©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Kinetic Characterization of the Proteinase Binding Defect in a Reactive Site Variant of the Serpin, Antithrombin
ROLE OF THE P1` RESIDUE IN TRANSITION-STATE STABILIZATION OF ANTITHROMBIN-PROTEINASE COMPLEX FORMATION (*)

Steven T. Olson(§) (1), Andrew W. Stephens (2)(¶), C. H. W. Hirs (2), Paul E. Bock (3)(**), Ingemar Björk (4)

From the (1) Center for Molecular Biology of Oral Diseases, University of Illinois-Chicago, Chicago, Illinois 60612, the (2) Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262, the (3) Department of Pathology, Vanderbilt University, Nashville, Tennessee 37232, and the (4) Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, S-75123 Uppsala, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To elucidate the role of the P1` residue of the serpin, antithrombin (AT), in proteinase inhibition, the source of the functional defect in a natural Ser-394 Leu variant, AT-Denver, was investigated. AT-Denver inhibited thrombin, Factor IXa, plasmin, and Factor Xa with second order rate constants that were 430-, 120-, 40-, and 7-fold slower, respectively, than those of native AT, consistent with an altered specificity of the variant inhibitor for its target proteinases. AT-Denver inhibited thrombin and Factor Xa with nearly equimolar stoichiometries and formed SDS-stable complexes with these proteinases, indicating that the diminished inhibitor activity was not due to an enhanced turnover of the inhibitor as a substrate. Binding and kinetic studies showed that heparin binding to AT-Denver as well as heparin accelerations of AT-Denver-proteinase reactions were normal, consistent with the P1` mutation not affecting the heparin activation mechanism. Resolution of the two-step reaction of AT-Denver with thrombin revealed that the majority of the defective function was localized in the second reaction step and resulted from a 190-fold decreased rate constant for conversion of a noncovalent proteinase-inhibitor encounter complex to a stable, covalent complex. Little or no effects of the mutation on the binding constant for encounter complex formation or on the rate constant for stable complex dissociation were evident. These results support a role for the P1` residue of antithrombin in transition-state stabilization of a substrate-like attack of the proteinase on the inhibitor-reactive bond following the formation of a proteinase-inhibitor encounter complex but prior to the conformational change leading to the trapping of proteinase in a stable, covalent complex. Such a role indicates that the P1` residue does not contribute to thermodynamic stabilization of AT-proteinase complexes and instead favors a kinetic stabilization of these complexes by a suicide substrate reaction mechanism.


INTRODUCTION

Antithrombin is the principal protein inhibitor of mammalian blood coagulation proteinases and a member of the serpin (serine proteinase inhibitor) superfamily of protein proteinase inhibitors (Huber & Carrell, 1989; Olson & Björk, 1994). Inhibitors of this family act by displaying a unique reactive bond in an exposed loop of the protein, which is recognized by the proteinase as a normal substrate to be cleaved. Rather than cleaving this bond, however, the proteinase becomes trapped in a highly stable complex with the inhibitor, in which the inhibitor-reactive bond is bound tightly at the active site of the proteinase (Huber and Carrell, 1989; Bode and Huber, 1992; Olson and Björk, 1994). An analogous stable complex appears to be formed between low molecular weight protein proteinase inhibitors of other families and their target enzymes (Laskowski and Kato, 1980; Read and James, 1986; Bode and Huber, 1992). Unique to the serpin mechanism, however, is that accompanying the formation of the stable inhibitor-proteinase complex, the inhibitor undergoes a substantial conformational change in which the exposed reactive center loop is thought to insert into the major -sheet of the inhibitor. This conformational change appears to be essential for arresting the cleavage of the reactive bond initiated by the proteinase and thus for trapping the enzyme in a stable inhibited complex (Engh et al., 1990; Carrell et al., 1991; Skriver et al., 1991; Björk et al., 1992a, 1992b; Björk et al., 1993; Hopkins et al., 1993).

The reactive bond of human antithrombin has been identified as the Arg-393-Ser-394 bond, consistent with the specificity of the target proteinases of antithrombin for cleaving at Arg- X bonds (Björk et al., 1981, 1982). Characterization of natural and engineered variants of antithrombin and other serpins have confirmed the importance of the reactive bond for inhibitor function and in determining the specificity of the inhibitor for its target proteinases. Mutation of the arginine residue of this bond, i.e. the P1 residue, to His, Cys, or Pro in natural variants of antithrombin thus results in a complete loss of inhibitor function (Erdjument et al., 1988a, 1988b, 1989; Owen et al., 1988; Lane et al., 1989a, 1989b). Similar consequences result from mutation of the P1 Arg residue of the related serpins, plasminogen activator inhibitor-1, and C1inhibitor, to residues other than Lys (Sherman et al., 1992; Elderling et al., 1992). Certain P1 mutations of other serpins produce a loss of function with natural target proteinases but the appearance of function with other nontarget proteinases (Owen et al., 1983; Derechin et al., 1990; Rubin et al., 1990), indicating that the P1 residue is a primary determinant of target proteinase specificity.

