Coupling of Conformational and Proteolytic Activation in the Kinetic Mechanism of Plasminogen Activation by Streptokinase

Abstract

Binding of streptokinase (SK) to plasminogen (Pg) induces conformational activation of the zymogen and initiates its proteolytic conversion to plasmin (Pm). The mechanism of coupling between conformational activation and Pm formation was investigated in kinetic studies. Parabolic time courses of Pg activation by SK monitored by chromogenic substrate hydrolysis had initial rates (v1) representing conformational activation and subsequent rates of activity increase (v2) corresponding to the rate of Pm generation determined by a specific discontinuous assay. The v2 dependence on SK concentration for [Lys]Pg showed a maximum rate at a Pg to SK ratio of ∼2:1, with inhibition at high SK concentrations. [Glu]Pg and [Lys]Pg activation showed similar kinetic behavior but much slower activation of [Glu]Pg, due to an ∼12-fold lower affinity for SK and an ∼20-fold lower kcat/Km. Blocking lysine-binding sites on Pg inhibited SK·Pg* cleavage of [Lys]Pg to a rate comparable with that of [Glu]Pg, whereas [Glu]Pg activation was not significantly affected. The results support a kinetic mechanism in which SK activates Pg conformationally by rapid equilibrium formation of the SK·Pg* complex, followed by intermolecular cleavage of Pg to Pm by SK·Pg* and subsequent cleavage of Pg by SK·Pm. A unified model of SK-induced Pg activation suggests that generation of initial Pm by SK·Pg* acts as a self-limiting triggering mechanism to initiate production of one SK equivalent of SK·Pm, which then converts the remaining free Pg to Pm. Binding of streptokinase (SK) to plasminogen (Pg) induces conformational activation of the zymogen and initiates its proteolytic conversion to plasmin (Pm). The mechanism of coupling between conformational activation and Pm formation was investigated in kinetic studies. Parabolic time courses of Pg activation by SK monitored by chromogenic substrate hydrolysis had initial rates (v1) representing conformational activation and subsequent rates of activity increase (v2) corresponding to the rate of Pm generation determined by a specific discontinuous assay. The v2 dependence on SK concentration for [Lys]Pg showed a maximum rate at a Pg to SK ratio of ∼2:1, with inhibition at high SK concentrations. [Glu]Pg and [Lys]Pg activation showed similar kinetic behavior but much slower activation of [Glu]Pg, due to an ∼12-fold lower affinity for SK and an ∼20-fold lower kcat/Km. Blocking lysine-binding sites on Pg inhibited SK·Pg* cleavage of [Lys]Pg to a rate comparable with that of [Glu]Pg, whereas [Glu]Pg activation was not significantly affected. The results support a kinetic mechanism in which SK activates Pg conformationally by rapid equilibrium formation of the SK·Pg* complex, followed by intermolecular cleavage of Pg to Pm by SK·Pg* and subsequent cleavage of Pg by SK·Pm. A unified model of SK-induced Pg activation suggests that generation of initial Pm by SK·Pg* acts as a self-limiting triggering mechanism to initiate production of one SK equivalent of SK·Pm, which then converts the remaining free Pg to Pm. Streptokinase (SK) 1The abbreviations used are: SK, streptokinase; Pg, plasminogen; [Glu]Pg, intact native plasminogen; [Lys]Pg, native Pg lacking the N-terminal 77 residues; Pm, plasmin; FFR-Pm, Pm inhibited with (d-Phe)-Phe-Arg-CH2Cl; 6-AHA, 6-aminohexanoic acid; pNA, p-nitroaniline; Pg*, nonproteolytically activated form of the plasminogen zymogen. is a protein from Streptococcus equisimilis that converts the zymogen, plasminogen (Pg), into the fibrin clot-dissolving serine proteinase, plasmin (Pm) (1Collen D. Lijnen H.R. Thromb. Haemostasis. 1995; 74: 167-171Crossref PubMed Scopus (93) Google Scholar). The domain organization of the circulating form of Pg ([Glu]Pg) consists of a 77-residue N-terminal peptide, five kringle domains, and a serine proteinase catalytic domain that is activated by cleavage at Arg561–Val562 (2Henkin J. Marcotte P. Yang H. Prog. Cardiovasc. Dis. 1991; 34: 135-164Crossref PubMed Scopus (68) Google Scholar, 3Ponting C.P. Marshall J.M. Cederholm-Williams S.A. Blood Coagul. Fibrinolysis. 