The Mechanism of a Bacterial Plasminogen Activator Intermediate between Streptokinase and Staphylokinase*

Irina Y. Sazonova, Aiilyan K. Houng, Shakeel A. Chowdhry, Brian R. Robinson, Lizbeth HedstromDagger , and Guy L. Reed§

From the Cardiovascular Biology Laboratory, Harvard School of Public Health and the § Massachusetts General Hospital, Boston, Massachusetts 02114 and the Dagger  Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02453

Received for publication, October 11, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The therapeutic properties of plasminogen activators are dictated by their mechanism of action. Unlike staphylokinase, a single domain protein, streptokinase, a 3-domain (alpha , beta , and gamma ) molecule, nonproteolytically activates human (h)-plasminogen and protects plasmin from inactivation by alpha 2-antiplasmin. Because a streptokinase-like mechanism was hypothesized to require the streptokinase gamma -domain, we examined the mechanism of action of a novel two-domain (alpha ,beta ) Streptococcus uberis plasminogen activator (SUPA). Under conditions that quench trace plasmin, SUPA nonproteolytically generated an active site in bovine (b)-plasminogen. SUPA also competitively inhibited the inactivation of plasmin by alpha 2-antiplasmin. Still, the lag phase in active site generation and plasminogen activation by SUPA was at least 5-fold longer than that of streptokinase. Recombinant streptokinase gamma -domain bound to the b-plasminogen·SUPA complex and significantly reduced these lag phases. The SUPA-b·plasmin complex activated b-plasminogen with kinetic parameters comparable to those of streptokinase for h-plasminogen. The SUPA-b·plasmin complex also activated h-plasminogen but with a lower kcat (25-fold) and kcat/Km (7.9-fold) than SK. We conclude that a gamma -domain is not required for a streptokinase-like activation of b-plasminogen. However, the streptokinase gamma -domain enhances the rates of active site formation in b-plasminogen and this enhancing effect may be required for efficient activation of plasminogen from other species.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Cleavage of the zymogen plasminogen (Pg)1 to the active enzyme plasmin by Pg activators is the first step in fibrinolysis, the process of blood clot dissolution. Two bacterial Pg activators, streptokinase (SK) and staphylokinase (SAK), initiate fibrinolysis in humans by forming a cofactor-enzyme complex with plasmin (1-4). In this complex, SK or SAK serves as a cofactor that redirects the substrate specificity of plasmin from the cleavage of fibrin to the cleavage of Pg. Despite their similarities, SAK and SK have fundamentally different mechanisms. SK binds with significantly greater affinity to plasmin than SAK (5). The SAK·plasmin complex, but not the SK·plasmin complex is inactivated by alpha 2-antiplasmin, the serine protease inhibitor of plasmin (2, 6). Among all of the Pg activators, SK has the unique ability to bind and nonproteolytically generate an active site in the Pg zymogen (i.e. convert Pg to Pg*) (7-9).

The mechanism by which SK nonproteolytically generates an active site in the zymogen Pg (Pg*) is controversial (10-12). Clues to this mechanism have come from comparisons of the structures of SK and SAK (13). SK has three sequential domains of roughly equal sizes (alpha , beta , and gamma ) linked by flexible loops (11). The SK gamma -domain binds near the autolysis loop of plasmin (11). On the basis of studies with recombinant (r) fragments Young et al. (10) concluded that the carboxyl terminus of SK (including all of gamma -domain) induced a conformational change in the active center of Pg leading to nonproteolytic activation of the molecule, a process that has been dubbed "binding or contact activation (13)." By analogy to the activation of tissue Pg activator, which has significantly greater activity as a zymogen than Pg, it has been suggested that the binding of gamma -domain to the autolysis loop of Pg induces the formation of a critical intramolecular salt bridge between Lys698 and Asp740 of Pg that productively structures the activation domain (11). Another hypothesis ("molecular sexuality") states that SK nonproteolytically activates Pg when the NH2-terminal isoleucine-1 (Ile1) of the alpha -domain forms a salt bridge with Asp740 of Pg (12). In this activation sequence, which is typical of the zymogens of the chymotrypsinogen family, salt bridge formation productively restructures the latent activation domain (14). Either mechanism, "gamma -domain contact activation" or molecular sexuality, is plausible given the current structural information for the microplasmin·SK complex (13).