While such studies have demonstrated a critical role for the P1 residue in serpin inhibitor function, the role of the P1` residue is less well defined. Thus, mutagenesis of the P1` residue of plasminogen activator inhibitor-1 has indicated a broad tolerance for different amino acids in this position (Sherman et al., 1992). In contrast, mutation of the P1` serine residue of antithrombin to leucine in the natural variant, antithrombin-Denver, drastically reduces the inhibitory activity toward thrombin, although it has a less pronounced effect on the inhibition of Factor Xa (Sambrano et al., 1986; Stephens et al., 1987; Theunissen et al., 1993). Further analysis of a number of P1` variants of antithrombin produced by site-directed mutagenesis has shown that the size and hydrophobicity of the P1` residue can significantly influence inhibitor function (Stephens et al., 1988; Theunissen et al., 1993).

Because of the contrasting findings with regard to the importance of the P1` residue in serpin function, we have further characterized the functional defect in the natural P1` variant, antithrombin-Denver. The results of these studies demonstrate the importance of the P1` residue in determining the specificity of antithrombin for inhibition of various target proteinases. They additionally establish a mechanism for the expression of this specificity in which an interaction between the inhibitor P1` residue and proteinase stabilizes the transition state and not the final ground state on the pathway to a covalent antithrombin-proteinase complex. Such findings support a mechanism for serpin inhibitor function different from that of low molecular weight protein proteinase inhibitors but similar to that of a suicide substrate (Fish and Björk, 1979; Olson, 1985; Patston et al., 1991).


EXPERIMENTAL PROCEDURES

Materials

Human antithrombin was purified from outdated plasma by chromatography on heparin-agarose, DEAE-Sepharose, and Sephacryl S-200 as described previously (Olson, 1988; Olson et al., 1993). Heparin was a size- and affinity-fractionated preparation of M7900 (±10%) described previously (Olson et al., 1992). Human -thrombin was generously provided by Dr. John Fenton of the New York State Department of Health, Albany, NY. Factor Xa, plasmin, and Factor IXa were obtained from the purified zymogens of these proteinases by activation with specific enzymes, followed by their purification as described previously (Björk et al., 1992a). All proteins were judged pure by SDS gel electrophoresis. Normal and variant antithrombin concentrations were determined from the absorbance at 280 nm using an absorption coefficient of 0.65 litersgcmand molecular weight of 58,000 (Nordenman et al., 1977). Concentrations of proteinases were determined by active site titration with 4-methylumbelliferyl p-guanidinobenzoate or fluorescein mono- p-guanidinobenzoate as described previously (Jameson et al., 1973; Bock et al., 1989). Comparison with the concentrations determined from the 280 nm absorbance using published absorption coefficients and molecular weights (Discipio et al., 1977, 1978; Barlow et al., 1969; Fenton et al., 1977) showed that all proteinases were >70% active.

Purification of Antithrombin-Denver

Antithrombin-Denver was purified from the plasma of a patient heterozygous for the mutation by modification of a previously described method, which exploits the differential reactivities of normal and mutant inhibitors with thrombin (Stephens et al., 1987). Normal and mutant antithrombins were first copurified by the procedure used to purify normal antithrombin, except that the Sephacryl S-200 step was omitted. The purified mixture of normal and abnormal antithrombins was treated at a concentration of 8 µM (0.5 mg/ml) with 0.55 molar equivalents of thrombin in the presence of 1 µg/ml Polybrene (Aldrich) at 25 °C. Neutralization of thrombin enzymatic activity was monitored by chromogenic substrate assay (see below) until the loss of activity had ceased (1 h). Residual active thrombin was then quenched with a 2-fold molar excess of D-Phe-L-Pro-L-Arg-chloromethyl ketone (Calbiochem). The sample was dialyzed and rechromatographed on heparin-agarose, which separated the antithrombin-thrombin complex and chloromethyl ketone-inactivated thrombin from unreacted antithrombin-Denver. The purified variant antithrombin (10 µM) was subsequently treated again with 0.02 molar equivalents of thrombin for 3 h to remove any residual traces of normal antithrombin. The reaction was quenched with chloromethyl ketone as above, dialyzed, and rechromatographed a third time on heparin-agarose. The resulting antithrombin was subsequently processed through the DEAE-Sepharose and Sephacryl S-200 steps as in the purification of normal antithrombin.

Inhibition Stoichiometry

Fixed levels of thrombin (5 µM) or Factor Xa (1 µM) were reacted with increasing levels of antithrombin or antithrombin-Denver ranging from 0.1 to 1.2 mol of inhibitor/mol of enzyme in 20 mM sodium phosphate, 0.1 M NaCl, 0.1 mM EDTA, 0.1% polyethylene glycol 8000, I 0.15, pH 7.4, 25 °C. Residual enzyme activity was assayed after 27 h for thrombin reactions and after 16 h for Factor Xa reactions. Assays were performed by diluting samples 1000-fold either into 100 µMD-Phe-L-Pip-L-Arg- p-nitroanilide (S-2238,() Chromogenix) for thrombin or 200 µM Spectrozyme FXa for Factor Xa, each containing 100 µg/ml Polybrene, and monitoring the initial rate of substrate hydrolysis from the absorbance increase at 405 nm. Similar titrations of 0.1 µM thrombin or Factor Xa with normal or variant antithrombins at molar ratios of inhibitor/enzyme ranging from 0.1 to 1.5 were also conducted in the presence of 0.3 µM heparin for 3-20 h and assayed for residual enzyme activity as above. Inhibitor titrations of Factor Xa in the absence of heparin were also done at the lower enzyme concentration using a 20-70-h incubation. The inhibition stoichiometry was obtained from the abscissa intercept of a linear regression fit of measured rates of substrate hydrolysis versus the molar ratio of inhibitor to enzyme.