1992; 3: 605-614Crossref PubMed Scopus (200) Google Scholar). Pm cleavage of [Glu]Pg releases the N-terminal peptide, yielding [Lys]Pg, which is more readily activated by plasminogen activators, including SK·Pg and SK·Pm (4Violand B.N. Byrne R. Castellino F.J. J. Biol. Chem. 1978; 253: 5395-5401Abstract Full Text PDF PubMed Google Scholar, 5Urano T. Sator de Serrano V. Chibber B.A. Castellino F.J. J. Biol. 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The mechanism of Pg activation by SK is unique in that SK binding to Pg results in expression of an active catalytic site on the zymogen by a conformational change, without the normally required peptide bond cleavage (12McClintock D.K. Bell P.H. Biochem. Biophys. Res. Commun. 1971; 43: 694-702Crossref PubMed Scopus (153) Google Scholar, 13Reddy K.N. Markus G. J. Biol. Chem. 1972; 247: 1683-1691Abstract Full Text PDF PubMed Google Scholar). Conformational activation is followed by proteolytic conversion of Pg to Pm by cleavage of Arg561–Val562, and SK bound to Pm propagates proteolytic Pg activation. Recognition of Pg as a substrate of SK·Pm is mediated by expression of a specific substrate recognition exosite on the SK·Pm complex (14Wang X. Lin X. Loy J. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). conformational and proteolytic activation to to SK-induced Pg but of the mechanism of conformational activation and its coupling to subsequent proteolytic Pm formation equilibrium binding and kinetic of conformational activation in the J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that by rapid and SK binding and activation of the catalytic The activation equilibrium is more for Pg with an [Lys]Pg and [Glu]Pg at concentration (4Violand B.N. Byrne R. Castellino F.J. J. Biol. Chem. 1978; 253: 5395-5401Abstract Full Text PDF PubMed Google Scholar, 5Urano T. Sator de Serrano V. Chibber B.A. Castellino F.J. J. Biol. Chem. 1987; 262: 15959-15964Abstract Full Text PDF PubMed Google Scholar, Castellino F.J. Biochemistry. 1995; 34: PubMed Scopus Google with [Glu]Pg in the J.M. Brown A.J. Ponting C.P. Biochemistry. 1994; 33: 3599-3606Crossref PubMed Scopus (105) Google Scholar, Lin V. PubMed Scopus Google Scholar, C.P. Cederholm-Williams S.A. Marshall J.M. Brown A.J. G. Biophys. 1992; PubMed Scopus Google due to the of lysine-binding site kinetic of Pm formation that Pm was by proteolytic activation of Pg the conformationally activated SK·Pg* complex Biochemistry. PubMed Scopus Google Scholar). The subsequent that SK·Pm not a of SK, Pg, and to Pm and the of SK·Pg* to Pg the cleavage Castellino F.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Biochemistry. PubMed Scopus Google Scholar). (14Wang X. Lin X. Loy J. PubMed Scopus Google Scholar, C.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Castellino F.J. Biochemistry. PubMed Scopus Google for an complex, that intermolecular cleavage of Pg bound to SK·Pg* in a substrate to initial Pm binding that the SK with and affinity to Pg J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Biochemistry. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and native Pg J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google with the initial The in affinity in of Pg from the SK·Pg* complex by Pm, the proteolytic Pg complex SK·Pm. The of Pm generation from SK·Pg* and the of SK·Pm in of for the and intermolecular The of lysine-binding sites on the kringle of Pg and Pm in the mechanism of SK-induced Pg activation is The 6-aminohexanoic binding affinity of SK for [Lys]Pg in with inhibition of conformational but not [Glu]Pg J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Biochemistry. PubMed Scopus Google and formation of Pm S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. Biochemistry. PubMed Scopus Google Scholar). that the affinity of SK binding to Pg and subsequent proteolytic formation of Pm inhibited by 6-AHA, but the between of inhibition and the for the on [Glu]Pg and [Lys]Pg activation not The of the was to the results of equilibrium binding and kinetic of and in with initial rate kinetic to the coupling of conformational Pg activation to the proteolytic activation of SK-induced Pm The the mechanism of proteolytic Pm the between by SK·Pg* and SK·Pm and of by lysine-binding site The of proteolytic Pg activation and inhibition of Pm formation by SK is by a mechanism in which Pm is by intermolecular cleavage of Pg by the SK·Pg* The binding and conformational activation equilibrium for [Lys]Pg, in the