Novel insights into the structural elements required for a staphylokinase versus a streptokinase mechanism of Pg activation may come from an analysis of a recently isolated Streptococcus uberis Pg activator (SUPA, also known as PauA) (15). SUPA is produced by a streptococcus that causes mastitis in cows and has no significant nucleotide or genomic homology to streptokinase or staphylokinase (16, 17). SUPA has a putative two-domain structure (29 kDa) that is intermediate between the one-domain structure of SAK (16 kDa) and the three-domain structure of SK (47 kDa). SUPA has been considered a new class of bacterial Pg activator that may act through a unique mechanism (16, 17) with Pg activation kinetics similar to streptokinase and a staphylokinase-like susceptibility to inhibition by alpha 2-antiplasmin (18). In this study we have investigated the properties of the activator complex formed by SUPA and bovine Pg to determine the structural elements required for nonproteolytic active site generation and for rendering the activator complex resistant to inhibitors such as alpha 2-antiplasmin.

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EXPERIMENTAL PROCEDURES
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Protein and Reagents-- -Bovine (b) Pg was prepared from fresh citrated b-plasma by affinity chromatography with lysine-substituted Sepharose and pretreated with aprotinin-agarose to remove contaminating plasmin (19). The purified b-Pg was assessed by 10% SDS-PAGE and active site titration after activation by urokinase (9 nM, 1 h at 37 °C, in 50 mM Tris buffer, 0.15 M NaCl, 20% glycerol) as described (5). The b-Pg contained less than 0.5% plasmin.

Cloning, Expression, and Purification of Recombinant (r) Proteins-- The SK gene was cloned from Streptococcus equisimilis, expressed in bacteria as a MBP fusion protein via the pMalc vector (New England Biolabs, Beverly, MA), and purified as described in detail (5). The gamma -domain of SK (residues 293-414) was cloned using the full-length cDNA of SK and the corresponding primers (5' sense primer catcagctgttcaccatcaaatacgttg, PvuII; 3' antisense primer gcctgcagtcattatttgtcgttagg, PstI). After double-stranded DNA sequencing it was ligated into pMALc vector at StuI and PstI sites. The SK gamma -domain fusion protein was purified by affinity chromatography on an amylose resin (New England Biolabs) and its purity was assessed by SDS-PAGE.

Eleven strains of Streptococcus uberis were tested for their ability to activate b-Pg. Genomic DNA was isolated as described (20). Polymerase chain reaction was performed with primers containing EcoRV and HindIII 5'-gatatcaccggttaygaywsngaytaytaygc and tctagattaaggtttataacttttyttngtdatnarrtayttytc. The amplified DNA from SUPA clone 70.2 was sequenced on both strands and ligated into the pMal-c vector (New England Biolabs) at the StuI site for expression as a fusion polypeptide with maltose-binding protein (MBP). Cloning at this site permits specific cleavage between MBP and Ile1 of SUPA, yielding intact SUPA with its native NH2 terminus (12). The MBP-rSUPA fusion protein was absorbed on DEAE Affi-Gel Blue-agarose and eluted between 110 and 150 mM NaCl by a linear gradient of 0 to 200 mM NaCl in 10 mM phosphate buffer, pH 7.2. The rSUPA was cleaved from MBP by treatment with Factor Xa (New England Biolabs) for 24 h at room temperature in 200 mM Tris-HCl buffer, pH 8.0, 100 mM NaCl, 2 mM CaCl2. The SUPA cDNA 70.2 was also subcloned into pProEX HTb expression vector (Life Technologies) using BamHI and PstI restriction enzymes. This resulted in the expression of SUPA with a short N-terminal fusion peptide containing a His6-tag. The His6-SUPA protein was absorbed on Ni-NTA affinity resin and eluted by 20 mM Tris-HCl, pH 8.5 (at 4 °C), with 100 mM KCl, 5 mM 2-mercaptoethanol, 10% glycerol, and 100 mM imidazole. The eluted sample was analyzed by SDS-PAGE and dialyzed against assay buffer (50 mM Tris-HCl, 100 mM NaCl, pH 7.4).

Active Site Titration-- The molar quantity of active sites generated by rSUPA in b-Pg was determined by active site titration with the fluorogenic substrate 4-methylumbelliferyl p-guanidinobenzoate (MUGB, Sigma) as described (5, 21).

