The Influence of Factor Va on the Active Site of Factor Xa*

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry end Molecular Biology, Inc.

Vol. 268, No. 19, Issue of July 5, pp. 13920-13929,1993 Printed in U.S.A.

The Influence of Factor Va on the Active Site of Factor Xa*

(Received for publication, December 15, 1992, and in revised form, February 9, 1993)

Randall K. Walker$ and Sriram KrishnaswamyQ From the Department of Medicine, Division of Hematobgy/Oncobgy, Emory University, Atlanta, Georgia 30322

The interaction of factor Xa with factor Va on the membrane surface results in a 3,000-fold increase in the kcat for the activation of prothrombin catalyzed by factor Xa. The reaction between the transition state irreversible inhibitor dansylglutamyl-glycyl-arginyl chloromethyl ketone (DEGRck) and factor Xa was characterized and employed to evaluate changes in the active site of factor Xa resulting from its interaction with factor Va, which may account for the increased catalytic efficiency of the enzyme complex. Inhibition studies indicated a two-step inhibition reaction involving a reversible binding step (Ki = 1.13 PM) and an irreversible alkylation step (ki = 0.65 s-’). The interaction between factors Va and Xa in solution or on membranes resulted in a small decrease in the overall second-order rate constant (ki/Ki) for the inhibition reaction. The incorporation of DEGRck into the active site of factor Xa results in a large change in the fluorescence intensity of the dansyl moiety. The fluorescence change was employed to study the reaction between enzyme and inhibitor directly by stopped-flow fluorescence measurements. The fluorescence traces were biphasic, indicating that the association of DEGRck with factor Xa and the subsequent covalent modification of the active site could be resolved because of differences in fluorescence intensities of the intermediate and product. This interpretation was ver- ified by rapid chemical quench experiments. The reaction between DEGRck and factor Xa was characterized by a second-order association rate constant of 8.38 2 0.28 X 10’ M-’ .s” and an intrinsic rate constant for the alkylation step of 1.67 2 0.25 s-*. The rate constant for the alkylation step was unchanged in the presence of factor Va and membranes, whereas the association rate constant was modestly decreased by -50%. The decrease in the association rate constant did not result from the partitioning of factor Xa to the membrane surface and could therefore be ascribed to an effect of factor Va on the protease. The data suggest that the interaction between factors Va and Xa on the membrane surface does not detectably alter the catalytic residues but may result in changes in the binding or

* This work was supported by National Institutes of Health Grants HL-38337 and HL-47465 ( t o S. K.). A preliminary account of this work was presented in poster form at the 65th meeting of the American Heart Association, New Orleans, Louisiana, November 16- 19, 1992 (1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ This work was completed in partial fulfillment of the require- ments for the Ph.D. degree at the Dept. of Biochemistry, University of Vermont, Given Health Sciences Center, Burlington, VT 05405.

§ To whom correspondence should be addressed Dept. of Medicine, Hematology/Oncology, Drawer AR, Emory University, Atlanta, GA 30322. Tel.: 404-727-3806; Fax: 404-727-3404.

accessibility of substrate to extended macromolecular recognition sites on the protease.

The proteolytic conversion of prothrombin to a-thrombin is catalyzed by the prothrombinase complex of blood coagulation (3-5). This macromolecular enzyme complex is composed of the serine protease, factor Xa, and the .cofactor protein, factor Va, reversibly associated on a membrane surface in the presence of calcium ions (4). Although factor Xa itself is capable of catalyzing the activation of prothrombin, assembly of the prothrombinase complex results in a dramatic increase in reaction rate by a factor of approximately 10S-fold (6). The acceleration of the rate of prothrombin activation as a consequence of prothrombinase assembly is considered a key event leading to the rapid formation of thrombin following initiation of the coagulation cascade (4, 7).

Studies of prothrombinase assembly and function using small unilamellar phospholipid vesicles composed of 75% L- a-phosphatidylcholine and 25% L-a-phosphatidylserine (w/ w) (PCPs)’ have indicated that the complex is composed of 1 mol of factor Xa bound tightly (Kd lo-’ M)/mOl of factor Va on the membrane surface (6, 8, 9). Complex assembly on PCPs membranes proceeds through the initial reversible binding of factor Xa (Kd 2 M ) and Va (Kd s lo-’ M) to free sites on the vesicle surface (10). Factors Xa and Va can however interact productively in solution with a 1,000-fold lower affinity (Kd s 1O“j M) than that observed on the membrane surface (11, 12). In addition to facilitating the kinetic and equilibrium aspects of complex assembly, the membrane surface is also considered to play an important role in the activation of prothrombin catalyzed by the assembled complex through the ability of prothrombin also to bind reversibly to these surfaces (13-15).

Steady-state kinetic studies of prothrombin activation have been conducted using factor Xa in solution, binary mixtures of prothrombinase constituents, and the fully assembled prothrombinase complex (11, 14, 16-19). The increased catalytic efficiency of prothrombinase over factor Xa alone derives from a 100-fold decrease in the apparent K,,, and a 3,000-fold increase in the kc, for the reaction (17). The decrease in Kmpp has been attributed to a result of the co-condensation of enzyme and substrate on the membrane surface, leading to the achievement of very high concentrations of these reac- tants in microscopic subspaces surrounding the vesicle (13,

The abbreviations used are: PCPs, phosphatidylcholine and phosphatidylserine; dansyl, 5 dimethylaminonaphthalene-1-sulfonyl; DEGRck, dansylglutamyl-glycyl-arginyl chloromethyl ketone; DEGR-Xa, factor Xa modified with dansylglutamyl-glycyl-arginyl chloromethyl ketone; EGRck, glutamyl-glycyl-arginyl chloromethyl ketone; S2222, benzoylisoleucyl-glutamyl-glycyl-arginyl p-nitroanilide; Spectrozyme Xa, cyclohexylglycyl-glycyl-arginyl p-nitroanilide; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liq- uid chromatography.

13920

The Active Site of Factor Xa 13921

14). The increase in kat however is not adequately explained by such a model and is presumed to result from the influence exerted by factor Va either on factor Xa and/or substrate (14). More recent kinetic studies of prothrombin activation catalyzed by the Xa-Va binary complex in solution have provided indications that the interaction between the cofactor and protease regardless of the membrane surface is sufficient to account for most, if not all, of the increased catalytic efficiency observed for the activation of prothrombin (11).

Two possible mechanisms through which factor Va could influence the hat for prothrombin conversion are: (i) by alter- ing the active site of factor Xa to increase its complementarity to the transition state or by increasing the reactivity of catalytically important residues; and (ii) by binding the substrate and stabilizing a conformation that more closely approximates the transition state.