J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google is by lysine-binding site whereas subsequent Pm formation is on the of [Glu]Pg conformational and proteolytic activation on lysine-binding sites for the substrate of the SK·Pg* complex for [Lys]Pg with The equilibrium binding and kinetic not support a for Pm formation the SK·Pg* The the kinetic of the of Pm formation by SK and the of in SK affinity for Pg activation and the of lysine-binding site in and the activation A unified kinetic mechanism of Pg activation suggests that the activation at of Pg SK is by a self-limiting initial of Pm formation by SK·Pg* that results in of SK in the SK·Pm complex, which conversion of free Pg to Pm. and [Lys]Pg, Pm, and SK and determined as in the J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). activation at by of SK in and with for at of and on with and protein with a of Pg by SK was by of the increase in of at The of formation by J. Biol. Chem. 1978; 253: Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google to the initial rate of substrate hydrolysis at the of the (v1) and the rate of increase in activity with time of the time courses was by by Biochemistry. PubMed Scopus Google Scholar). for activation of [Glu]Pg and [Lys]Pg determined from the of v2 rates on the Pg concentration at SK and Pm. of the to SK·Pm Pm and SK·Pm to but Pm not Pg J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the initial rates of Pm generation in of Pg and SK by of SK and for and the by of and to the SK·Pg* complex and to formation of Pm. of chromogenic substrate of by of and the of Pm from of the The substrate concentration was that free Pm and Pm had to the of SK concentration J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of Pm generation with time at to Pg and by to the in which and in the in rates of The of with increase in activity The the Pm concentration in of Pg SK of Pm. of by of and v2 on SK concentration by the mechanism in and SK to Pg with to form the conformationally activated SK·Pg* complex, which chromogenic substrate hydrolysis with and Binding of Pg as a substrate of SK·Pg* with the complex that Pm with the catalytic rate in the SK·Pm complex Pg activation to Pm, and SK·Pm the chromogenic substrate with and The high affinity of SK for Pm with [Glu]Pg and [Lys]Pg is to in SK·Pm as the initial of the Pm is as SK·Pm, whereas at SK with Pm as the the that Pg activation by SK·Pm not significantly to the initial and in with [Lys]Pg at the of activity formation is by is by the in the J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and the initial rate of Pm generation by the SK·Pg* complex (v2) is by v2 by in of is by the J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and which to J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google to and the proteolytic activation as with rate is by the in and in the J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). v2 rates as by and as determined for the SK·Pm complex J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The v2 from activation as a of SK and Pg by the and the binding the of Pg much lower for activation of Pg as a substrate of the SK·Pm complex, that the proteolytic in as with rate the for proteolytic in and by of the rate Pm by the SK·Pg* complex with rate is as whereas Pm by the SK·Pm complex with rate is The binding and to to the proteolytic and of and of and the rate to the in Pm by was to the affinity for SK and to Pg activation with the rate The concentration of Pm was as courses of the with and initial in the of v2 to the of Pm of Pg activation with the initial rate (v1) representing substrate by the conformationally activated SK·Pg* complex, followed by an increase in rate (v2) to proteolytic Pm formation J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). A discontinuous specific for Pm was to that v2 to the rate of Pm The was specific for Pm and was not by the of Pg SK·Pg* as by a of Pm formation from a of and SK determined by the discontinuous and Pm formation as by the discontinuous Pm was for to with a of Pm The initial Pm concentration was of and in Pm formation the of of Pm The v2 from of of and SK as a of of as with to The dependence of v2 on chromogenic substrate concentration was with inhibition of Pm formation with an inhibition of the for inhibition by the of Pm formation was Pm in with the rate by