Kinetic Assays-- The amidase kinetic parameters of human plasmin (h-plasmin), b-plasmin, and the activator complexes were studied with H-D-valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride (S2251, Chromogenix, Sweden) as previously described (22). The h-plasmin·rSK and b-plasmin·rSUPA complexes were prepared by mixing 10 nM h-Pg (>= 95% Glu-Pg; Chromogenix, Sweden) or b-Pg with 20 nM rSK or rSUPA for 5 min at 37 °C. The h-plasmin·rSUPA complex was formed by mixing 10 nM h-plasmin (Sigma) and 20 nM rSUPA for 10 min on ice. The enzymes were transferred to a thermostatically regulated (37 °C) quartz cuvette containing assay buffer (50 mM Tris-HCl, 100 mM NaCl, pH7.4) and various concentrations of S2251 (80-800 µM) in a total volume of 300 µl and the change in absorbance was monitored at 405 nm for 5 min in a Cary 100-Bio spectrophotometer. Less than 10% of H-D-Val-Leu-Lys-nitroanilide was consumed during the course of the reaction. The data were plotted as V/S and analyzed by hyperbolic curve fitting with the Sigma Plot program. An epsilon M at 405 nm of 10,000 was used for p-nitroanilide.

Steady-state Pg Activation Kinetic Parameters-- The kinetics of Pg activation by either free rSK and rSUPA or activator complexes were studied as described (23). Stoichiometric activator complexes were formed by mixing h-Pg (>= 95% Glu-Pg; Chromogenix, Sweden) and rSK or b-Pg and rSUPA on ice for 30 min. The stoichiometric h-plasmin·rSUPA activator complex was formed at 37 °C for 5 min. Activators were added to a quartz cuvette containing assay buffer, 0.5 mM S2251, and Pg (50-900 nM) in a total volume of 300 µl at 37 °C as described. Initial reaction rates were obtained from the first 300 s by plotting A405/min2, and the apparent Michaelis and catalytic rate constants were calculated by fitting the data to a hyperbolic curve as described (23) using the Sigma Plot program.

Continuous Assay for Determination of the Rates of Active Site Generation in Pg-SK Complexes-- The first-order rate constant for the appearance of an amidolytic active center in the hPg·SK complex was determined as described by Chibber and Castellino (24) at 4 °C, in a final volume of 0.3 ml, with a low ionic strength buffer consisting of 10 mM Hepes/NaOH, pH 7.4. Then h-Pg (100 nM; >= 95% Glu-Pg; Chromogenix, Sweden) or b-Pg (300 nM; <= 0.5% plasmin) was added to a cuvette in the presence of 0.5 mM S2251. After incubation for 5 min at 4 °C, native SK (100 nM, Sigma) or rSUPA (300 nM) was added, and the release of p-nitroanilide was monitored. The rate constant for active site appearance (k) was measured as described (24) by the equation: [P] = K[Pg-SK]ot-K[Pg-SK]o/k as described where K = kcat[S]/(Km + [S]) and was essentially constant because substrate depletion was <= 10%.

Binding Assays-- Wells of microtiter plate were coated with 50 µl of b-Pg (5 µg/ml). The wells were washed and nonspecific protein-binding sites were blocked with 1% bovine serum albumin. After that 50 µl of rSUPA (50 µg/ml) were added for 1 h. After washing 50 µl of SK gamma -domain (0-50 µg/ml) or MPB (0-50 µg/ml) were added. After a 1-h incubation and washing, anti-MBP monoclonal antibody was added for 1 h. After washing bound antibody was detected by 125I-goat anti-mouse antibody (50,000 cpm) followed by gamma -counting.

Influence of SUPA on the Bovine Plasmin-- alpha 2-Antiplasmin reaction kinetic measurements were carried out in a 1-ml cuvette in filtered, dust-free assay buffer (50 mM Tris, 100 mM NaCl, pH 7.4) at 37 °C using a thermostatted Cary 100-Bio spectrophotometer. The rate constant k1 of the reaction between b-plasmin and alpha 2-antiplasmin was measured as described (6). Briefly b-plasmin (14 nM) was added to cuvettes containing S2251 (500 nM), SUPA (0-500 nM), and the change in absorbance at 405 nm recorded for 90 s prior to the addition of human alpha 2-antiplasmin (Calbiochem; 90 nM). The residual enzyme activity was measured by the first derivative (dA/dt) of the curve before 60% of the enzyme was inactivated. The apparent rate constant (k1, app) of the inhibition of the enzyme activity by alpha 2-antiplasmin in the presence of various concentrations of SUPA was calculated from the classical second-order rate equation as described (6).