Evidence supporting the first possibility is derived from steady-state fluorescence measurements using the active-site- directed fluorophore, dansylglutamyl-glycyl-arginyl chloromethyl ketone (DEGRck) covalently incorporated into factor Xa (8). Alterations in steady-state fluorescence intensity and anisotropy in the fluorescent adduct of factor Xa (DEGR-Xa) are observed following its interaction with factor Va on PCPs membranes (8, 9, 20, 21). These fluorescence changes in the reporter group have been used extensively to examine the kinetic and equilibrium aspects of complex assembly and have also been interpreted to reflect perturbations in the active- site environment of factor Xa induced following the interaction between factors Xa and Va on the membrane surface (8- 10,20,21). Whether these inferred perturbations in the active site of the protease reflect changes in the catalytic residues uersus extended macromolecular binding sites and directly correspond to changes that result in increased catalytic efficiency cannot be presently resolved. However, the fluorescence changes do correlate well with the increase in catalytic activity following the interaction between factors Xa and Va on the membrane surface or in solution (9, 11).

The second possible explanation for the increased catalytic efficiency of prothrombinase toward prothrombin is supported by evidence indicating that the substrate can interact reversibly with the cofactor through its fragment 2 domain (22-24). Kinetic studies of the reaction catalyzed by the Xa- Va binary complex in solution have provided good evidence for a key contribution of the cofactor-substrate interaction toward prothrombin activation (11). Additional evidence supporting this possibility lies in the fact that the cleavages of the two bonds in prothrombin are characterized by different kinetic constants and are differentially influenced by factor Va (16, 25, 26). The rate of conversion of meizothrombin to thrombin (cleavage at Ar$74-Thr275) proceeds rapidly in the absence of cofactor and is further increased by a modest extent (-5-fold) by saturating concentrations of factor Va (25, 26). In contrast, the rate of cleavage at Arg3'3-Ile3'4 by factor Xa proceeds very slowly and is accelerated by approximately IO3- fold at saturating concentrations of cofactor (25, 26). One interpretation of these observations is that the increased catalytic efficiency of prothrombinase does not result entirely from a direct effect of factor Va on the active site of factor Xa in the prothrombinase complex.

Changes in the active site of the protease component resulting from its interaction with the cofactor have been doc- umented using synthetic peptidyl substrates in other analo- gous macromolecular complexes of coagulation. Since small peptidyl substrates are assumed to lack the structural ele- ments of the macromolecular substrates which permit interactions with the cofactor, this approach permits the evalua-

tion of changes in catalytic efficiency due to effects exerted by the cofactor at the level of the enzyme separately from the effects exerted at the level of the substrate.

The interaction between the serine protease, a-thrombin with its cofactor protein, thrombomodulin, results in a change in its catalytic efficiency toward a variety of synthetic peptidyl substrates (27, 28). These types of observations along with fluorescence measurements using active site-directed reporter groups and studies of macromolecular substrate specificity have provided reasonable evidence for an allosteric modula- tion of the active site of thrombin following its interaction with thrombomodulin (29-31). Similarly, the interaction of factor VIIa with the cofactor protein, tissue factor, results in a substantial increase in the catalytic efficiency of the protease toward synthetic peptidyl substrates (32-35), leading to the suggestion that the interaction between factor VIIa and tissue factor results in an alteration at the active site of factor VIIa (34).

Comparable evidence for changes in the catalytic center of factor Xa induced by factor Va is lacking. The rate of hydrolysis of the synthetic peptidyl substrate benzoyl isoleucyl- glutamyl-glycyl-arginylp-nitroanilide (S2222) by factor Xa is slightly decreased upon prothrombinase assembly, resulting from a modest increase in K,,, with no change in kc, (13). Although these data are suggestive of a minimal effect of factor Va on the active site of factor Xa, the changes in the steady-state kinetic constants have been interpreted on a purely hydrodynamic basis reflecting the partitioning of the enzyme to the membrane surface to which the synthetic substrate cannot bind (13). Thus, the mechanism(s) through which factor Va increases the kat for prothrombin activation by factor Xa remains unknown.

We have used the modification of factor Xa by DEGRck as a model reaction system to evaluate changes in the active-site environment of the protease as a consequence of its incorporation into the prothrombinase complex. The tripeptidyl sequence corresponds to PI-P3 residues' preceding the scissile bond in prothrombin and alkylation of the active-site histidine of serine proteases by halomethyl ketones involves the participation of the active site serine through the formation of a tetrahedral hemiketal which is stereochemically analo- gous to the tetrahedral transition state in the hydrolysis of peptide bonds (36). Thus, kinetic measurements of the binding and subsequent alkylation of factor Xa by this irreversible transition state inhibitor in the presence or absence of other constituents of prothrombinase provide information relevant to perturbations in the primary and extended substrate binding sites (s1-S~) and the catalytic residues of factor Xa resulting from its interaction with factor Va on the membrane surface.

EXPERIMENTAL PROCEDURES

Materials-Hepes, Tris base, L-a-phosphatidylserine (bovine brain), and L-a-phosphatidylcholine (hen egg) were purchased from Sigma. Cyclohexylglycyl-glycyl-arginyl p-nitroanilide (Spectrozyme fXa) was from American Diagnostica. The peptidyl chloromethyl ketones DEGRck and glutamyl-glycyl-arginyl chloromethyl ketone (EGRck) were from Calbiochem. Initial solutions of DEGRck were prepared in 10 mM HCl based on dry weight, and the concentration was determined by amino acid composition following acid hydrolysis. Absorbance measurements assuming 1 mol of fluorophore/mol of glutamic acid and glycine yielded = 3,500 M" . cm" for the dansyl moiety in 20 mM Hepes, 0.15 M NaCl, pH 7.4. Phospholipid vesicles composed of 75% phosphatidylcholine (w/w) and 25% phosphatidylserine (w/w) were prepared by sonication and differential centrifu- gation as described previously (37). The concentrations of phospholipid are expressed as the concentration of the monomeric species

Nomenclature of Schechter and Berger (2).

13922 The Active Site of Factor Xa based on a colorimetric phosphate assay (37, 38).

Proteins-Factor X was isolated from bovine plasma as described (9, 39). Factor Xa was prepared by activation using the purified activator from Russell's viper venom followed by purification using benzamidine-Sepharose as described (26, 40). Bovine factor Va was purified following activation of partially purified factor V as described previously (41). The isolated covalent adducts of factor Xa with DEGRck or EGRck used for some experiments were prepared by reacting the protease with between 15- and 25-fold excess of the chloromethyl ketone followed by separation from excess modifier by gel filtration using Sephadex G-25-150 as described (10). The purity of all proteins was assessed by SDS-PAGE before and after treatment with dithiothreitol followed by staining with Coomassie Brilliant Blue (42). Protein concentrations were determined using the following molecular weights and extinction coefficients (E;&%): bovine factor Va, 168,000, 1.74 (41, 43); factor X, 55,600, 1.24 (44, 45); and factor Xa, 45,300, 1.24 (44, 45). Active-site titration of factor Xa with p- nitrophenyl-p' guanidinobenzoate (46) yielded 1.1 mol of active site/ mol of Xa.

