent proteolytic activator of plasminogen. SK binding to the catalytic domain of Pm, with a dissociation constant of 12 pm, is assisted by SK Lys414 binding to a Pm kringle, which accounts for a 11-20-fold affinity decrease when Pm lysine binding sites are blocked by 6-aminohexanoic acid (6-AHA) or benzamidine. The pathway of SK·Pm catalytic complex formation was characterized by stopped-flow kinetics of SK and the Lys414 deletion mutant (SKΔK414) binding to Pm labeled at the active site with 5-fluorescein ([5F]FFR-Pm) and the reverse reactions by competitive displacement of [5F]FFR-Pm with active site-blocked Pm. The rate constants for the biexponential fluorescence quenching caused by SK and SKΔK414 binding to [5F]FFR-Pm were saturable as a function of SK concentration, reporting encounter complex affinities of 62-110 nm in the absence of lysine analogs and 4900-6500 and 1430-2200 nm in the presence of 6-AHA and benzamidine, respectively. The encounter complex with SKΔK414 was ∼10-fold weaker in the absence of lysine analogs but indistinguishable from that of native SK in the presence of 6-AHA and benzamidine. The studies delineate for the first time the sequence of molecular events in the formation of the SK·Pm catalytic complex and its regulation by kringle ligands. Analysis of the forward and reverse reactions supports a binding mechanism in which SK Lys414 binding to a Pm kringle accompanies near-diffusion-limited encounter complex formation followed by two slower, tightening conformational changes. Binding of the fibrinolytic proteinase plasmin (Pm) to streptokinase (SK) in a tight stoichiometric complex transforms Pm into a potent proteolytic activator of plasminogen. SK binding to the catalytic domain of Pm, with a dissociation constant of 12 pm, is assisted by SK Lys414 binding to a Pm kringle, which accounts for a 11-20-fold affinity decrease when Pm lysine binding sites are blocked by 6-aminohexanoic acid (6-AHA) or benzamidine. The pathway of SK·Pm catalytic complex formation was characterized by stopped-flow kinetics of SK and the Lys414 deletion mutant (SKΔK414) binding to Pm labeled at the active site with 5-fluorescein ([5F]FFR-Pm) and the reverse reactions by competitive displacement of [5F]FFR-Pm with active site-blocked Pm. The rate constants for the biexponential fluorescence quenching caused by SK and SKΔK414 binding to [5F]FFR-Pm were saturable as a function of SK concentration, reporting encounter complex affinities of 62-110 nm in the absence of lysine analogs and 4900-6500 and 1430-2200 nm in the presence of 6-AHA and benzamidine, respectively. The encounter complex with SKΔK414 was ∼10-fold weaker in the absence of lysine analogs but indistinguishable from that of native SK in the presence of 6-AHA and benzamidine. The studies delineate for the first time the sequence of molecular events in the formation of the SK·Pm catalytic complex and its regulation by kringle ligands. Analysis of the forward and reverse reactions supports a binding mechanism in which SK Lys414 binding to a Pm kringle accompanies near-diffusion-limited encounter complex formation followed by two slower, tightening conformational changes. The central event in the fibrinolytic system is dissolution of fibrin clots by the serine proteinase, plasmin (Pm), 2The abbreviations used are: Pm[Lys]plasminμPmmicro-Pmthe Pm catalytic domainFFR-PmPm inhibited with d-Phe-Phe-Arg-CH2Cl[5F]FFR-Pm[5-(acetamido)fluorescein]-d-Phe-Phe-Arg-PmSKstreptokinaseSKΔK414SK lacking the COOH-terminal Lys414 residuePgplasminogen[Glu]Pgintact native plasminogen[Lys]Pgnative Pg lacking the NH2-terminal 77 residuesFFR-CH2Cld-Phe-Phe-Arg-CH2Cl6-AHA6-aminohexanoic acidPg*nonproteolytically