    RESULTS
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Primary Structure of SUPA-- The lysates of 11 b-isolates of S. uberis were tested for their ability to activate b-Pg. The SUPA gene was cloned from the S. uberis isolate (number 70) that showed the greatest Pg activation. The deduced amino acid sequence predicted a mature peptide mass of 30.7 kDa (GenBankTM accession number AF283574). In pairwise alignments by ClustalW (Table I), the putative alpha -domain of SUPA showed 11.9% identity with SAK, 30.9% with SK alpha , 10.3% with SUPA beta , 16.7% with SK beta , and 9.5% with SK gamma  (25). The SUPA beta -domain showed 11.9% identity with SK alpha , 15.6% with SAK, 27.4% with SK beta , and 12.2% with SK gamma . By comparison, SAK showed 17.7% identity with SK alpha , 14% with SK beta , and 20.6% with SK gamma . In contrast to previous reports, a comparison of the three sequences by ClustalW (Fig. 1) showed that SUPA contained an Ile-Thr-Gly terminus which is similar to the Ile-Ala-Gly sequence that has been suggested to mediate nonproteolytic activation of Pg by SK (12, 16, 17). In the crystal structure SK alpha -domain residues Glu39 and Glu134 form salt bridges with Arg719 of h-plasmin (which is also conserved in b-Pg) (11, 13). In the ClustalW alignment these residues are not conserved in staphylokinase, although in an ALSCRIPT alignment SK Glu39 is conserved as Glu43 of SAK (26). In SUPA there is modest conservation of these residues as His43 and Glu113. The ClustalW alignment also highlights conservation of residues in SUPA that have been shown to be important for SK function: SK Leu42 (27) which is SUPA Leu46; SK Lys257 (28), which is SUPA Lys234; SK Arg248, Glu249 which are SUPA Arg225, Gln226, and SK Lys282 which is SUPA Lys260 (29).

                              
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Table I
Pairwise sequence identities between domains of SUPA, SK, and SAK
The pairwise sequence identity between domains was determined by the ClustalW program (25). The sequences used for these analyses included the following residues: SUPAalpha -(1-126), SUPAbeta -(127-261), SKalpha -(1-147), SK beta -(148-283), and SKgamma -(284-414).


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Fig. 1.   Alignment of SAK, SUPA, and SK by domains. A ClustalW alignment of the three proteins is shown. Residues that are identical between SUPA and SK or SAK are outlined in black. Residues that are similar between all proteins in the alignment are boxed.

Active Site Generation-- A critical distinction between a SAK and SK mechanism is the ability of SK to generate an active site in Pg under conditions in which proteolysis is blocked. Consequently, the ability of SUPA to generate an active site generation in b-Pg was studied in the presence of a titrant (MUBG) that rapidly acylates the active site of plasmin, as originally described by McClintock and Bell (7). Under these conditions trace plasmin is rapidly inactivated and SK, but not SAK, can generate an active site in Pg. When b-Pg was added to the active site titrant only minimal numbers of preformed active sites were detected (Fig. 2A) indicating that only trace quantities of plasmin were present in this preparation (21). When b-Pg was preincubated with MUGB for 5 min (to inhibit trace amounts of plasmin), followed by the addition of His6-rSUPA or MBP-rSUPA minimal active site generation occurred (~5-12% of expected). However, when rSUPA was added 5 min after b-Pg to the cuvette, there was rapid and complete active site formation. Examination of the reaction between SUPA and b-Pg by SDS-PAGE showed that active site generation occurred in the b-Pg molecule without proteolytic generation of plasmin (Fig. 2B). Thus SUPA, like SK but not SAK (3), nonproteolytically generated an active site in b-Pg. However, rSUPA was unable to generate an active site in h-Pg and rSK could not generate one in b-Pg (Fig. 4B and not shown).