Steady-state Fluorescence Measurements-Spectra1 measurements were conducted with an SLM 8000 or SLM 8OOOC fluorescence spectrophotometer both with adapted hardware and software provided by OLIS, Bogart, GA. Samples (2 ml) for technical spectra contained 2 p~ DEGRck or 2 p~ DEGRck plus 3 p~ Xa in 50 mM Tris, 0.15 M NaC1, 2 mM CaC12, pH 7.4, in 1 X 1-cm stirred cuvettes maintained at 25 "C. Ratiometric spectra were collected by scanning the emission monochromator between 380 and 620 nm (band pass = 16 nm) using = 330 nm (band pass = 2 nm) with a long pass filter (Schott KV-370) in the emission beam. Excitation spectra were collected between 270 and 420 nm (band pass = 2 nm) by monitoring fluorescence at 550 nm (band pass = 16 nm). Corrected emission spectra were obtained by monitoring fluorescence intensity with a vertically oriented polarizer in the emission beam and using correc- tion factors provided by SLM instruments. Quantum yields were determined by integrating the corrected spectra of samples with known absorbance at 330 nm by comparison with the values obtained with quinine sulfate in 0.1 M HzSO,. Quantum yields were calculated using the equations described and assuming a value of 0.71 for quinine under these conditions (47).

Dynamic Fluorescence Measurements-Fluorescence lifetimes were measured by single photon counting using a frequency-doubled, dye- pumped, cavity-dumped YAG laser as described by Lee et a1 (48). Excitation was at 372 nm with samples maintained at 25 "C, and fluorescence intensity decays were monitored at 540 nm using an emission polarizer a t the magic angle. Decays were fit to multiexpo- nential processes as described (49), and the values reported represent intensity-weighted averages of the observed lifetimes.

Steady-state Inhibition Measurements-The reaction between DEGRck and factor Xa was evaluated indirectly (50-52) by monitoring the inactivation of factor Xa in the presence of the synthetic substrate, Spectrozyme E a . The reaction between DEGRck and factor Xa was measured using an HP8452A diode array spectrophotometer with software provided by OLIS. Reaction mixtures (1 ml) contained 160 p~ Spectrozyme fXa and increasing concentrations of DEGRck (0-16 PM) in 20 mM Hepes, 0.15 M NaCl, 2 mM CaC12, 0.1% polyethylene glycol 8000. Progress curves were initiated with variable concentrations of enzyme, and the rates of inactivation of factor Xa by DEGRck were inferred from exponential decays in the rate of product formation by monitoring the absorbance at 406 nm at am- bient temperature. The instrument time constant ranged between 0.1 and 0.3 s to provide adequate response times for the data collection interval used and to ensure that the response time was at least 10 times shorter than the ts for the reaction being measured. Inhibition of factor Xa in the presence of other constituents of prothrombinase was measured at low concentrations of DEGRck (c0.2 pM) in a V,. kinetic plate reader (Molecular Devices) using 96-well plates (Corning Assay Plate). Reaction mixtures (100 pl) containing 600 p M Spectro- zyme fXa and increasing concentrations of DEGRck in 20 mM Hepes, 0.15 M NaCl, 2 mM CaClZ, 0.1% polyethylene glycol 8000, pH 7.4, were initiated with 100 pl of enzyme solution in the same buffer. The final concentrations of enzyme components were: 0.5 nM Xa; 0.5 nM Xa plus 50 p M PCPs; 0.5 nM Xa plus 21 p M Va; and 0.5 nM Xa, 50 p~ PCPs, 10 nM Va. Exponential decays in the rate of product accumulation were measured by monitoring absorbance at 405 nm.

Fluorescence Stopped Flow-The reaction between DEGRck and factor Xa was measured directly using stopped-flow instrumentation (Kinetic Instruments, Ann Arbor, MI) attached to an SLM 8000C fluorescence spectrophotometer equivalent to that described previ-

ously (10). Rapid kinetic measurements in the ratiometric mode were performed at 25 "C, using Lx = 280 nm and monitoring broad band fluorescence (2520 nm) with a long pass filter (Schott KV-520) in the emission beam. Reactions were initiated by rapidly mixing equal volumes (100 pl) of the contents of the two driving syringes. Syringe A contained enzyme solution (Xa, Xa-PCPS, or Xa-Va-PCPS) at the indicated concentrations in 20 mM Hepes, 0.15 M NaC1, 2 mM CaC12, pH 7.4, and syringe B contained various concentrations of DEGRck. Following initiation, 400 points were collected per trace using two collection intervals to permit the adequate description of the rapid and slow phases of the fluorescence change ( e . g 200 points in 0-200 ms, datum interval = 1 ms and 200 points in 200-4200 ms, datum interval = 20 ms). The concentrations were selected to make the reaction pseudo-first-order in DEGRck. Between six and nine replicate traces were collected for each experimental condition.

Possible fluorescence changes caused by the modification of protein by trace contaminating dansylchloride in the DEGRck preparations was a significant concern. This possibility was eliminated by repeating experiments in 20 mM Tris- uersus 20 mM Hepes-containing buffers to provide a nucleophile to scavenge NHp-reactive contaminants prior to initiation of the reaction. Stopped-flow and steady-state fluorescence measurements under a wide variety of conditions were repeated in both buffer systems and yielded indistinguishable results. Thus, the results obtained in Tris or Hepes buffers are presented inter- changeably throughout. The introduction of systematic error caused by chloromethyl ketone hydrolysis at pH 7.4 during the 15 min required to acquire the replicates for each set of stopped-flow experiments was a concern. This possibility was eliminated by the coinci- dence of stopped-flow traces obtained following deliberate aging of the chloromethyl ketone solution for 15 and 30 min following loading of the driving syringes.

The contribution of the inner filter effect to stopped-flow measurements conducted in the presence of factor Va was evaluated by measuring the kinetics of modification of factor Xa by DEGRck in the presence or absence of glycyl-tryptophan at a concentration (-50 pM) yielding the same absorbance (at 280 nm) as 0.75 p M factor Va. All fitted parameters from these measurements were independent of the presence of the chromophore, suggesting that inner filter considerations do not account for the observed effects of factor Va on the kinetic measurements.