activated form of the plasminogen zymogenLBSlysine binding sitesPEGpolyethylene glycol 8000Mes4-morpholineethanesulfonic acid. formed by activation of the zymogen, plasminogen (Pg). The thrombolytic drug and streptococcal pathogenicity factor, streptokinase (SK) activates Pg to Pm through a unique mechanism (1Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2004; 279: 36633-36641Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 2Boxrud P.D. Bock P.E. J. Biol. Chem. 2004; 279: 36642-36649Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). SK acts by binding Pg and Pm in stoichiometric SK·Pg* and SK·Pm catalytic complexes that bind Pg as a specific substrate and proteolytically convert it into Pm by cleavage of Arg561-Val562 (1Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2004; 279: 36633-36641Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 2Boxrud P.D. Bock P.E. J. Biol. Chem. 2004; 279: 36642-36649Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 3McClintock D.K. Bell P.H. Biochem. Biophys. Res. Commun. 1971; 43: 694-702Crossref PubMed Scopus (153) Google Scholar, 4Wohl R.C. Summaria L. Arzadon L. Robbins K.C. J. Biol. Chem. 1978; 253: 1402-1407Abstract Full Text PDF PubMed Google Scholar, 5Reddy K.N. Markus G. J. Biol. Chem. 1972; 247: 1683-1691Abstract Full Text PDF PubMed Google Scholar, 6Schick L.A. Castellino F.J. Biochem. Biophys. Res. Commun. 1974; 57: 47-54Crossref PubMed Scopus (66) Google Scholar, 7Bajaj A.P. Castellino F.J. J. Biol. Chem. 1977; 252: 492-498Abstract Full Text PDF PubMed Google Scholar, 8Davidson D.J. Higgins D.L. Castellino F.J. Biochemistry. 1990; 29: 3585-3590Crossref PubMed Scopus (47) Google Scholar). The active site of Pg is conformationally induced in the catalytic SK·Pg* complex through the molecular sexuality mechanism, without the typically required proteolytic cleavage (3McClintock D.K. Bell P.H. Biochem. Biophys. Res. Commun. 1971; 43: 694-702Crossref PubMed Scopus (153) Google Scholar, 5Reddy K.N. Markus G. J. Biol. Chem. 1972; 247: 1683-1691Abstract Full Text PDF PubMed Google Scholar, 6Schick L.A. Castellino F.J. Biochem. Biophys. Res. Commun. 1974; 57: 47-54Crossref PubMed Scopus (66) Google Scholar, 9Wang S. Reed G.L. Hedstrom L. Biochemistry. 1999; 38: 5232-5240Crossref PubMed Scopus (59) Google Scholar, 10Wang S. Reed G.L. Hedstrom L. Eur. J. Biochem. 2000; 267: 3994-4001Crossref PubMed Scopus (42) Google Scholar, 11Boxrud P.D. Verhamme I.M. Fay W.P. Bock P.E. J. Biol. Chem. 2001; 276: 26084-26089Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). [Lys]Pm binds SK with ∼830-fold tighter affinity than unlabeled [Lys]Pg and with ∼3500-fold tighter affinity than fluorescently labeled [Lys]Pg (12Boxrud P.D. Fay W.P. Bock P.E. J. Biol. Chem. 2000; 275: 14579-14589Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 13Boxrud P.D. Bock P.E. Biochemistry. 2000; 39: 13974-13981Crossref PubMed Scopus (42) Google Scholar). Fluorescently labeled [Glu]Pg and [Lys]Pg analogs bind SK with ∼5-fold lower affinity than the native proteins, whereas the affinity for [Lys]Pm is unaffected by labeling. The magnitudes of the decreases in affinity induced by 6-AHA are not affected by active-site labeling. [Lys]plasmin Pm the Pm catalytic domain Pm inhibited with d-Phe-Phe-Arg-CH2Cl [5-(acetamido)fluorescein]-d-Phe-Phe-Arg-Pm streptokinase SK lacking the COOH-terminal Lys414 residue plasminogen intact native plasminogen native Pg lacking the NH2-terminal 77 residues d-Phe-Phe-Arg-CH2Cl 6-aminohexanoic acid nonproteolytically activated form of the plasminogen zymogen lysine binding sites polyethylene glycol 8000 4-morpholineethanesulfonic acid. Our equilibrium binding and steady-state kinetic studies describe