the discontinuous assay. of Pm generation determined by the discontinuous and in the of for activation of by SK showed a SK concentration The results rates of Pm formation from the Pg activation by the and discontinuous of Pm by the between v2 and Pm formation and inhibition of the by SK, the of of Pg and SK by in of with SK for showed an increase in Pm formation to SK, followed by inhibition at SK as by an increase in Pg and a in of the Pm and of activation of for with SK similar results that inhibition of Pm formation required SK with cleavage of SK to form the SK is the complex by cleavage of streptokinase at Castellino F.J. Biochemistry. PubMed Scopus Google at SK concentration and at high SK with results J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). results that v2 the rate of proteolytic Pm which was inhibited by high SK concentrations. of the of SK-induced Pm kinetic of Pg activation by SK that Pm was by cleavage of Pg the SK·Pg* complex Biochemistry. PubMed Scopus Google Scholar, Castellino F.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Biochemistry. PubMed Scopus Google by intermolecular cleavage of Pg by SK·Pg* (14Wang X. Lin X. Loy J. PubMed Scopus Google Scholar, C.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Castellino F.J. Biochemistry. PubMed Scopus Google Scholar). the intermolecular model of Pg activation without of Pg activation the SK kinetic of of Pg and to the form of model by the in SK to Pg with the the conformationally activated SK·Pg* complex, which and Pg to Pm with rate in the J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that of the formation of SK·Pg* as a binding and activation and the proteolytic as v2 from of of with SK by and the binding for with the that and as of the dependence of on SK concentration a to the with for J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the v2 in a of for which was lower the from the and of for of with SK of [Lys]Pg and [Glu]Pg activation by dependence of v2 from of of and from of and with SK in the of as a of SK concentration the to the by the intermolecular activation and as and kinetic determined for conformational and proteolytic Pg in a with [Glu]Pg showed much slower and inhibition by of from of for J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google whereas of the v2 in of and of and binding of SK to [Glu]Pg with [Lys]Pg, as in binding J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and a in Pm generation by v2 for [Lys]Pg activation by with the maximum in more at SK concentrations. The v2 from activation of [Glu]Pg, in to [Lys]Pg not significantly by and comparable with of [Lys]Pg in results that inhibition of Pm formation by was due to of the affinity of SK for [Lys]Pg and inhibition of subsequent proteolytic activation. the affinity of SK for [Glu]Pg and its proteolytic conversion to Pm not significantly lysine-binding of the in the was that Pg activation not significantly to the initial rate of Pm was by of the SK·Pg* and SK·Pm in by of the rate The kinetic for cleavage of [Lys]Pg and in determined in v2 of [Lys]Pg activation by SK·Pm with an of Pm not Pm not Pg J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). and for and was the Pg concentration used the proteolytic to as with rate as in the the rate and and the for the SK·Pm complex at determined whereas was a of comparable with determined for [Lys]Pg from of J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and for v2 the in for the time of the increase in Pm which with the rates by the and discontinuous v2 of the the of showed that the rate was a of with the by a the of the and Pg activation was the of Pm formation a of SK and [Lys]Pg and for to substrate of the time courses of the and of the at [Lys]Pg and SK with the the results in a free by SK·Pg* and The free and to a whereas the concentration showed a with an increase to a rate that was for by formation of with the The Pm concentration the rate of and as was of the initial rate of formation that the rate of as from the of the rate of formation at for to Pg rates that by a maximum of with the initial rates with rates to the of v2 determined of the time at SK concentration with the the results in the rate of Pm formation was and with the initial rate for the increase in concentration from of the with SK concentration that inhibition of the rate by