Rapid Chemical Quench-Quenched-flow methodology was used to determine independently the rate constant for the irreversible alkylation of factor Xa by DEGRck. Rapid quench experiments were performed using a stepper motor-controlled five-syringe instrument (QFM-5, Bio-Loqic, Eschiorelles, France). Temperature was controlled at 25 "C. The instrument was operated in the interrupted mode, and the reaction was initiated by rapid mixing of 60 pl of Xa solution (syringe 1: 8 p M Xa in 50 mM Tris, 0.15 M NaC1, 2 mM CaCl2, pH 7.4) with an equal volume of DEGRck (syringe 2: 200 pM DEGRck in the same buffer). After appropriate aging times the reaction was quenched with 120 pl of glacial acetic acid and expelled from the instrument. Detection and quantitation of alkylation were accomplished by HPLC or by SDS-PAGE. For the HPLC method, quenched samples were applied directly to a reversed phase C-8 cartridge column (3-cm Aquapore RP-300, Brownlee) equilibrated in water containing 0.05% (v/v) trifluoroacetic acid. Factor Xa was eluted with a linear gradient (0-60% CH3CN plus 0.05% trifluoroacetic acid) over 20 min at a flow rate of 1 ml/min and detected by absorbance (280 nm) and fluorescence (Aex = 280, L, > 500 nm). Reaction extent was determined by dividing the height of the fluorescence peak by the height of the absorbance peak and expressed as a fraction of the value determined from extrapolation to infinite time.

For the SDS-PAGE method, the quenched samples were dialyzed against 0.1 M CH3COOH, evaporated to dryness, dissolved in 75 r l of sample buffer (62.5 mM Tris, 2% (w/w) SDS, 10% (v/v) glycerol, 0.01% (w/w) bromphenol blue, 35 mM dithiothreitol, pH 6.8) and heated at 90 "C for 5 min. The samples were subject to SDS-PAGE (42) using a gel composed of 9.5% acrylamide and 0.5% N,N'- methylenebisacrylamide. Following electrophoresis, the fluorescent bands were visualized by UV illumination and photographed (Pola- roid type 55 film). Protein bands were visualized by staining with Coomassie Brilliant Blue R-250. Quantitation was achieved by scanning densitometry analysis (Shimadzu CS-930) of the negative film (fluorescence) and the stained gel (protein). The reaction extent was determined and normalized as before by dividing the integrated areas obtained by the two methods. Control experiments established that the response of both film and stained gel was linear over the range chosen.

The Active Site of Factor X u 13923

Data Analysis-All data sets were analyzed according to the indicated equations by nonlinear least squares analysis to obtain estimates of the parameters and the 95% confidence limits (53). The quality of the fits were evaluated on the basis of the errors associated with the fitted parameters, the correlation between parameters, the root mean squared deviation, and the random distribution of residuals (54). Some of the absorbance data yielded excellent fits with systematic runs in the residuals. These fits were viewed to describe the data adequately because in many cases the extent of deviation from the fitted line was within digital precision of the data or resulted from a slow excursion in signal stability.

Progress curves of Spectrozyme fXa hydrolysis by factor Xa in the presence of increasing concentrations of DEGRck were fitted to a single exponential rise

s i g n a h = offset + amp. (1 - e-kb.') (Eq. 1)

in which the observed absorbance (signah.) measured as a function of time ( t ) was analyzed to extract the observed pseudo-first-order rate constant (kobs), the base-line offset (offset) and the amplitude of the change (amp). The dependence of kobs on the concentration of DEGRck was analyzed assuming a complexing type of inhibition reaction (see Scheme I) using Equation 2,

in which k; is the intrinsic first-order rate constant for the alkylation reaction (see Scheme I), K; is the dissociation constant for the initial reversible interaction of DEGRck with factor Xa (see Scheme I), I and S refer to the concentrations of DEGRck and Spectrozyme fXa, and K,,, is the Michaelis constant for the hydrolysis of Spectrozyme fXa by the enzyme. Determined values of kobs as a function of DEGRck were analyzed according to Equation 2 to yield fitted values of K; and ki. The K,,, for Spectrozyme fXa was determined to be 54 f 5 NM under the present experimental conditions.

For the determination of the overall second-order rate' constant (k;/K;), the concentration of DEGRck (I) was varied over a range (0- 160 nM) such that I << Ki(1 + S/K,,,). Under these conditions, the term I can be eliminated from the denominator of Equation 2 to yield Equation 3.

The slope of the linear dependence of kobs on DEGRck was divided by the term (1 + S/K,,,) to yield the overall second-order rate constant for the inhibition reaction.

Stopped-flow fluorescence traces were analyzed according to Scheme I1 assuming consecutive first-order reactions (55), according to Equations 4-6,

in which the observed fluorescence (signa1,b.) collected as a function of time ( t ) is related to the fraction of total enzyme (Emt) present as E.1 or E'.I through the observed first-order rate constant for steps 1 and 2 ( kl,,,bs and kZ+,ba) and the fluorescence contributions of E . I and E'. I (Q1 and Qz). Fluorescence data was analyzed to obtain fitted values for the two observed rate constants, base-line offset (offset) and the quantum yield factors (Q1 and Q 2 ) . Because of the "ill- conditioned nature of multiexponentials (56), the quality of the fits was substantially improved by averaging replicate stopped-flow traces to reduce random noise prior to fitting the data.

Both first-order rate constants obtained from the fluorescence measurements are apparent rate constants describing the reaction steps in Scheme 11. The rate constant for the first step (kl+bs) is a pseudo-first-order rate constant that is related to the intrinsic rate constant (k+1) by the relationship kl,obs = (I. k+J + k-l where I is the

concentration of inhibitor which is in excess of the concentration of enzyme. The rate constant for the second step (kz,obs) is also dependent on the concentration of inhibitor, reaching a limiting value at saturating concentrations of I (57).

RESULTS

Steady-state Measurements of Inhibition of Factor Xu by DEGRck-Because of the rapid inactivation of factor Xa by DEGRck, initial kinetic studies involved continuous measurements of the inhibition reaction in the presence of the synthetic substrate, Spectrozyme fXa. Concentrations of enzyme and substrate were chosen to ensure that the concentration of product increased linearly with time in the absence of added DEGRck. In the presence of inhibitor, the accumulation of product could be described adequately by a single exponential rise (Fig. 1). The observed rate constant and the limiting amplitude of the rise were dependent on the concentration of DEGRck present in the reaction mixture (Fig. 1, inset).