a unified model for SK-Pg activation in which the conformationally activated SK·Pg* complex initially binds Pg as a specific substrate and cleaves it to Pm in a self-limiting, triggering mechanism (1Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2004; 279: 36633-36641Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 2Boxrud P.D. Bock P.E. J. Biol. Chem. 2004; 279: 36642-36649Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Upon formation of slightly more than 1 SK eq of Pm, Pg is displaced from the SK·Pg* complex, and the much tighter SK·Pm catalytic complex is formed which propagates Pg activation through expression of a Pg binding exosite, converting the remaining free Pg to Pm in a second catalytic cycle (2Boxrud P.D. Bock P.E. J. Biol. Chem. 2004; 279: 36642-36649Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Previous biophysical studies have shown that SK in solution behaves as independently folded domains linked by flexible segments, resulting in a highly mobile structure (14Damaschun G. Damaschun H. Gast K. Gerlach D. Misselwitz R. Welfle H. Zirwer D. Eur. Biophys. J. 1992; 20: 355-361Crossref PubMed Scopus (39) Google Scholar). When bound to micro-Pm (μPm), the isolated catalytic domain of Pm, however, the three SK β-grasp domains rearrange into a well defined crater-like structure surrounding the Pm active site (15Wang X. Lin X. Loy J.A. Tang J. Zhang X.C. Science. 1998; 281: 1662-1665Crossref PubMed Scopus (219) Google Scholar). This large change in SK structure is accompanied by expression of the Pg substrate binding exosite (12Boxrud P.D. Fay W.P. Bock P.E. J. Biol. Chem. 2000; 275: 14579-14589Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The circulating form of the Pg zymogen, [Glu]Pg, consists of a 77-residue NH2-terminal peptide, five kringle domains (K1-K5), some of which contain lysine binding sites (LBS), and a COOH-terminal serine proteinase catalytic domain (16Henkin J. Marcotte P. Yang H. Prog. Cardiovasc. Dis. 1991; 34: 135-164Crossref PubMed Scopus (68) Google Scholar, 17Ponting C.P. Marshall J.M. Cederholm-Williams S.A. Blood Coagul. Fibrinolysis. 1992; 3: 605-614Crossref PubMed Scopus (200) Google Scholar). K1, K2, K4, and K5 exhibit varying degrees of affinity for lysine analogs and small aromatic ionic ligands (18Marti D.N. Hu C.K. An S.S. von Haller P. Schaller J. Llinas M. Biochemistry. 1997; 36: 11591-11604Crossref PubMed Scopus (61) Google Scholar). K1, K4, and K5 mediate Pg and Pm binding to COOH-terminal lysine residues on fibrin and to lysine and arginine residues of other proteins (19Castellino F.J. McCance S.G. Ciba Found. Symp. 1997; 212: 46-60PubMed Google Scholar, 20Christensen U. FEBS Lett. 1985; 182: 43-46Crossref PubMed Scopus (60) Google Scholar, 21Lucas M.A. Fretto L.J. McKee P.A. J. Biol. Chem. 1983; 258: 4249-4256Abstract Full Text PDF PubMed Google Scholar, 22Wiman B. Lijnen H.R. Collen D. Biochim. Biophys. Acta. 1979; 579: 142-154Crossref PubMed Scopus (229) Google Scholar, 23Bok R.A. Mangel W.F. Biochemistry. 1985; 24: 3279-3286Crossref PubMed Scopus (75) Google Scholar, 24Clemmensen I. Petersen L.C. Kluft C. Eur. J. Biochem. 1986; 156: 327-333Crossref PubMed Scopus (176) Google Scholar). The LBS located in K5 is thought to be primarily responsible for interactions with the NH2-terminal peptide in [Glu]Pg, keeping the zymogen in a spiral, compact α-conformation (25Cockell C.S. Marshall J.M. Dawson K.M. Cederholm-Williams S.A. Ponting C.P. Biochem. J. 1998; 333: 99-105Crossref PubMed Scopus (72) Google Scholar, 26Ponting C.P. Holland S.K. Cederholm-Williams S.A. Marshall J.M. Brown A.J. Spraggon G. Blake C.C. Biochim. Biophys. Acta. 1992; 1159: 155-161Crossref PubMed Scopus (42) Google Scholar, 27Marshall J.M. Brown A.J. Ponting C.P. Biochemistry. 