SDS Gel Electrophoresis of Antithrombin-Proteinase Reaction Products

Antithrombin-proteinase complexes were formed by incubating 6 µM antithrombin or antithrombin-Denver with 2 µM proteinase. Reactions with thrombin were done in the presence of 6 µM heparin for 15 min at 25 °C in 0.02 M sodium phosphate buffer, pH 7.4, containing 0.25 M NaCl ( I 0.3), conditions that both avoided the degradation of the inhibitor-proteinase complex by free proteinase that occurs in the absence of polysaccharide and minimized the salt-dependent substrate reaction of the inhibitor that occurs in the presence of heparin (Olson, 1985). Factor Xa reactions were done in the absence of heparin for 1 h at 25 °C in I 0.15, pH 7.4 sodium phosphate buffer. Reactions containing 25 µl were quenched with an equal volume of SDS sample buffer, boiled for 2 min, and then electrophoresed under nonreducing conditions in 10% polyacrylamide gels using the Laemmli buffer system (Laemmli, 1970). Proteins bands were visualized by Coomassie Blue R-250 staining.

Kinetics of Inhibitor-Proteinase Complex Formation

Reactions of antithrombin or antithrombin-Denver with proteinases in the absence or presence of heparin were done in total reaction volumes of 40 or 50 µl in polystyrene cuvettes. The buffer was 20 mM sodium phosphate, 0.10 M NaCl, 0.1 mM EDTA, 0.1% polyethylene glycol 8000, and 1 mg/ml bovine serum albumin, pH 7.40, at 25 °C (except for Factor IXa reactions, where bovine serum albumin was omitted, and EDTA was present at 1 mM). Plasmin reactions were done in the presence of 20 mM -aminocaproic acid to stabilize the enzyme against autolysis. Reactions were conducted under pseudo-first order conditions by employing at least a 10-fold molar excess of antithrombin or antithrombin-heparin complex over proteinase. A molar excess of antithrombin over heparin was also used to fully saturate (>90%) the polysaccharide chains and minimize antagonism of the reaction by free heparin (Olson, 1988). Measured equilibrium binding constants were used to calculate concentrations of antithrombin-heparin complex. Proteinase concentrations were 0.1-100 nM for thrombin, 10-20 nM for Factor Xa, 10-100 nM for plasmin, and 200 nM for Factor IXa. Heparin-accelerated reactions of normal antithrombin with thrombin and Factor Xa were done in the presence of 2-5 mM p-aminobenzamidine to slow reactions sufficiently for manual sampling. Reaction rates were corrected for the effect of the competitive inhibitor as described previously (Olson and Björk, 1991; Olson et al., 1992). The addition of bovine serum albumin served to minimize proteinase adsorption and was found not to significantly affect the rates of proteinase inhibition. For antithrombin-Factor IXa reactions, 1 nM hirudin (Sigma) was included to neutralize a 0.006% thrombin contamination detectable from the hirudin-dependent loss in enzymatic activity observed with the fluorogenic substrate assay for Factor IXa activity. Reactions were quenched at various times with 0.8-1.0 ml 50 µM Tosyl-Gly-Pro-Arg-7-amido-4-methyl coumarin (Sigma) (thrombin and Factor IXa), 100 µM S-2238 (thrombin), 200 µM Spectrozyme FXa (American Diagnostica) (Factor Xa), or 200 µMD-Val-L-Leu-L-Lys- p-nitroanilide (Chromogenix) (plasmin), all containing 100 µg/ml Polybrene. Residual enzyme activity was measured from the initial rate of substrate hydrolysis, either from the increase in fluorescence at 380 nm, 440 nm for thrombin and Factor IXa reactions, or from the absorbance increase at 405 nm for thrombin, Factor Xa, and plasmin reactions. Control incubations in the absence of inhibitor were used to establish the rate corresponding to fully active enzyme, as well as the stability of the activity over the time course of the reactions. Observed pseudo-first order rate constants ( k) were obtained from the slopes of semilog plots of enzyme activity versus time or by nonlinear regression analysis of data by a single exponential decay function (Olson et al., 1993). Second order rate constants were obtained by linear regression analysis of plots of k versus the antithrombin or antithrombin-heparin complex concentrations, after correction in the latter instance for the free inhibitor reaction (Olson, 1988).