The relationship between kobs and inhibitor concentration is predicted to be linear for a one-step noncomplexing type inhibitor or hyperbolic for a two-step reaction mechanism that involves the initial reversible binding of the inhibitor to the enzyme to form a dissociable binary complex, prior to the irreversible alkylation step (51, 52). The nonlinear dependence of k,bs on the concentration of DEGRck illustrated in Fig. 2 is consistent with the second type of inhibition mechanism. Thus, relevant kinetic steps describing the inhibition of factor Xa by DEGRck in the presence of Spectrozyme fXa are appropriately accounted for by Scheme I

Km $at

E + S + ES + E + P +I I t Ki

k,

SCHEME I E.1 + E*.I

10 20

Time (min) 0.00 I I

0.0 0.2 0.4 0.6 0.8 1 .o Time (min)

FIG. 1. Kinetics of inhibition of factor Xa by DEGRck. A reaction mixture containing Spectrozyme Xa (161 p ~ ) and DEGRck (1.12 pM) in 20 mM Hepes, 0.15 M NaC1, 2 mM CaC12, pH 7.4, was initiated by the addition of factor Xa (21 nM). Spectrozyme fXa hydrolysis was measured by an increase in absorbance at 405 nm. The data were analyzed according to Equation 1 to extract kobs = 7.5 f 0.02 min". The residuals to the fitted line are illustrated in the upper panel. The inset illustrates representative progress curves obtained using 5 nM Xa and increasing concentrations of DEGRck (0, 44,88, 225, and 562 nM).

13924 The Active Site of Factor X a

0 . 0 8 I

[DEGRck] pM 0 4 8 12 18

FIG. 2. Dependence of koa on the concentration of DEGRck. Values for kb. were extracted from progress curves at the indicated concentrations of DEGRck as illustrated in Fig. 1. The line is drawn according to Equation 2 with the fitted constants Ki = 1.13 f 0.16 p M and ki = 0.65 f 0.04 s-’.

in which E, S, and I represent factor Xa, Spectrozyme m a , and DEGRck, respectively. Ki represents the dissociation constant for the formation of the dissociable E . I binary complex, and ki represents the intrinsic first-order rate constant for the alkylation step yielding the covalently inactivated adduct ET. I. The dependence of k b s on the concentration of DEGRck was adequately described by Equation 2, presented by Tsou (51) for this type of inhibition reaction, and yielded ki = 0.65 k 0.04 s-l and Ki = 1.13 f 0.16 FM. Because of experimental difficulties associated with measuring rapid decays (t% < 2 s) in open cuvette measurements, both constants could be potentially underestimated by this experimental approach.

The influence of the other constituents of prothrombinase on the reaction between factor Xa and DEGRck was assessed by comparing the overall second-order rate constant for inhibition by DEGRck for factor Xa alone using binary and ternary mixtures of factor Xa, PCPs, and factor Va. For these measurements, experiments such as those illustrated in Fig. 1 were conducted using DEGRck concentrations (0-160 nM) that were well below the determined Ki. As described under “Data Analysis,’’ this experimental design yields a linear dependence of koba on the concentration of DEGRck and reliably permits the calculation of the overall second-order rate constant (ki/Ki) for the inhibition reaction according to Equation 3.

The results obtained from these measurements are listed in Table I. The overall second-order rate constant for the inhibition of factor Xa alone is within 2-fold of the value that can be calculated from the constants obtained from Fig. 2. This observation implies that potential underestimates in either ki or Ki determined in Fig. 2 are not large. The addition of PCPs to bind saturably all of the added factor Xa on the basis of previously described equilibrium parameters yielded no significant change in the overall rate constant for inhibition by DEGRck. In contrast, the incorporation of factor Xa into prothrombinase by the addition of PCPs and factor Va resulted in a modest but reproducible decrease in ki/Ki. These data suggest that the efficiency of inhibition of factor Xa is decreased slightly upon its incorporation into the enzyme complex. These results were compared with those obtained using the Xa-Va binary complex to distinguish further between the effects of membranes and cofactor on the active site of the enzyme. In these experiments, the concentration of factor Va (21 p ~ ) was chosen to yield > 90% saturation of

TABLE I Effect of factor Va and PCPs on the overall second-order rate

constant for the inhibition of factor Xa by DEGRck The overall second-order rate constants for inhibition by DEGRck

were determined by measurements of Spectrozyme Xa hydrolysis as described under “Experimental Procedures.” The value of ki/Ki was determined according to Equation 3.

Enzyme species“ ki/K; * S.D.L X 10’ “1.8-1

Xa 3.28 f 0.01 Xa-PCPS 3.60 f 0.02 Xa-Va 2.53 f 0.05 Xa-Va-PCPs 2.46 f 0.03

“Reaction mixtures contained 0.5 nM Xa, 0.5 nM Xa plus 50 pM PCPs, 0.5 nM Xa plus 21 p~ Va or 0.5 nM Xa, 10 nM Va plus 50 p~ PCPS.

~~

* The constants are listed f 95% confidence limits.

1 .o 1

- 300 350 400 450 500 550 600

Wavelength (nm) FIG. 3. Fluorescence spectra for DEGRck in the absence or

presence of factor Xa. Measurements were made in 50 mM Tris, 0.15 M NaCl, 2 m M CaC12, pH 7.4, at 25 “c with samples containing 2.0 p~ DEGRck (spectra B and D) or 2.0 pM DEGRck plus 3.0 p~ factor Xa (spectra A and C). Excitation spectra ( A and B ) were collected by monitoring emission at 530 nm, and technical emission spectra (C and D) were recorded by excitation at 280 nm.

the factor Xa present on the basis of the published dissociation constants (& = 0.8-2.7 pM) for the Xa-Va interaction in solution (11, 12). The value for ki/Ki obtained under these conditions is comparable to that obtained with the fully assembled prothrombinase (Table I). These data imply that it is the interaction between factors Va and Xa which causes the observed decrease in the efficiency of inhibition of factor Xa by DEGRck following the assembly of prothrombinase.

The data presented in Table I permit the preliminary conclusion that the interaction of factor Xa with factor Va in the prothrombinase complex results in modest changes in the efficiency of its inhibition by DEGRck. Since the overall second-order rate constant is a ratiometric term, modest changes in kJKi could arise from large but offsetting changes in the individual rate constants for the inhibition reaction. Therefore, further studies of the inhibition of factor Xa by DEGRck were conducted by direct fluorescence measurements of this reaction to evaluate the effects of prothrombinase assembly on the individual reaction steps.