1994; 33: 3599-3606Crossref PubMed Scopus (105) Google Scholar, 28Mangel W.F. Lin B.H. Ramakrishnan V. Science. 1990; 248: 69-73Crossref PubMed Scopus (159) Google Scholar). Removal of the NH2-terminal peptide by Pm cleavage results in formation of [Lys]Pg, which assumes a partially extended β-conformation that is more rapidly activated by tissue-type plasminogen activator and urokinase and by the SK·Pg* and SK·Pm catalytic complexes (2Boxrud P.D. Bock P.E. J. Biol. Chem. 2004; 279: 36642-36649Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 29Hoylaerts M. Rijken D.C. Lijnen H.R. Collen D. J. Biol. Chem. 1982; 257: 2912-2919Abstract Full Text PDF PubMed Google Scholar, 30Wohl R.C. Summaria L. Robbins K.C. J. Biol. Chem. 1980; 255: 2005-2013Abstract Full Text PDF PubMed Google Scholar, 31Violand B.N. Byrne R. Castellino F.J. J. Biol. Chem. 1978; 253: 5395-5401Abstract Full Text PDF PubMed Google Scholar, 32Violand B.N. Castellino F.J. J. Biol. Chem. 1976; 251: 3906-3912Abstract Full Text PDF PubMed Google Scholar). The lysine analog 6-aminohexanoic acid (6-AHA) binds with high affinity to K1 (KD 11-13 μm) and to lower affinity sites on K4 (KD 20-60 μm) and K5 (KD 95-140 μm) based on studies of the isolated kringle domains (17Ponting C.P. Marshall J.M. Cederholm-Williams S.A. Blood Coagul. Fibrinolysis. 1992; 3: 605-614Crossref PubMed Scopus (200) Google Scholar, 18Marti D.N. Hu C.K. An S.S. von Haller P. Schaller J. Llinas M. Biochemistry. 1997; 36: 11591-11604Crossref PubMed Scopus (61) Google Scholar, 33Lerch P.G. Rickli E.E. Lergier W. Gillessen D. Eur. J. Biochem. 1980; 107: 7-13Crossref PubMed Scopus (183) Google Scholar, 34Markus G. DePasquale J.L. Wissler F.C. J. Biol. Chem. 1978; 253: 727-732Abstract Full Text PDF PubMed Google Scholar). Benzamidine binds isolated K5 with highest affinity (KD 290 μm), interacts weaker with K1 (KD 12.5 mm) and K2 (KD 33 mm), and displays insignificant affinity for K3 and K4 (18Marti D.N. Hu C.K. An S.S. von Haller P. Schaller J. Llinas M. Biochemistry. 1997; 36: 11591-11604Crossref PubMed Scopus (61) Google Scholar, 35Thewes T. Constantine K. Byeon I.J. Llinas M. J. Biol. Chem. 1990; 265: 3906-3915Abstract Full Text PDF PubMed Google Scholar, 36Varadi A. Patthy L. Biochem. Biophys. Res. Commun. 1981; 103: 97-102Crossref PubMed Scopus (43) Google Scholar). Benzamidine also induces a partially extended β-conformation of [Glu]Pg but, unlike 6-AHA, does not induce the transition of [Lys]Pg to the fully extended γ-form (25Cockell C.S. Marshall J.M. Dawson K.M. Cederholm-Williams S.A. Ponting C.P. Biochem. J. 1998; 333: 99-105Crossref PubMed Scopus (72) Google Scholar, 27Marshall J.M. Brown A.J. Ponting C.P. Biochemistry. 1994; 33: 3599-3606Crossref PubMed Scopus (105) Google Scholar). The affinity of SK for native and fluorescently labeled [Glu]Pg in the compact conformation is relatively low and is unaffected by 6-AHA, whereas expression of LBS in the β-conformation of [Lys]Pg and [Lys]Pm increases affinity for SK, which is weakened ∼13-20-fold by 6-AHA in interactions mediated by the COOH-terminal Lys414 residue of SK (1Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2004; 279: 36633-36641Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 13Boxrud P.D. Bock P.E. Biochemistry. 2000; 39: 13974-13981Crossref PubMed Scopus (42) Google Scholar, 37Panizzi P. Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2006; 281: 26774-26778Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Pm retains significant affinity for SK when LBS interactions are blocked (KD 200-300 pm) through its interactions with the catalytic domain of Pm. LBS interactions, thus, play a critical role in formation and stabilization of the SK·Pm catalytic complex. The present study describes the first rapid-reaction kinetics investigation of active site fluorescently labeled Pm binding to SK, characterizes the binding and conformational intermediates on the pathway, and identifies LBS-dependent steps. Stopped-flow fluorescence kinetics of SK binding to fluoresce-in-labeled [Lys]Pm identified the forward reactions of complex stabilization, whereas the reverse reactions were examined by competitive displacement of fluorescently labeled Pm from SK by unlabeled, active site-blocked Pm. Combined analysis of forward and reverse reactions allowed the sequence of elementary reaction steps of the mechanism to be delineated for the first time. Our results support the hypothesis that LBS-dependent near-diffusion-limited SK binding to form an SK·Pm encounter complex is followed by two consecutive, favorable conformational changes. The much slower competitive displacement presents as an apparent first-order process but is governed by an off-rate that is a function of the forward and reverse rate constants for the second and third steps. The conformational changes are hypothesized to reflect in part, changes in SK from its flexible conformation in solution to its ordered structure when it binds to Pm and/or a conformational change affecting the Pm catalytic site in processes that involve specific LBS interactions that are differentially affected by benzamidine and 6-AHA. Interaction of SK Lys414 with a Pm kringle contributes primarily to stabilizing the SK·Pm encounter complex in the first step of the pathway. Protein Purification and Characterization—Human [Glu]Pg carbohydrate form 2 ([Glu]Pg2) was purified from plasma by published procedures (38Deutsch D.G. Mertz E.T. Science. 1970; 170: 1095-1096Crossref PubMed Scopus (1670) Google Scholar, 39Castellino F.J. Powell J.R. Methods Enzymol. 1981; 80: 365-378Crossref PubMed Scopus (180) Google Scholar). Activation of 10 μm [Glu]Pg2 to [Lys]Pm2 with 90 units/ml urokinase (Calbiochem) was performed in 10 mm Mes, 10 mm Hepes, 0.15 m NaCl, 20 mm 6-AHA, and 1 mg/ml polyethylene glycol 8000 (PEG) at pH 7.4 and 25 °C. Pm was purified by affinity chromatography on soybean trypsin inhibitor-agarose (12Boxrud P.D. Fay W.P. Bock P.E. J. Biol. Chem. 2000; 275: 14579-14589Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Pm was dialyzed against 5 mm Hepes, 0.3 m NaCl, 10 mm 6-AHA, and 1 mg/ml PEG at pH 7.0 and 4 °C. The active site of Pm (10-15 μm) was blocked with a 5-fold molar excess of d-Phe-Phe-Arg-CH2Cl (FFR-CH2Cl) in 0.1 m Hepes, 0.3 m NaCl, 1 mm EDTA, 10 mm 6-AHA, 1 mg/ml PEG, pH 7.0 buffer at 25 °C for 30-60 min, reducing the initial rate of hydrolysis of d-Val-Leu-Lys-para-nitroanilide to <0.1%, as described previously (12Boxrud P.D. Fay W.P. Bock P.E. J. Biol. Chem. 2000; 275: 14579-14589Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 13Boxrud P.D. Bock P.E. Biochemistry. 2000; 39: 13974-13981Crossref PubMed Scopus (42) Google Scholar). Excess inhibitor was removed by dialysis against >250 volumes of 50 mm Hepes, 0.3 m NaCl, 1 mm EDTA, pH 7.0 at 4 °C. Native SK (Diapharma) was obtained as outdated therapeutic material and purified as described (40Bock P.E. Day D.E. Verhamme I.M. Bernardo M.M. Olson S.T. Shore J.D. J. Biol. Chem. 1996; 271: 1072-1080Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The SKΔK414 mutant was prepared as described previously (37Panizzi P. Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2006; 281: 26774-26778Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). All proteins were quick-frozen in 2-propanol/dry ice and stored at -70 °C. Protein concentrations were determined by absorbance at 280 nm using the following absorption coefficients ((mg/ml)-1cm-1) and molecular weights: Pm, 1.9 and 84,000 (39Castellino F.J. Powell J.R. Methods Enzymol. 