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Fig. 2.   Active site generation in h-Pg by SK and b-Pg by SUPA. A, active site development detected by a fluorescent active site titrant. Human Pg (100 nM) or b-Pg (200 nM) was added to a cuvette containing 2 µM MUGB in filtered buffer (50 mM Tris-HCl, 0.15 M NaCl, pH 7.4) at 25 °C. After 5 min 220 nM SK, 440 nM rSUPA, 440 nM MBP-rSUPA, 440 nM His6-rSUPA, or buffer alone (control) was added to separate reactions. The development of fluorescence was monitored continuously with excitation at 365 nm and emission at 445 nm. B, reducing SDS-PAGE analysis of the reaction between b-Pg and SUPA. Equimolar complex of b-Pg and rSUPA at 25 °C, pH 7.4, incubated in the presence of MUGB for 1 min (lane 4), 10 min (lane 5), or 25 min (lane 6). Controls include SUPA (lane 2), b-Pg (lane 3), and b-plasmin (lane 7) formed by incubation for 60 min at 37 °C of a catalytic ratio of SUPA (1:30) in the absence of MUGB.

Temperature Dependence and Rate of Active Site Formation-- When compared with rSK and h-Pg under similar conditions, active site generation in b-Pg by rSUPA was slower (Fig. 2A) and rSUPA also required a higher reaction temperature (19 °C) than SK (4 °C) for any active site generation to be detected (not shown). Similarly, when rSUPA was added to b-Pg it generated an amidolytic active site with a rate constant of 0.18 min-1 while rSK generated an active site in h-Pg with a rate of 0.55 min-1 at 4 °C (data not shown). In Pg activation studies, rSUPA showed a greater delay or lag than rSK in the development of plasmin generation. For example, at 37 °C the delay or lag phase before the onset of Pg activation by rSUPA was >5-fold longer than rSK (Fig. 3). Pg activation by rSUPA was more temperature-dependent than rSK (Fig. 3), the lag phase for Pg activation by rSUPA lengthened twice as much as rSK for every degree drop in reaction temperature. To determine whether the lag phase in the onset of b-Pg activation by rSUPA was related in part to its lack of a gamma -domain, we examined the effects of MBP-gamma -domain of SK or MBP alone on b-Pg activation by rSUPA (Fig. 4). Binding experiments confirmed that the MBP-SK gamma -domain, but not MBP alone bound to the formed SUPA·b-Pg complex (Fig. 4A). In activation experiments, the MBP-gamma -domain reduced the lag phase in b-Pg activation by rSUPA by about half (from ~10 to 5 min) while MBP or catalytic amounts of rSK had no effect (Fig. 4B). Additional active site titration studies were performed to determine whether this reduced "lag phase" in Pg activation induced by the gamma -domain was due to an acceleration of active site exposure in the b-Pg·rSUPA complex. When compared with MBP as a control, the MBP-gamma -domain reduced the time necessary for active site generation by rSUPA in b-Pg (from ~240 to 120 s; Fig. 4C).


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Fig. 3.   Effects of temperature on Pg activation. A, activation of h-Pg by rSK at different temperatures. B, activation of b-Pg by rSUPA at different temperatures. rSK (10 nM) or rSUPA (20 nM) were added to quartz cuvettes containing h-Pg (300 nM) or b-Pg (300 nM) with 0.5 mM S2251 in 50 mM Tris-HCl, 0.15 M NaCl, pH 7.4, at various temperatures. The rate of substrate cleavage was monitored by the change in absorption at 405 nm.


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Fig. 4.   Interaction of the SK gamma -domain with the b-Pg·rSUPA activator complex. A, binding of gamma -domain to b-Pg·rSUPA complex. To form the b-Pg·rSUPA complex, SUPA (50 µl/ml; 50 µg/ml) was added to wells coated with b-Pg (5 µg/ml) and blocked with 1% bovine serum albumin. After a 1-h incubation and washing, 50 µl of MBP-gamma -domain (0-50 µg/ml) or MBP (0-50 µg/ml) was added for 1 h. After washing the anti-MBP monoclonal antibody was added. Bound antibody was detected by 125I-goat anti-mouse antibody followed by gamma -counting. B, influence of the gamma -domain on the lag-phase of activation of b-Pg by rSUPA. b-Pg (300 nM) in 50 mM Tris-HCl, 0.15 M NaCl, pH 7.4, was preincubated with the MBP-gamma -domain (300 nM), MBP (300 nM), assay buffer or SK (20 nM) for 30 min at 25 °C. Then rSUPA (20 nM) was added and the rate of substrate cleavage was monitored at 37 °C. C, effect of gamma -domain on active site generation in b-Pg by rSUPA. The MBP-gamma -domain or MBP (300 nM) was preincubated with b-Pg (300 nM) in the presence of MUGB (2.5 µM) for 850 s prior to the addition of SUPA (450 nM). Active site generation was monitored as described in the legend to Fig. 2.