Fluorescence Properties of DEGRck-Fluorescence excitation and emission spectra for DEGRck in the presence and absence of factor Xa are illustrated in Fig. 3. The excitation spectrum of the free probe is characterized by a maximum at 330 nm, corresponding to the absorbance maximum of the dansyl moiety. The technical emission spectrum of DEGRck is characterized by a maximum at 560 nm. The incorporation

The Active Site of Factor Xa 13925

of DEGRck into factor Xa results in a large increase in fluorescence intensity with a significant blue shift (-20 nm) in the emission maximum. The excitation spectrum of DEGR- Xa is characterized by an increase in the fluorescence observed by excitation at 330 nm and by a prominent increase centered at 280 nm because of resonance energy transfer from intrinsic fluorophores in factor Xa to the dansyl moiety.

Some of the determined fluorescence properties of DEGRck and DEGR-Xa are listed in Table 11. The incorporation of DEGRck into the active site of factor Xa results in a 2.5-fold increase in the quantum yield of the dansyl moiety, which is matched by a proportional increase in the intensity-weighted average lifetime of the excited state. The quantum yield, lifetime and spectral measurements suggest that the fluorophore is shielded from solvent and quenching effects when it is covalently incorporated into the active site of factor Xa.

The large fluorescence change in the dansyl moiety and in the energy transfer between intrinsic protein fluorophores and the dansyl moiety therefore provides a convenient signal to monitor continuously the reaction between DEGRck and factor Xa. On the basis of the data illustrated in Fig. 3 and in Table 11, further studies of this reaction were conducted by monitoring broad band fluorescence (Aex > 500 nm) with excitation at 280 nm.

Measurements of the Reaction between DEGRck and Factor Xa by Stopped-flow Fluorescence-Fluorescence stopped-flow techniques were used to evaluate the rapid reaction between DEGRck and factor Xa. Experiments were conducted using high concentrations of DEGRck and limiting concentrations of factor Xa to ensure that the bimolecular combination of inhibitor with enzyme was pseudo-first-order with respect to DEGRck. A representative stopped-flow trace is illustrated in Fig. 4. Following initiation, the fluorescence increase is biphasic with approximately 80% of the fluorescence change occurring in the first 100 ms, followed by a slower subsequent rise accounting for the remaining 20% of the observed fluorescence. Reactions initiated under identical experimental conditions except containing factor Xa modified at the active site with the nonfluorescent peptidyl chloromethyl ketone (EGRck) showed no change in fluorescence intensity (Fig. 4, inset). The data therefore demonstrate that the fluorescence change requires the binding of DEGRck to the active site of factor Xa and/or alkylation of the active-site histidine.

The biphasic increase in fluorescence (Fig. 4) suggests that the inhibition of factor Xa by DEGRck involves at least two reaction steps with well separated time constants, yielding species that are differentially fluorescent. The stopped-flow measurements can be interpreted in terms of a two-step reaction that is consistent with the complexing type of inhi-

TABLE I1 Fluorescence measurements of DEGRck and DEGR-Xa

Parameter DEGRck" DEGR-Xa

F/Fo ( L = 280)* 1.00 7.32 F/Fo ( L = 330) 1.00 2.91 Quantum yield' 0.060 0.165 T d 2.64 5.54

a Fluorescence measurements were conducted using the free pep- tidy1 chloromethyl ketone (DEGRck) or its covalent adduct with factor Xa (DEGR-Xa) in 20 mM Hepes, 0.15 M NaCl, 2 mM CaC12, pH 7.4, at 25 'C.

'Normalized fluorescence intensities were measured at 540 nm using excitation wavelengths of 280 or 330 nm.

e Quantum yields were measured from corrected emission spectra obtained by excitation at 330 nm and by comparison with quinine sulfate in 0.1 M H2S0,.

Intensity-weighted average of the excited-state lifetime in nano- seconds.

0.02

-0.02 1 .o

Q) 0 !f) 0.9

E 0 v)

0 0.8 3 L -

0.7 0.00 0.05 0.10 0.15 0.20 1 2 3 4

Time (s) FIG. 4. Stopped-flow fluorescence measurements of the re-

action between DEGRck and factor Xa. Reactions were initiated using 2.0 pM factor Xa in 50 mM Tris, 0.15 NaC1, 2 mM CaC12, pH 7.4 (syringe A), and 160 PM DEGRck in the same buffer (syringe B). Fluorescent intensity was monitored (LX = 280 nm, L,,, > 500 nm) at 25 "C, and 400 points were acquired using two collection intervals as described under "Experimental Procedures." The line is drawn according to Equations 4-6 using the fitted parameters kl,oba = 64.4 f 1.49 s-', k2,0bs= 1.53 f 0.08 s-', offset = 0.698 f 0.003, Q1 = 0.238 f 0.003, and Q2 = 0.303 f 0.003. The residuals to the fitted line are illustrated in the upper panel. Inset, control experiment in which syringe A contained factor Xa that had been previously inactivated with EGRck.

bition mechanism inferred from the steady-state kinetic measurements shown in Scheme 11,

k+l k2

k-1

E + I + E.1 + E*.I

SCHEME I1

in which E and I refer to factor Xa and DEGRck, E .I is the dissociable binary complex between enzyme and inhibitor, and ET. I is the covalently modified species (DEGR-Xa). The data are consistent with the interpretation that E I and E'. I are distinguishable on the basis of their relative fluorescence intensities. Because of experimental design, the bimolecular association reaction would be a pseudo-first-order reaction. Thus, Scheme I1 simplifies to two consecutive first-order reactions that can be analyzed according to Equations 4-6 to obtain the observed first-order rate constants for the two steps along with factors (Q1 and Q 2 ) that provide some measure of the contributions of E.1 and F.1 to the observed fluorescence change.

The stopped-flow traces were adequately described by Equations 4-6 to yield a pseudo-first-order rate constant of 64.5 s-l for the bimolecular association reaction and an observed first-order rate constant of 1.71 s-' for the covalent modification step. The first-order alkylation rate constant is in reasonable agreement with the value for k2,0bs determined from the steady-state kinetic measurements (Fig. 2) and sup- ports the use of Scheme I1 and Equations 4-6 to describe the stopped-flow fluorescence traces. The fitted fluorescence am- plitudes (normalized Q1 = 0.8, Q 2 = 1.0) imply that the dissociable E. I complex has 80% of the fluorescence intensity of the covalent E'. I adduct. These data allow the reasonable speculation that the irreversible alkylation reaction results in rearrangements in factor Xa or the peptidyl chloromethyl ketone bound to factor Xa that is detected by changes in fluorescence intensity.

Rapid Chemical Quench Measurements of the Formation of DEGR-Xu-Quenched-flow studies were undertaken to pro-

13926 The Active Site of Factor Xa

vide independent verification of the stopped-flow fluorescence studies by direct measurements of the alkylation rate constant. The logic behind this approach is that quenching of the reaction with glacial acetic acid followed by a determination of the accumulation of the fluorescent Xa under denaturing conditions permits the calculation of the rate constant for the formation of the covalently modified species ( E ' . I, Scheme 11) without considering the dissociable E . I binary complex.