SK was due to of the free Pg concentration by formation of the SK·Pg* complex, with inhibition of the for the was from results that the in the rate of Pm generation from [Lys]Pg at of SK not in the was that to a of the of the rate at of the to the initial rate of Pm formation by the SK·Pg* was due to the the of the proteolytic similar rate by and the of inhibition by for the of the v2 dependence for [Lys]Pg activation on SK concentration of for showed that the lower of the at whereas a of a to the at the in the of the v2 and the lower from with the of the dependence on SK A similar of the SK dependence of [Glu]Pg activation showed complex behavior not the [Glu]Pg of Pg was the initial rate The kinetic determined for [Glu]Pg activation by SK·Pm in Pm a of and a lower corresponding to an lower rate of with [Lys]Pg as a substrate The for [Glu]Pg activation in the initial rates of SK·Pg* of Pg activation with from the much slower of from of and v2 in for [Glu]Pg by the model lacking the A model in which Pm was by cleavage of as Biochemistry. PubMed Scopus Google Scholar, Castellino F.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Biochemistry. PubMed Scopus Google not the v2 for [Lys]Pg, inhibition in v2 with SK of the with an of including the of proteolytic conversion of SK·Pg* to SK·Pm, not the v2 dependence on SK concentration as and required a rate of intermolecular cleavage to for the generation of SK·Pm in rates of Pm formation in SK the generation of SK·Pm was not inhibited by of free Pg by SK results cleavage as a in activation of [Lys]Pg by The results of equilibrium binding and kinetic and in the J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google support a unified for the mechanism of SK-induced Pg activation to Pm. in rapid equilibrium binding of SK to Pg is by conformational activation of the catalytic site in the [Lys]Pg SK with ∼12-fold affinity [Glu]Pg due to the of lysine-binding site and the of for lysine-binding sites with 6-AHA, SK and conformationally activates [Glu]Pg and [Lys]Pg with equivalent affinity J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The complex with bound in the catalytic free Pg in a substrate to form the Binding of substrate Pg is mediated by a specific recognition exosite on the complex, as for J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the model free Pg binding to the complex in the substrate binding in the exosite is not on free SK, Pg, Pm J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). suggests that the complex as by binding of Pg in the catalytic followed by Pg binding as a proteolytic cleavage of Pg in the catalytic Pm, which from the Pg substrate binding and by the rate was of for [Glu]Pg, whereas [Lys]Pg activation was inhibited of [Lys]Pg was to a rate from that for [Glu]Pg activation lysine-binding sites that the for activation of [Lys]Pg [Glu]Pg by is for by lysine-binding site and [Lys]Pg and [Glu]Pg equivalent in conformational activation and in the initial proteolytic activation The mechanism the inhibition of Pm formation by high SK concentration and the inhibition for [Lys]Pg with The proteolytic is inhibited by high SK concentration of the of Pg as the catalytic of and as substrate for high SK proteolytic Pg activation is inhibited the free Pg concentration is by formation of the by model for the of [Lys]Pg for which the affinity for SK is the maximum rate at SK, the required for formation of the complex [Glu]Pg, the is inhibited by high SK concentration but inhibition is of the lower affinity of SK for The results that the rate of Pm formation from [Lys]Pg at high SK concentrations. A mechanism generation of Pm by cleavage the complex Biochemistry. PubMed Scopus Google Scholar, Castellino F.