1981; 80: 365-378Crossref PubMed Scopus (180) Google Scholar); SK and SKΔK414, 0.95 and 47,000 (41Taylor Jr., F.B. Botts J. Biochemistry. 1968; 7: 232-242Crossref PubMed Scopus (49) Google Scholar, 42Jackson K.W. Tang J. Biochemistry. 1982; 21: 6620-6625Crossref PubMed Scopus (121) Google Scholar). The active Pm concentration (90% of total protein) was determined by active-site titration with fluorescein mono-p-guanidinobenzoate (43Bock P.E. Craig P.A. Olson S.T. Singh P. Arch. Biochem. Biophys. 1989; 273: 375-388Crossref PubMed Scopus (83) Google Scholar). Active-site Labeling of Pm—The active-site specific inhibitor, Nα-[(acetylthio)acetyl]-(d-Phe)-Phe-Arg-CH2Cl (ATA-FFR-CH2Cl), prepared by published procedures (44Bock P.E. J. Biol. Chem. 1992; 267: 14963-14973Abstract Full Text PDF PubMed Google Scholar, 45Bock P.E. J. Biol. Chem. 1992; 267: 14974-14981Abstract Full Text PDF PubMed Google Scholar, 46Bock P.E. Methods Enzymol. 1993; 222: 478-503Crossref PubMed Scopus (31) Google Scholar), was incubated at a 5-fold molar excess with 10-15 μm [Lys]Pm2 in 0.1 m Hepes, 0.3 m NaCl, 1 mm EDTA, 10 mm 6-AHA, 1 mg/ml PEG, pH 7.0, at 25 °C for 60 min until inhibition was complete (<0.1% activity). Excess inhibitor was removed by dialysis against 50 mm Hepes, 0.3 m NaCl, 1 mm EDTA, 1 mg/ml PEG, pH 7.0 at 4 °C. Quantitation of inhibitor incorporation measured from the amplitude of the burst reaction of NH2OH-generated thiol with 5,5′-dithiobis(2-nitrobenzoic acid) (46Bock P.E. Methods Enzymol. 1993; 222: 478-503Crossref PubMed Scopus (31) Google Scholar) indicated 0.8-1.4 mol thioester/mol of Pm active sites. The thiol group on the incorporated inhibitor generated by treatment of ATA-FFR-Pm with NH2OH was labeled with 5-(iodoacetamido)fluorescein to yield [5F]FFR-Pm as described (12Boxrud P.D. Fay W.P. Bock P.E. J. Biol. Chem. 2000; 275: 14579-14589Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 13Boxrud P.D. Bock P.E. Biochemistry. 2000; 39: 13974-13981Crossref PubMed Scopus (42) Google Scholar). Excess dye was removed by chromatography on Sephadex G25 superfine followed by extensive dialysis. [5F]FFR-Pm concentration and probe incorporation (87%) were determined from the probe and protein absorbance in 6 m guanidine as described (44Bock P.E. J. Biol. Chem. 1992; 267: 14963-14973Abstract Full Text PDF PubMed Google Scholar). Proteins were homogeneous by SDS gel electrophoresis. Stopped-flow Kinetics of SK and SKΔK414 Binding to [5F]FFR-Pm—Time traces of SK and SKΔK414 binding to [5F]FFR-Pm were acquired with an Applied Photophysics SX-18MV stopped-flow spectrofluorometer in single mixing mode. Fluorescence was measured with excitation at 500 nm and an emission cut-on filter (Melles-Griot) with 50% transmission at 515 nm. The reaction volume was 200 μl, path length was 2 mm, and experiments were performed at 25 °C. SK or SKΔK414 (0.025-15 μm) and [5F]FFR-Pm (5-20 nm) were reacted under pseudo-first order conditions (SK ≥ 5-fold over labeled Pm at the lowest concentration) in 50 mm Hepes, 0.125 m NaCl, 1 mm EDTA, 1 mg/ml PEG, 1 mg/ml bovine serum albumin, 1 μm FFR-CH2Cl, pH 7.4, in the absence and presence of 50 mm 6-AHA or in 50 mm Hepes, 0.075 m NaCl, 1 mm EDTA, 1 mg/ml PEG, 1 mg/ml bovine serum albumin, 1 μm FFR-CH2Cl, pH 7.4, containing 50 mm benzamidine to maintain constant ionic strength. Time traces (1000 data points) of near-complete (>95%) exponential fluorescence decreases ranged from 0.5 to 30 s, depending on the SK concentration and the presence of