Reaction progress curves for the accumulation of the covalent adduct between DEGRck and factor Xa determined by rapid quench are illustrated in Fig. 5 . The extent of formation of DEGR-Xa was evaluated following quenching either by SDS-PAGE or by reversed phase HPLC as described under "Experimental Procedures." Relative amounts of DEGR-Xa determined in several experiments by the two independent methods of analysis were analyzed to yield a first-order rate constant of 0.9 f 0.1 s-'. This rate constant is in good agreement with the values of ki determined from steady-state measurements (Fig. 2). These experiments also demonstrate that the covalent modification of factor Xa by DEGRck proceeds on a time scale that is comparable to the second phase of the fluorescent transient. The data provide independent support for the interpretation of the stopped-flow fluorescence measurements according to Scheme 11.

Effect of Factor Vu and PCPs on the Binding of DEGRck to Factor Xu-Fluorescence stopped-flow measurements of the rapid phase were conducted at increasing concentrations of DEGRck to obtain the intrinsic second-order rate constant for the formation of the dissociable E . I complex ( k+l, Scheme 11). In reactions between factor Xa and DEGRck, the rate constant for the rapid fluorescence change (kl+,bs) increased linearly with increasing concentrations of inhibitor (Fig. 6) and was also found to be independent of the fixed concentration of factor Xa (not shown). Thus, the observed rate constant is first-order with respect to DEGRck and zero-order with respect to factor Xa, consistent with Scheme I1 and with the experimental design (I > > E ) used in these measurements. The intrinsic second-order rate constant (k+l = 8.38 & 0.28 X lo6 M - ' . s-') was extracted from the slope of this plot.

Similar experiments were conducted by reacting DEGRck with Xa saturated with PCPs or with factor Xa saturated

x

c 0 .-

0.4 0

0.0 p ' I 0 4 8 12 16 20

Time (s) FIG. 5. Quenched-flow measurements of the covalent mod-

ification of factor Xa by DEGRck. Reactions were initiated by mixing equal volumes of 8 pM Xa in 50 mM Tris, 0.15 M NaCl, 2 mM CaClz, pH 7.4, and 200 p~ DEGRck in the same buffer. Following aging for various times at 25 "C, reactions were quenched with glacial acetic acid. The concentration of covalently modified Xa was determined in separate experiments by SDS-PAGE and densitometry (V, V) or by reversed phase HPLC (0, 0, 0) as described under "Exper- imental Procedures." The data were analyzed according to a first- order rise to yield kz,obs = 0.9 * 0.1 s-'.

120 -

100 -

n

I 7

0 20 40 60 80 100 120 140

[DEGRck] pM FIG. 6. Influence of factor Va and PCPs on the binding of

DEGRck to the active site of factor Xa. The pseudo-first-order rate constant (kl,abs) for the association of DEGRck with Xa was determined by stopped-flow fluorescence measurements using the indicated final concentrations of DEGRck and 0.5 p~ Xa (0), 0.5 p M Xa plus 50 pM PCPs (n), and 0.5 p M Xa, 50 p M PCPs plus 0.75 pM factor Va (V) in 50 mM Tris, 0.15 M NaCl, 2 mM CaC12, pH 7.4. The values determined from between six and eight replicate traces are illustrated along with error bars denoting 95% confidence limits. The lines are drawn by linear regression analysis to obtain the intrinsic second-order rate constant for the association reaction from the slope of the line. The values of k, are 8.38 +. 0.28 X 10' M-'. s-' for Xa, 8.94 +. 0.45 x lo5 M-'. s-l for Xa-PCPs, and 4.68 * 0.28 X lo6 M". s-' for Xa-Va-PCPS.

with factor Va and PCPs (Fig. 6). The formation of the Xa- PCPs binary complex had no detectable effect on the association of DEGRck with factor Xa, whereas the incorporation of Xa into prothrombinase by the addition of saturating concentrations of factor Va and PCPs resulted in a 1.8-fold decrease in the intrinsic second-order rate constant (k+' = 4.68 f 0.28 X lo5 M-'.s- ' ) for the association reaction. Cal- culations using previously published equilibrium parameters (10) indicated that at least 85% of the added factor Xa was bound to the membrane surface in the experiments conducted using Xa and PCPs in the absence of factor Va. The fact that the binding of factor Xa to the membrane surface had no effect on kl,obs eliminates the possibility that the decreased rate constant observed with prothrombinase arises from hydrodynamic considerations related to the partitioning of factor Xa to the membrane surface to which the inhibitor cannot bind (13). Therefore, the observations suggest that the interaction between factors Xa and Va on the membrane surface decreases the frequency of productive collisions between DEGRck and the active site of the protease. This decrease in productive collisions could arise from alterations in the accessibility in the active site of factor Xa resulting from changes in the orientation of the protein on the membrane surface following its interaction with factor Va or from a cofactor- induced change in the catalytic or extended substrate binding sites in the protease.

Factor Va is also a substrate for factor Xa, which catalyzes cleavages in both chains of the cofactor (58). Although the relationship between the interactions between Xa and Va which lead to cleavages in factor Va versus its function as a cofactor are unknown, it is possible that the modest reduction in k,' for the reaction of DEGRck with prothrombinase can be accounted for by inhibition resulting from the competing effects of factor Va serving as an alternate substrate for factor Xa (59). This possibility was tested by repeating the stopped- flow measurements at three concentrations of DEGRck using

The Active Site of Factor Xu 13927

different concentrations of factor Va and Xa so as to vary either the total concentration of factor Va by %fold or the free concentration of the cofactor by the same magnitude. The values of kl,obs obtained from these experiments were experimentally indistinguishable from the results presented for prothrombinase in Fig. 6. These data along with the independence of kz,obs on the presence of factor Va (Table 111) and the linear dependence of kl,,,h on the concentration of DEGRck (Fig. 6) all suggest that potential contributions of factor Va acting as an alternate substrate are experimentally negligible.

The first-order rate constant for the dissociation of the E . I binary complex (kl) can be estimated from the ordinate intercept of the data illustrated in Fig. 6 (60). However, in all three cases, estimates of the intercept were near zero with a large standard error, therefore indicating that k-l could not be determined reliably from the available data. Estimates of this rate constant were however obtained from the measured values of ki/Ki (Table I), k+l (Fig. 6), and kz (below) for the three conditions and are summarized in Table 111. Within experimental error, the first-order rate constant for the dissociation of the E .I binary complex was not influenced by the presence of PCPs and/or factor Va.