Kinetics of Inhibitor-Proteinase Complex Dissociation

Antithrombin-proteinase complex dissociation rate constants were measured essentially by the method of Jesty (1979). Complexes were prepared by incubating proteinase (0.5 µM thrombin or 1 µM Factor Xa) with 5 µM antithrombin and 5 µM heparin at 37 °C in I 0.15 sodium phosphate buffer for 10 min. Dissociation was then induced by diluting 5-27 µl of the incubation mixture into 1.0 ml (final volume) chromogenic substrate consisting of 400 µM S-2238 or 400 µM Spectrozyme FXa plus 100 µg/ml Polybrene (final concentrations) in I 0.15 sodium phosphate buffer at 37 °C. Complex concentrations after dilution ranged between 2.5 and 27 nM. The initial rate of complex dissociation (<0.5% dissociation) was continuously monitored for 1 h at 37 °C from the parabolic increase in absorbance at 405 nm due to the reappearance of enzyme activity. The absorbance changes were fit by a second order polynomial, and the dissociation rate constant was obtained from the fitted parameters using the measured turnover number at 37 °C and the total concentration of complex as described previously (Danielsson and Björk, 1983; Olson et al., 1993). Measured rate constants were not appreciably affected by changing the antithrombin or proteinase concentrations, by including heparin to accelerate complex formation, or by increasing the substrate concentration employed to monitor complex dissociation, consistent with the measured rate constant not containing a significant contribution from the association process. Reported time-dependent decreases in the dissociation rate constant due to complex aggregation were minimized by the short reaction time of 10 min employed for complex formation (Danielsson and Björk, 1983).

Heparin Binding Titrations

Binding of high affinity heparin to antithrombin or antithrombin-Denver was measured by fluorescence titrations in which the enhancement in protein tryptophan fluorescence accompanying polysaccharide binding was used to monitor the interaction (Olson and Shore, 1981; Olson et al., 1993). Titrations were done with 50 nM inhibitor in 20 mM sodium phosphate, 0.10 M NaCl, 0.1 mM EDTA, 0.1% polyethylene glycol 8000, pH 7.4. Fluorescence measurements were made at 280 nm, 340 nm on an SLM 8000 spectrofluorometer. Relative fluorescence changes, F/ F= ( F F)/ F(where Fand Fare observed and starting fluorescence values, respectively), were computer fit by the quadratic equation describing the equilibrium binding process from which values of the dissociation constant, stoichiometry, and maximum fluorescence change were obtained (Olson et al., 1993).


RESULTS

Mutation of the reactive center P1` serine residue to leucine in the natural variant, antithrombin-Denver, was previously reported to drastically reduce the rate of inhibition of thrombin in the presence of the activator, heparin (Sambrano et al., 1986; Stephens et al., 1987). Antithrombin-Denver retained the ability to inhibit thrombin as well as Factor Xa at a slow but measurable rate also in the absence of heparin (Fig. 1). The observed inhibition was not due to contaminating heparin, since Polybrene had no effect on the rates (not shown). Fitting the progress curves in Fig. 1by a first order exponential decay indicated that antithrombin-Denver inhibited thrombin with a 430-fold reduced pseudo-first order rate constant ( k) as compared with antithrombin. By contrast, the variant antithrombin inhibited Factor Xa with only a 6.5-fold reduced kas compared to the normal inhibitor. Reactions of antithrombin-Denver with thrombin were characterized by monophasic inactivation curves and indistinguishable second order rate constants when inhibitor/proteinase ratios were varied from 20,000 to as low as 20, indicating that the slow inactivation of this proteinase by the variant inhibitor was not due to contaminating wild-type inhibitor. The greatly different reductions in the rates of proteinase inactivation by antithrombin-Denver relative to antithrombin for thrombin and Factor Xa provided additional evidence that the inactivation of these proteinases by the variant inhibitor was not the result of contaminating normal antithrombin.


Figure 1: Reactions of antithrombin and antithrombin-Denver with proteinases in the absence of heparin. Reactions of 5 µM inhibitor with 10 nM thrombin (, ), 1 nM thrombin (), or 20 nM Factor Xa (, ) were conducted at I 0.15, pH 7.4, 25 °C as described under ``Experimental Procedures.'' In control incubations, the inhibitor was omitted (). Solid lines are computer fits to a single exponential decay function.



The reactions of antithrombin-Denver and wild-type antithrombin with both thrombin and Factor Xa required approximately equimolar levels of inhibitor and proteinase for complete inhibition (Fig. 2, ). These findings indicated that the observed inactivation of proteinases by antithrombinDenver was due to the formation of 1:1 stoichiometric complexes like those formed in normal antithrombin-proteinase reactions. Heparin increased the inhibition stoichiometries of both normal and variant inhibitors with the two proteinases (), due to the polysaccharide promoting a competing reaction of antithrombin with the enzymes as a substrate (Olson, 1985; Olson et al., 1992). Somewhat greater inhibition stoichiometries were observed for the variant as compared with the normal inhibitor both in the absence and presence of polysaccharide, suggesting the P1` mutation increased the amount of inhibitor reacting as a substrate. However, such effects were small relative to those on the rate of proteinase inhibition. SDS gel electrophoresis of mixtures of thrombin or Factor Xa with a molar excess of normal or mutant inhibitors confirmed that equimolar enzyme-inhibitor complexes were the primary reaction products for both normal and variant inhibitor-proteinase reactions (Fig. 2).