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Biochemistry. PubMed Scopus Google not the the generation of Pm not inhibited by A mechanism inhibition by results support the that Pm is by intermolecular cleavage of free Pg by The inhibition of Pm formation for [Lys]Pg that proteolytic formation of Pm by on is at a The of Pm formation for [Glu]Pg and [Lys]Pg as a of SK and Pg concentration by the intermolecular which inhibition at SK initial was for of the rates of Pm generation by of chromogenic substrate The rates inhibited by with an inhibition of The inhibition was the as for the complex J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that inhibition of the of Pm formation to the rate of Pm generation is that between chromogenic substrate binding to and the affinity of SK binding and conformational activation for the inhibition J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the showed that the rate of Pm generation was to in not at a substrate and the v2 rates for inhibition at The of was by the of the and rates with the inhibition the not to inhibition by was and the was for of the not the of the in the kinetic the the Pm and in the catalytic free SK in the catalytic with high of equilibrium binding for the native of for [Glu]Pg, for [Lys]Pg, and for Pm J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google representing and affinity of SK for Pm with [Lys]Pg and [Glu]Pg, from in and as in the J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Pm readily Pg from the that the initial Pm SK in the bound The complex initiates the lower catalytic in which consists of Pg binding as a substrate and its proteolytic activation to Pm J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of for [Glu]Pg and [Lys]Pg as of an lower rate for [Glu]Pg, due to a and to a lower the of the complex for [Glu]Pg with [Lys]Pg to due to lower affinity binding of [Glu]Pg as a a in lysine-binding site which not the of the and the determined of Pm formation not the for the dependence of v2 on Pg and SK concentration was by a model including the catalytic and the subsequent conversion of Pg to Pm by the for the was with the that proteolytic the of Pg concentration was for Pg cleavage from the of the rates of conformational activation (v1) as a of SK and Pg concentration J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The as on the Pg much lower the determined and the determined kinetic of the time courses of the the behavior for the [Lys]Pg the of Pm formation at of SK and at concentrations. of the that at the rates by a maximum of with the rates for the model including the model the v2 as a of SK and [Lys]Pg concentration but with an affinity for the conformational activation equilibrium of with determined from of The in the of the at for and that the was more in with the of and results of the equilibrium binding studies. The rate for [Lys]Pg activation by and by The of for the proteolytic with similar rate and the mechanism of inhibition of the rate by of free Pg in the and by the mechanism including Pg activation. The for [Glu]Pg activation was and the determined from of and v2 by of the model the in was due to the lower affinity of SK for binding and conformational activation of [Glu]Pg and the lower rate for by with in a of the to Pm of the rate for and [Glu]Pg and [Lys]Pg activation an ∼20-fold slower rate of [Glu]Pg activation by whereas activation of [Glu]Pg with an slower results and the similar rate for [Lys]Pg activation that and for [Glu]Pg and similar for [Lys]Pg as The of the more of [Glu]Pg activation by is not but in the catalytic site SK binding that in the lower chromogenic substrate for with J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is that [Glu]Pg substrate binding is more for of the time courses of the and of the in the mechanism including and catalytic and a and unique of the The time courses for [Glu]Pg activation with the determined kinetic in The was at SK and [Glu]Pg, representing for the conformational activation that SK was with one of the catalytic results in a of Pm formation representing more one SK which is to of the SK in the complex and the concentration of the complex to of [Lys]Pg activation by SK and [Lys]Pg showed similar the catalytic SK of Pm of the SK was in due to the of Pm in catalytic with [Glu]Pg generation of of Pm in of SK was required in [Lys]Pg activation to of the was due to the and affinity of SK for Pm with [Lys]Pg and [Glu]Pg, and the slower rate of activation of [Glu]Pg with results support the that Pm formation by conformationally activated in the complex acts as a triggering to Pm to of the SK, which the initial catalytic The bound complex then propagates conversion of the remaining Pg to Pm. and binding and conformational activation of Pg by SK and an initial of intermolecular Pg activation by is a self-limiting that one SK equivalent of the activated complex, which the in the conversion of Pg to Pm. to R. for and to for of