Influence of rSUPA on the b-Plasmin/alpha 2-Antiplasmin Reaction-- Another property that distinguishes SK from SAK is the resistance of the SK activator complex to inhibition by alpha 2-antiplasmin (2, 6). The second-order rate constant (k1) for inhibition of plasmin by alpha 2-antiplasmin was 3.15 ± 0.61 × 105 M-1 s-1. Increasing concentrations of rSUPA inhibited the inactivation of b-plasmin (14 nM) by an excess of alpha 2-antiplasmin (90 nM) in a dose-dependent fashion, with a 50% reduction seen at a concentration of ~30 nM rSUPA (Fig. 5).


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Fig. 5.   Influence of rSUPA on the inactivation of b-plasmin by alpha 2-antiplasmin reaction. Various amounts of rSUPA (0-300 nM) were mixed with b-plasmin (14 nM) and S2251 (500 nM) in assay buffer (50 mM Tris-HCl, 100 mM NaCl, pH 7.4) at 37 °C. The A405 was continuously recorded and after 90 s human alpha 2-antiplasmin (90 nM) was added. The residual plasmin activity was determined at different time intervals and the apparent inhibition rate constant (k1, app) was calculated (see "Experimental Procedures"). The data are expressed as a percentage of the k1 value obtained in the presence of a given SUPA concentration versus that in the absence of rSUPA. The data represent the mean ± S.E.

Steady State Amidase Parameters and Kinetics of Pg Activation-- In amidolysis assays h-plasmin and h-plasmin-SK showed similar catalytic parameters (Table II). However, the catalytic efficiency (kcat/Km) of b-plasmin-rSUPA was 3.6-fold less than free b-plasmin. This difference was largely due to a 2.8-fold increase in the Km for amidolysis when rSUPA was bound to plasmin. The b-plasmin·SUPA complex was 2.9-fold less efficient (kcat/Km) in amidolysis than the h-plasmin·SK complex because of a slightly higher Km and lower kcat.

                              
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Table II
Kinetic constants for amidolysis
Amidolytic experiments were carried out at 37 °C in a total volume of 300 µl as described under "Experimental Procedures." The values represent the mean ± S.E.

The b-Pg·rSUPA complex and free rSUPA showed similar kinetic parameters for Pg activation (Table III). The h-Pg·SK complex was slightly more catalytically efficient (2-fold) as an activator of h-Pg than the b-plasmin·rSUPA complex was as an activator of b-Pg, largely because of a lower Km (1.7-fold). The b-plasmin·SUPA complex also activated h-Pg, but was 7.9-fold less efficient than h-Pg-rSK, chiefly because of a 25-fold lower kcat.

                              
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Table III
Kinetic constants for plasminogen activation
Activation experiments were carried out at 37 °C in a total volume of 300 µl and kinetic parameters were determined as described under "Experimental Procedures." The values represent the mean ± S.E.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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The unique amino acid and genomic structure of SUPA had suggested that it activated Pg through a different mechanism (16, 17) with properties typical of both SAK and SK (18). However, in the presence of plasmin, SAK and SK mechanisms are indistinguishable (12). In the present experiments, active site generation by SUPA was examined under classical experimental conditions where excess acylating agent was present to inhibit trace amounts of plasmin (7, 8). These studies indicate that SUPA nonproteolytically activates b-Pg through a SK- and not a SAK-type of mechanism. Moreover, like the SK activator complex, and unlike the SAK·plasmin complex, the SUPA activator complex resists inactivation by alpha 2-antiplasmin. These findings indicate that for the activation of b-Pg by SUPA, a gamma -domain is not required for a SK-type of mechanism.