Figure 2: Stoichiometry and products of normal and variant antithrombin-proteinase reactions. A, titrations of fixed levels of thrombin (5 µM) or Factor Xa (1 µM) with antithrombin () or antithrombin-Denver (). Experimental details are given under ``Experimental Procedures.'' Solid lines are linear regression fits from which the reaction stoichiometry was obtained from the abscissa intercept. B, SDS gels of the products of the reactions of antithrombin or antithrombin-Denver with thrombin ( left gel) and with Factor Xa ( right gel). Conditions are detailed under ``Experimental Procedures.'' Lane 1, 2.3 µg of thrombin or 1.8 µg of Factor Xa; lanes 2 and 3, 8.7 µg of antithrombin and antithrombin-Denver; lanes 4 and 5, 8.7 µg of normal and variant antithrombins reacted with 2.3 µg of thrombin or 1.8 µg of Factor Xa. Protein standards with molecular weights of 97,000, 67,000, 45,000, 30,000, and 20,000 are included on the left- hand side of each gel.



The equilibrium binding of high-affinity heparin ( M 7900) to antithrombin-Denver or antithrombin was compared by titrations of the 40% protein fluorescence enhancement that accompanies this binding (Fig. 3). The averaged results from two titrations gave Kvalues (± range) of 12 ± 1 and 16 ± 5 nM, stoichiometries of 0.9 ± 0.1 and 1.0 ± 0.1 mol of heparin/mol of inhibitor, and maximum relative fluorescence changes of 40 ± 1 and 36 ± 2% for antithrombin and antithrombin-Denver, respectively. The similar binding affinities and fluorescence enhancements indicated that heparin binding to the mutant inhibitor, and the conformational change induced by this binding, were largely unaffected by the mutation.


Figure 3: Equilibrium binding of heparin to antithrombin and antithrombin-Denver. Antithrombin () or antithrombin-Denver () (50 nM) was titrated with the indicated molar ratios of heparin to inhibitor at I 0.15, pH 7.4, 25 °C and heparin binding monitored from the relative change in protein fluorescence, F/ F, as described under ``Experimental Procedures.'' Solid lines are nonlinear regression fits to the quadratic equation for ligand binding to a protein with equivalent, noninteracting sites.



Second order rate constants were measured for the reaction of antithrombin-Denver with a number of target proteinases under pseudo-first order conditions in the absence and presence of heparin levels sufficient to saturate the inhibitor (). The results showed that the effect of the antithrombin-Denver mutation strongly depended on the proteinase inhibited, although for each proteinase the effect was similar in the absence or presence of heparin. Thus, the reduction in the inhibition rate constant was greatest for the thrombin reaction (430-fold without heparin and 310-fold with heparin), somewhat less for the Factor IXa reaction (120-fold without heparin and 240-fold with heparin), significantly less for the plasmin reaction (40-fold without heparin and 28-fold with heparin), and least for the Factor Xa reaction (6.8-fold without heparin and 3.8-fold with heparin). As a consequence of the similar reductions in second order rate constants for heparin-catalyzed and uncatalyzed reactions, heparin enhanced the rate of all antithrombin-Denver-proteinase reactions to an extent similar to that of normal antithrombin. Of note is the substantially lower second order rate constant observed for the heparin-dependent inhibition of Factor IXa by plasma antithrombin in this study as compared with previous work (Jordan et al., 1980). Direct monitoring of the formation of antithrombin-Factor IXa complexes by SDS gel electrophoresis and densitometric analysis confirmed the validity of the inhibition rate constant measured by loss of activity. The extent of heparin acceleration of this reaction may thus be much smaller than previously reported.

Previous studies have demonstrated that the reactions of antithrombin with thrombin and Factor Xa occur in two steps. An initial encounter between inhibitor and proteinase thus produces a loose, noncovalent complex and is followed by conversion of this encounter complex to a tight, probably covalent, complex (Fig. S1) (Olson and Shore, 1982; Craig et al., 1989). To determine which of the two steps of the inhibitor-proteinase reaction was affected by the mutation, we measured the dependence of observed pseudo-first order inhibition rate constants on inhibitor concentration. Only the heparin-accelerated reactions were examined because saturation of the encounter complex with normal antithrombin in the absence of heparin requires millimolar inhibitor concentrations. The dependence of kon antithrombin-heparin complex concentration for the reactions of antithrombin-Denver with thrombin and Factor Xa is shown in Fig. 4. Saturation of koccurred in an experimentally accessible range of inhibitor-heparin complex concentration for the thrombin reaction but not for the Factor Xa reaction. The linear dependence of kfound for the latter reaction indicated that only a second order inhibition rate constant could be obtained (Table II). Nonlinear least squares analysis of the hyperbolic saturation curve for the thrombin reaction yielded values of 0.23 ± 0.02 µM for the dissociation constant for the ternary encounter complex interaction and 0.027 ± 0.001 sfor the first order rate constant for conversion of the encounter complex to a stable complex (Fig. S1). Comparison with values previously determined for the reaction of normal antithrombin-heparin complex with thrombin, using the same heparin fraction (Olson and Björk, 1991), clearly revealed that the P1` mutation results in a minor, 1.9-fold increased dissociation constant for the initial encounter complex interaction but a substantial 190-fold reduction in the rate constant for conversion of the encounter complex to a stable complex (Fig. S1). The effect of the P1` mutation is thus nearly all in the second, covalent reaction step.


Figure S1: Scheme 1.




Figure 4: Inhibitor concentration dependence of pseudo-first order rate constants for reactions of antithrombin-heparin complex with proteinases. Rate constants were measured as detailed under ``Experimental Procedures.'' Solid lines are computer fits of data by a rectangular hyperbola or a straight line.