effector. Typically, 10 time traces were averaged for each SK concentration. Control reactions containing buffer and SK only, and buffer and [5F]FFR-Pm only, were performed to quantitate background and initial probe fluorescence, respectively, and to permit transformation of raw data into the fractional change in initial fluorescence ((Fobs - Fo)/Fo = ΔF/Fo). Averaged time traces were analyzed using Equation 1, ΔF/Fo=(Fo-FM)(A1e(-kobs1t)+(1-A1)e(-kobs2t))+FM(Eq. 1) where Fo is the starting fluorescence, FM is the final fluorescence, A1 is the fractional amplitude of the first exponential, (1 - A1) is the fractional amplitude of the second exponential, and kobs 1 and kobs 2 are the observed first-order rate constants. The observed pseudo-first order rate constants for each process were analyzed as a function of the total SK concentration ([SK]o) using Equation 2, kobs=klim[SK]oK1app+[SK]o+koff(Eq. 2) where klim is the maximal value for the rate constant, K1 app is the apparent dissociation constant for the SK·Pm encounter complex, and koff is the overall reverse rate constant for binding. In addition, arrays of time traces at varying SK concentrations were analyzed simultaneously using the numerical integration programs Dynafit (47Kuzmic P. Anal. Biochem. 1996; 237: 260-273Crossref PubMed Scopus (1357) Google Scholar) and KinTek Explorer (KinTek Corp.), combining numerical solution of the differential equations generated from the mechanism with nonlinear least-squares analysis. Competitive Dissociation of [5F]FFR-Pm from Its Complex with SK or SKΔK414 by FFR-Pm—Measurements were performed with an SLM 8100 spectrofluorometer or a Photon Technology International QuantaMaster 6000 fluorometer using acrylic cuvettes coated with polyethylene glycol 20,000. The excitation and emission wavelengths were 500 and 516 nm, respectively, with 4/8 nm and 2/8 nm excitation/emission band passes on the SLM and the Photon Technology International instruments, respectively. [5F]FFR-Pm (10-22 nm) was incubated with excess SK or SKΔK414 (15-100 nm) at 25 °C in the absence and presence of 50 mm 6-AHA or in buffer containing 0.075 m NaCl and 50 mm benzamidine until the fluorescence was stable (∼15 min). The reverse reaction was initiated by the addition of 100-1000 nm FFR-Pm, such that any remaining free SK was saturated with FFR-Pm, and the dissociation of SK from the SK·[5F]FFR-Pm complex by the remaining FFR-Pm was apparently first order in SK·[5F]FFR-Pm complex concentration. The increase in fluorescence was monitored until it was >90% complete, typically 4 h. Corrections were made for background scattering by subtraction of blanks containing all reactants except [5F]FFR-Pm. Corrections for instrument drift and probe photobleaching were done using cuvettes containing [5F]FFR-Pm and SK to form the complex, with the addition of buffer instead of FFR-Pm. Results expressed as ΔF/Fo versus time were fit by a single exponential to obtain estimates of koff. Least-squares fitting was performed with SCIENTIST Software (MicroMath), and the progress curves were also analyzed by numerical integration with Dynafit and Kintek Explorer. The latter program allowed simultaneous analysis of the competitive dissociation experiments and the array of forward reaction time traces at varying SK concentrations. All reported estimates of error represent ± 2 S.D. Equation 3 shows the analytical expression for koff in a 3-step binding reaction (48Tsodikov O.V. Record Jr., M.T. Biophys. J. 1999; 76: 1320-1329Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), rearranged as an expression of rate constants for the three steps, koff=k-1k-2k-3k-1(k-2+k3+k-3)+k-2(k-3+k2k-3)+k2k3(Eq. 3) Equilibrium Binding