The b-plasmin·SUPA complex activates b-Pg with kinetic parameters that resemble (within a factor of 3) the parameters of Pg·SK for h-Pg activation. When compared with the h-plasmin·SK complex, the b-plasmin·SUPA complex showed comparable kinetics for amidolysis with a slightly higher Km (1.8-fold) and lower kcat (1.7-fold). Although SUPA could not generate an active site in h-Pg, the b-plasmin·SUPA complex cleaved h-Pg, albeit with a 3.0-fold lower Km and an 25.1-fold lower kcat. This paralleled the observation that SK cannot generate an active site in b-Pg, although the hPg·SK complex can efficiently activate b-Pg substrate. Thus the process of active site formation in Pg by SK molecules appears species-specific. However, the substrate-cofactor function is conserved because h-plasmin·SK (23, 30) and b-plasmin·SUPA complexes (this study) can cleave Pg substrates from other species, albeit with different efficiencies. The fact that the b-plasmin·SUPA complex can cleave h-Pg substrate indicates that a gamma -domain per se is also not required for substrate processing by this activator complex.

The mechanism by which SK nonproteolytically generates an active site in Pg has been perplexing since its original description 30 years ago (7, 8). Pg remains a zymogen until its activation domain is productively rearranged by a salt bridge interaction between the amino group of the cleaved, neo-NH2 terminus of plasmin (Val562) and carboxylate group of Asp740. In the contact activation hypothesis, SK generates this active site through binding of the gamma -domain to the autolysis loop of Pg which facilitates the formation of a salt bridge between Asp740 and the counterion Lys698 of Pg. Alternatively the molecular sexuality hypothesis suggests that the NH2-terminal Ile1 of SK provides the counterion for the salt bridge with Asp740 of Pg in a manner analogous to activation of the zymogens of the chymotrypsinogen family (12). Mutagenesis studies aimed at identifying the counterion have indicated that Ile1 is required for nonproteolytic activation of Pg, while Lys698 appears to be required for normal binding interactions during the formation of the initial SK·Pg complex (12, 31). The conservation of the NH2-terminal motif (Ile-Xxx-Gly) between SUPA and all other reported SKs underlines the importance of this residue (12). As predicted by studies of SK, efficient active site generation in b-Pg in the presence of an acylating agent only occurred when the Ile1 of SUPA was free and not when it was tethered in fusion proteins.

What role does the gamma -domain play if SUPA can activate b-Pg nonproteolytically, protect b-plasmin from inhibition from by alpha 2-antiplasmin, and form an activator complex that cleaves different species Pgs? The gamma -domain is found in streptokinases isolated from humans, pigs, and horses suggesting that there is an evolutionary pressure to maintain it which may relate to the fact that there are species differences in the structural requirements for Pg activation. When compared with SK, SUPA generated an active site more slowly in b-Pg and required higher ambient temperatures for function. Exogenous gamma -domain bound to the SUPA activator complex and accelerated the process of active site formation, suggesting that the gamma -domain may serve as an enhancer of these processes, perhaps through its interactions with the autolysis loop of b-Pg (which is 88% identical to h-Pg (32)). Although this enhancer function is not required by b-Pg, it appears to be required by human Pg, because a SUPA-like mutant of SK lacking the gamma -domain is unable to activate human Pg.2 Analyses of the crystal structures of µPg have indicated that generation of a functional active site requires several intramolecular rearrangements that may be facilitated by interactions with the gamma -domain (32, 33). For example, by binding to the autolysis loop, the gamma -domain could promote the formation of a salt bridge between Ile1 of SK and Asp740 of Pg by destabilizing the hydrogen bonds between Asp740 and residues 685 and 686 that secure Pg in the zymogen conformation.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant HL-57314 (to G. L. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF283574.

To whom correspondence should be addressed: Cardiovascular Biology Laboratory, HSPH II-127, 677 Huntington Ave., Boston, MA 02115. Tel.: 617-432-4992; Fax: 617-432-0033; E-mail: reed@cvlab.harvard.edu.

Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M009265200

2 I. Sazonova and G. L. Reed, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: Pg, plasminogen; b, bovine; h, human; r, recombinant; SUPA, Streptococcus uberis plasminogen activator; MBP, maltose-binding protein; SK, streptokinase from Group C streptococcus; SAK, staphylokinase; S2251, H-D-valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride; PMSF, phenylmethylsulfonyl fluoride; MUGB, 4-methylumbelliferyl p-guanidinobenzoate; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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