To determine whether the mutation affected the stability of the final antithrombin-proteinase complex, we examined the kinetics of dissociation of the complexes with thrombin or Factor Xa. This was done by forming complex with a molar excess of inhibitor over proteinase (to minimize complex degradation by free proteinase) and then extensively diluting the complex into a substrate solution and following the regeneration of proteinase activity. Measurements were made at 37 °C to increase the rate of the dissociation reaction. Indistinguishable rate constants of (8.0 ± 0.7) 10sand (7.6 ± 1.0) 10swere found for the dissociation of complexes of thrombin with antithrombin or antithrombin-Denver, respectively. Equivalent rate constants (within experimental error) of (6.8 ± 0.6) 10sand (7.3 ± 1.6) 10swere also measured for the dissociation of Factor Xa complexes with antithrombin and antithrombin-Denver, respectively. These results indicate similar stabilities of antithrombin and antithrombin-Denver complexes with proteinases.


DISCUSSION

The results of the present study have shown that the P1` residue of antithrombin functions in determining the specificity of the inhibitor for its target proteinases and have provided insight into how this specificity is expressed. The contribution of the P1` residue to inhibitor specificity is indicated by the variable effects of the P1` Ser to Leu mutation in antithrombin-Denver on the rates of inhibition of different target proteinases (). In all cases, the specificity of the mutant inhibitor is reduced, indicating that all target proteinases prefer the native inhibitor P1` side chain, but the extent of this reduction is least for Factor Xa and greatest for thrombin. As a consequence, the P1` mutation in antithrombin-Denver results in a specific Factor Xa inhibitor, with Factor Xa being inhibited 13-130-fold faster than the other proteinases examined in the absence of heparin and 4-12,000-fold faster in the presence of heparin. Previous studies aimed at engineering a recombinant antithrombin with specificity for Factor Xa similarly found that mutation of the P1` residue of the inhibitor to a bulky hydrophobic group was sufficient to selectively reduce thrombin inhibitory activity with only modest effects on anti-Factor Xa activity. However, the extent of the selectivity achieved was not quantified in terms of inhibition rate constants, and changes in inhibitor specificity were only examined with thrombin and Factor Xa in the presence of heparin (Theunissen et al., 1993). The altered specificity of antithrombin-Denver for proteinases can be explained by the different specificities of the S1` substrate binding sites of proteinases for the mutant P1` residue differentially affecting the substrate-like recognition of the mutant inhibitor by proteinase. The observation that the S1` site of thrombin is occluded by a lysine residue in the x-ray structure of the proteinase has suggested a restricted specificity of this site for substrate P1` residues (Bode et al., 1989). Molecular modeling studies have confirmed the limited accessibility of large hydrophobic side chains to the S1` site of thrombin (Theunissen et al., 1993), consistent with the drastic effects of mutating the P1` residue of antithrombin to such side chains on thrombin inhibitory activity (Stephens et al., 1988; Theunissen et al., 1993). By contrast, modeling of the S1` site of Factor Xa has suggested a greater tolerance for accommodating large side chains, in keeping with the relatively smaller effects of engineered P1` mutations in antithrombin on Factor Xa inhibition (Theunissen et al., 1993). The differential effects of the P1` mutation in antithrombin-Denver on Factor IXa and plasmin inhibition similarly imply a restricted accessibility of the S1` site of Factor IXa but a greater permissiveness of the S1` site in plasmin for the P1` leucine side chain of the variant inhibitor, in agreement with the more restricted substrate specificity of Factor IXa as compared with plasmin.

Comparison of the effects of the P1` mutation in antithrombin-Denver on both heparin-catalyzed and uncatalyzed reactions with proteinases has shown that the mutation reduces the second order rate constants for proteinase inhibition to similar extents in the absence and presence of the polysaccharide. This is consistent with the reactive center mutation resulting in just a defect in proteinase recognition and not affecting heparin binding or conformational activation of the inhibitor. The mechanism of heparin acceleration of antithrombin-proteinase reactions differs for thrombin and Factor Xa reactions (Olson et al., 1992). With Factor Xa, heparin acts mostly as an allosteric activator of antithrombin by inducing a conformational change in the reactive center region, which makes the inhibitor more reactive toward the proteinase. With thrombin, the conformational change does not greatly influence the reactivity of antithrombin with the proteinase. Instead, heparin acts primarily as a bridge to promote the binding of thrombin to antithrombin through adjacent polysaccharide binding sites for the enzyme and inhibitor (Olson and Björk, 1991). Our findings that heparin binding to antithrombin-Denver is normal and that the promotion of thrombin binding to the heparin-bound mutant inhibitor to form an intermediate ternary complex is normal explain why the heparin accelerating mechanism for the antithrombin-Denver-thrombin reaction is unaffected. Likewise, the similar conformational changes induced in mutant and normal inhibitors, together with the greater tolerance of the S1` binding site of Factor Xa for binding large hydrophobic side chains, explain the modest effects of the P1` mutation on the heparin accelerating mechanism for the antithrombin-Factor Xa reaction. The previous suggestion that the differential effects of the P1` mutation on thrombin and Factor Xa inhibition reflect the different mechanisms of heparin activation is thus not supported by our results (Theunissen et al., 1993).