of [5F]FFR-Pm and FFR-Pm to SK and SKΔK414 in the Presence of Benzamidine—[5F]FFR-Pm was titrated with SK or SKΔK414 at 25 °C in the above-described buffer containing 50 mm benzamidine. Fluorescence titrations were performed with the Photon Technology International fluorometer at excitation and emission wavelengths of 500 and 516 nm, respectively, with 2/8-nm excitation/emission band passes. Fluorescence changes were measured after equilibration for 5-10 min. In the competitive binding experiments, SK or SKΔK414 was titrated into a mixture of labeled Pm and FFR-Pm. Concentrations of FFR-Pm were 5 and 15 nm in the titrations with SK and 10 nm in the titrations with SKΔK414. Measurements were corrected for background (≤10%) by subtraction of blanks lacking [5F]FFR-Pm. Simultaneous nonlinear least-squares fitting with SCIENTIST software was performed of the [5F]FFR-Pm titrations with SK or SKΔK414 in the absence and presence of fixed FFR-Pm concentrations by the cubic equation for tight competitive binding of a single ligand (SK or SKΔK414) to labeled ([5F]FFR-Pm) and non-labeled acceptor (FFR-Pm) (49Bock P.E. Olson S.T. Björk I. J. Biol. Chem. 1997; 272: 19837-19845Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 50Lindahl P. Raub-Segall E. Olson S.T. Björk I. Biochem. J. 1991; 276: 387-394Crossref PubMed Scopus (29) Google Scholar, 51Olson S.T. Bock P.E. Sheffer R. Arch. Biochem. Biophys. 1991; 286: 533-545Crossref PubMed Scopus (42) Google Scholar). The cubic equation is an exact solution for competitive binding of one ligand to two acceptors, typically one fluorescently labeled that reports the interaction and the non-labeled competitor, under conditions where the assumption cannot be made that free and total ligand concentrations are approximately equal (49Bock P.E. Olson S.T. Björk I. J. Biol. Chem. 1997; 272: 19837-19845Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 50Lindahl P. Raub-Segall E. Olson S.T. Björk I. Biochem. J. 1991; 276: 387-394Crossref PubMed Scopus (29) Google Scholar, 51Olson S.T. Bock P.E. Sheffer R. Arch. Biochem. Biophys. 1991; 286: 533-545Crossref PubMed Scopus (42) Google Scholar). This analysis gave the dissociation constant (KC) and stoichiometric factor (m) for competitive binding of SK or SKΔK414 to FFR-Pm as well as the maximum fluorescence intensity change (ΔFmax/Fo), the dissociation constant (KD), and the stoichiometric factor (n) for binding of SK or SKΔK414 to [5F]FFR-Pm (49Bock P.E. Olson S.T. Björk I. J. Biol. Chem. 1997; 272: 19837-19845Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 50Lindahl P. Raub-Segall E. Olson S.T. Björk I. Biochem. J. 1991; 276: 387-394Crossref PubMed Scopus (29) Google Scholar, 51Olson S.T. Bock P.E. Sheffer R. Arch. Biochem. Biophys. 1991; 286: 533-545Crossref PubMed Scopus (42) Google Scholar). Stopped-flow Kinetics of SK and SKΔK414 Binding to [5F]FFR-Pm—Changes in fluorescein fluorescence after rapid mixing of excess SK or SKΔK414 with [5F]FFR-Pm were biexponential in the absence of effectors but less distinctly so in the presence of saturating (50 mm) 6-AHA or benzamidine as illustrated by the respective logarithmic transformations of ΔF/Fo (Figs. 1, A and B). However, single exponential analysis of the time traces in the presence of 6-AHA and benzamidine consistently gave poorer fits with non-random residuals; hence, all time traces were analyzed with the biexponential Equation 1. The first-order rate constants k" @default.
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• W2021471987 title "Rapid-reaction Kinetic Characterization of the Pathway of Streptokinase-Plasmin Catalytic Complex Formation" @default.
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