While the evidence presented in this and other studies favors the idea that the P1` mutation in antithrombin-Denver results in defective proteinase recognition, it was surprising to find that the mutation did not affect the initial encounter complex interaction between inhibitor and proteinase but instead affected the subsequent transformation of the encounter complex to a stable complex. An additional significant finding was the lack of an effect of the P1` mutation on the rate of dissociation of the stable proteinase-inhibitor complex, implying that the mutation does not influence complex stability. An understanding of these effects is best appreciated in the context of the suicide substrate mechanism by which serpins are thought to inhibit their target proteinases () (Fish and Björk, 1979; Olson, 1985; Patston et al., 1991; Hopkins et al., 1993; Schechter et al., 1993; Olson and Björk, 1994).

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

According to this mechanism, the reaction of inhibitor (I) with a proteolytic enzyme ( E) proceeds initially as a normal substrate reaction with the formation of a noncovalent encounter complex ( EI) in which the inhibitor reactive bond is bound at the enzyme active site. This is followed by an attack of the catalytic serine of the enzyme on the inhibitor reactive bond to produce a covalent tetrahedral or acyl intermediate complex ( E-I). The fate of the intermediate complex is governed by the relative rates of continued reaction along the substrate pathway to release cleaved inhibitor (I) and active proteinase ( E) or of the arrest of the substrate reaction by an inhibitor conformational change, which traps the proteinase in a stable complex ( E-I*). The stable complex can subsequently dissociate to cleaved inhibitor and active proteinase, but at an extremely slow rate (Jesty, 1979; Danielsson and Björk, 1983).

Our results have shown that the reduced inhibitory activity of antithrombin-Denver with various proteinases is not due to increased turnover of the variant along the substrate pathway, as has been observed with other reactive center variants (Caso et al., 1991; Austin et al., 1991; Skriver et al., 1991; Hopkins et al., 1993; Hood et al., 1994; Lawrence et al., 1994). Mutations in the reactive center loop are thought to promote a substrate reaction by interfering with the inhibitor conformational change required for the trapping of proteinases in stable complexes. While increases in the inhibition stoichiometry reflecting increased turnover were observed for the variant inhibitor, these increases were modest relative to the effects on the inhibition rate constants and suggest relatively small changes (3-fold) in the ratio of rate constants for partitioning of the intermediate E-I complex along substrate or inhibition pathways (Hood et al., 1994). The predominant effect of the P1` mutation is therefore not to interfere with the inhibitor conformational change in which the reactive center loop is thought to insert into -sheet A, consistent with the localization of the the P1` residue far from the inserted ``hinge'' region of the loop (Skriver et al., 1991; Carrell et al., 1991; Björk et al., 1992a, 1992b; Hopkins et al., 1993; Björk et al., 1993; Hood et al., 1994; Lawrence et al., 1994). Since the P1` mutation in antithrombin-Denver affects the rate of conversion of the encounter complex to stable complex but does not significantly affect the reaction of the inhibitor as a substrate or the rate of stable complex dissociation, it follows that the mutation primarily affects the rate of formation of the common intermediate, E-I, which precedes the partitioning step ().

Fig. 5 illustrates how the P1` mutation may affect the rate of formation of the partitioning intermediate. According to this scheme, the initial encounter complex formed between antithrombin and proteinase involves a recognition of just the P1 residue and not the P1` residue. This proposal is consistent with the primary role of the P1 residue in proteinase recognition (Owen et al., 1983; Erdjument et al., 1988a, 1988b; Owen et al., 1988; Lane et al., 1989a, 1989b; Erdjument et al., 1989; Derechin et al., 1990; Rubin et al., 1990; Sherman et al., 1992; Elderling et al., 1992) and with rapid kinetic evidence that weak interactions of antithrombin with the proteinase S1 site are involved in the formation of encounter complexes (Olson and Shore, 1982; Craig et al., 1989). The encounter complex is then proposed to be transformed by a subsequent attack of the catalytic serine of the proteinase on the inhibitor reactive bond, as with a normal substrate. This attack induces the P1 carbonyl carbon from a trigonal to a tetrahedral configuration, which promotes the interaction of the P1` residue with the S1` site of the proteinase in the transition state leading to the formation of a tetrahedral intermediate. The formation of the tetrahedral adduct is envisaged as then triggering the conformational change, which leads to insertion of the reactive site loop into the A -sheet of the inhibitor and the consequent trapping of proteinase at the tetrahedral intermediate stage of the substrate pathway. This trapping is proposed to disrupt the P1` interaction and thereby explains why the P1` mutation does not affect the rate of complex dissociation. An alternative mechanism in which trapping of the proteinase occurs at the acyl-intermediate stage of reactive bond cleavage is also possible.


Figure 5: Proposed model for participation of the antithrombin P1` residue in the inhibition of proteinases. According to the model, reaction of antithrombin ( I) with a proteolytic enzyme ( E) proceeds initially like a normal substrate reaction by the formation of an encounter complex in which the inhibitor P1 residue interacts with the enzyme S1 site. Attack of the catalytic serine residue of the enzyme on the inhibitor reactive bond subsequently induces the P1` residue to interact with the enzyme S1` site in the transition state leading to the tetrahedral intermediate complex. Proton transfer from the Ser to His and Asp residues of the catalytic triad occurs concomitant with this attack. Formation of the tetrahedral intermediate then triggers the inhibitor conformational change, which arrests the substrate reaction and kinetically traps the enzyme in the tetrahedral intermediate complex. Completion of the substrate reaction competes with this conformational trapping, resulting in proteolytically cleaved inhibitor and free enzyme.



According to the proposed mechanism of Fig. 5, the binding energy of the P1` residue interaction with proteinase is used to stabilize the transition state complex leading to the tetrahedral intermediate and not the final ground state complex; i.e. the P1` residue functions to lower the activation energy and thereby to increase the rate of formation of the tetrahedral intermediate, but it does not contribute to the stability of the trapped intermediate. This mechanism thus accounts for the primary effect of the P1` Ser to Leu substitution in antithrombin-Denver on the rate of conversion of the encounter complex to a stable complex and the absence of an effect on the rate of dissociation of the stable complex. Such a mechanism contrasts with that of low molecular weight proteinase inhibitors, which interact noncovalently with their target proteinases through an extended contact region typically involving the P6-P3` residues of the reactive center loop and the proteinase active site (Laskowski and Kato, 1980; Read and James, 1986; Bode and Huber, 1992). Diagnostic of the thermodynamic stabilization, which characterizes the latter inhibitor-proteinase complexes, is the primary effect of alterations of the reactive center loop residues on dissociation rather than on association rate constants for complex formation (Empie and Laskowski, 1982). Our observation that the P1` mutation in antithrombin-Denver does not affect the rate constant for dissociation of the stable complex but only the rate constant for complex formation thus argues against a similar thermodynamic stabilization of antithrombin-proteinase complexes by such an extended complementary interaction. Our results therefore do not support proposals that insertion of the serpin reactive center loop into -sheet A induces an active ``canonical'' conformation in the loop like that of low molecular weight proteinase inhibitors which forms an extended contact interaction with proteinase that is responsible for stabilizing the inhibitor-enzyme complex (Carrell et al., 1991; Bode and Huber, 1992; Hopkins et al., 1993). Our findings instead argue that the stability of the final complex arises from a kinetic rather than thermodynamic trapping mechanism as is characteristic of the suicide substrate mechanism of Fig. 5. The kinetic trapping may involve the reactive center loop conformational change disrupting the interactions of the inhibitor reactive bond with the catalytic residues of the proteinase in the tetrahedral intermediate; e.g. by displacing the P1 carbonyl oxygen from the oxyanion hole of the enzyme. The resulting loss of enzyme catalysis could account for the slow dissociation of the covalently linked inhibitor-proteinase complex to cleaved inhibitor and free proteinase (Danielsson and Björk, 1983) and thus explain the trapping of proteinase in a stable complex. Moreover, the kinetically stabilized complex would not require any additional interactions other than the covalent interaction linking inhibitor and proteinase to account for its stability.

In summary, the results of this study provide additional support that serpins act as suicide substrates by coupling a normal substrate reaction between inhibitor and proteinase to an inhibitor conformational change that arrests the substrate reaction and thereby kinetically traps proteinase in a stable complex. In keeping with such a mode of action, our results have shown that the binding energy of the inhibitor P1` interaction with proteinase is used to lower the activation energy for conversion of an initial enzyme-inhibitor complex to a covalent tetrahedral or acyl-intermediate complex, as is typical of a normal substrate. The proposed mechanism for participation of reactive center residues other than P1 in the interaction with proteinase through transition state rather than ground state complementarity may be a general feature of serpin-proteinase interactions. Further kinetic investigations of the step or steps affected by such reactive center mutations similar to those reported in the present study will be required to evaluate this possibility.

  
Table: Inhibition stoichiometries for antithrombin-proteinase reactions

Inhibition stoichiometries were measured from titrations of proteinase with antithrombin (AT) or antithrombin-Denver (AT-Denver) in I 0.15 sodium phosphate buffer, pH 7.4, at 25 °C as in Fig. 2 (see ``Experimental Procedures''). Average values from two titrations ± range are reported except for titrations of Factor Xa in the presence of heparin, in which case the results of a single titration are reported.


  
Table: 0p4in Value obtained using a single inhibitor concentration.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant HL-39888 (to S. T. O.) and Swedish Medical Research Council Grant 4212 (to I. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Center for Molecular Biology of Oral Diseases, Rm. 530E, Dentistry (m/c 860), University of Illinois-Chicago, 801 S. Paulina St., Chicago, IL 60612. Tel.: 312-996-1043; Fax: 312-413-1604.

Present address: Nexagen, Inc., 2860 Wilderness Pl., Boulder, CO 80301.

**
Supported by National Institutes of Health Research Career Development Award HL-02832.

The abbreviation used is: S-2238, D-Phe-L-Pip-L-Arg- p-nitroanilide.


ACKNOWLEDGEMENTS

We thank Roberta Sheffer, Ann-Marie Frances-Chmura, Cathy Ruiz, and Rick Swanson for technical assistance and Dr. Peter Gettins of the University of Illinois-Chicago for critical comments on the manuscript.


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