Effect of Factor Va and PCPs on the Alkylation of Factor Xu by DEGRck-The dependence of the observed rate constant for the slower second step (kz,&) on the concentration of DEGRck is illustrated in Fig. 7. Because of the ability of E.1 to dissociate (Scheme 11), the observed rate constant for the alkylation step is expected to increase saturably, to a limiting value equal to kz, with increasing I (57). The observed data (Fig. 7) are consistent with this prediction. When rapid equilibrium assumptions are met ( k+l. I >> kz and >> kz ) the dependence of k2,0bs on I is described by a rectangular hyperbola determined by kz and the equilibrium constant for the formation of E . I. However, numerical integration of the differential equations according to Scheme I1 followed by fitting to Equations 4-6 indicated that for the present kinetic parameters, kz,obs at nonsaturating concentrations of I is sig- nificantly underestimated, resulting in deviation from a rectangular hyperbola. Thus, the intrinsic alkylation rate constant ( k z ) for the modification of factor Xa was estimated from the mean of the observed rate constants determined at high concentrations of DEGRck (Fig. 7). Using values of kz,obs

TABLE 111 Influence of factor Va and PCPs on the reaction between DEGRck

and factor Xa Enzyme species" k+l k S.D.b X 106 k-l S.D.' k, k S.D.d

"1. s-1 S -1 s-1

Xa 8.38 f 0.28 4.27 f 0.79 1.67 f 0.25 Xa-PCPS 8.94 f 0.45 3.92 f 0.96 1.58 f 0.30 Xa-Va-PCPs 4.68 f 0.28 3.10 f 1.08 1.63 f 0.45 ' The rate constants were measured using 0.5 p~ Xa (Xa), 0.5 p~

Xa plus 50 pM PCPs (Xa-PCPs), and 0.5 pM Xa, 50 p~ PCPs plus 0.75 pM Va (Xa-Va-PCPS) in 20 mM Hepes, 0.15 M NaCl, 2 mM CaC12, pH 7.4, at 25 "C.

* The bimolecular association rate constants f95% confidence limit were determined from the data illustrated in Fig. 6.

The dissociation rate constant was estimated from measured values of k+l, kz, and ki/Ki (Table I), assuming Scheme 11 and using the relationship ki/Ki = (kz.k+l)/k"l. The errors in the calculated values were determined by propagation of error analysis (53).

The limiting alkylation rate constant was determined from the mean of measured values of kZ,oba obtained at concentrations of DEGRck greater than 60 p~ as illustrated in Fig. 7. The mean values are presented f 1 S.D. from n = 14 (Xa), n = 7 (Xa-PCPs), and n = 8 (Xa-Va-PCPS) concentrations of inhibitor.

2.0

1.5

1 .o t i

I o

0.0 I I 0 20 40 60 80 100 120 140

[DEGRck] pM FIG. 7. Dependence of k2,0b. on DEGRck. The observed alkyl-

ation rate constant obtained from stopped-flow experiments conducted in the presence of solution-phase factor Xa are plotted as a function of the fixed concentration of DEGRck. The line is drawn to indicate the mean of the values obtained above 60 p~ inhibitor and estimate the limiting value for kz = 1.67 f 0.25 s-I.

obtained at greater than 60 PM DEGRck yielded a limiting value for kz = 1.67 f 0.25 s-'.

The limiting values of kz obtained from stopped-flow measurements conducted at high concentrations of DEGRck following stopped-flow measurements using factor Xa in solution, the Xa-PCPs binary complex and the ternary Xa-Va- PCPs complex are presented in Table 111. For all three enzyme species, the observed alkylation rate constant was essentially identical. Thus, factor Va or the membrane surface has no effect on the covalent modification of the active-site histidine by DEGRck. Since the alkylation reaction yields a tetrahedral adduct involving both the active-site histidine and serine (36), small perturbations in the geometry or environment of the catalytic triad could have a significant influence on this modification reaction. The lack of significant effect of the cofactor on the rate constant for this step implies that factor Va does not detectably alter the catalytic residues of factor Xa.

The measured values of k+l, kz and the overall second-order rate constant for inhibition of factor Xa determined from the steady-state experiments (Table I) permit an estimation of k 1 assuming Scheme I1 and is presented in Table 111. Collec- tively, the kinetic constants demonstrate that the only detectable effects of factor Va on factor Xa are related to the binding of this transition state irreversible inhibitor to the active site of the enzyme and not to changes in the catalytic residues.

DISCUSSION

We have characterized the kinetics of the reaction between DEGRck and factor Xa by measurements of the inhibition of enzymatic activity, by fluorescence measurements of the reaction using stopped-flow techniques, and by direct measurements of the covalent modification step by rapid chemical quench techniques. The results of all three approaches are consistent with a two-step inhibition reaction (Scheme 11) involving the bimolecular association of DEGRck and factor Xa to form a dissociable binary complex followed by a first- order reaction yielding the covalently modified species. The ability to examine the rate constants separately for the binding of DEGRck to factor Xa and the alkylation of the active- site histidine has permitted the use of this model reaction system to assess changes in the catalytic residues and ex-

13928 The Active Site of Factor Xu tended substrate binding sites of factor Xa which result from its interaction with factor Va on the membrane surface.

Our findings can be summarized as follows. (i) The incorporation of factor Xa into prothrombinase results in a modest decrease in the bimolecular association rate constant for the binding of DEGRck to the active site of factor Xa. (ii) The decrease in the association rate constant is unrelated to hydrodynamic considerations arising from the binding of factor Xa to the membrane surface and therefore suggests subtle changes in the substrate recognition sites or the accessibility of these sites in the protease following its interaction with the cofactor, factor Va. (iii) The rate constant for the covalent modification step is unchanged following prothrombinase assembly, indicating that the interaction between factor Xa and Va does not lead to dramatic improvements in catalytic efficiency by detectable alterations in the catalytic residues of factor Xa.

The determined kinetic mechanism of the reaction of DEGRck with factor Xa (Scheme 11) indicates that DEGRck is a "complexing-type" irreversible inhibitor (51). Th' 1s conclusion is in good agreement with the intended mechanism of action of this fluorescent tripeptidyl chloromethyl ketone containing the Pl-P, sequence found in the physiological substrate for factor Xa (61,62). The overall second-order rate constant for the inhibition of factor Xa by DEGRck determined from steady-state kinetic measurements 3.5 X lo7 "'. min" (Fig. 1) or 2.0 X lo7 M". min" (Table I) are in excellent agreement with values reported previously from more limited measurements under slightly different conditions (62).

The mechanism of inactivation of serine proteases includ- ing thrombin by chloromethyl ketones has been established through x-ray crystallography (36, 63). Following complexa- tion, covalent modification involves the alkylation of the active-site histidine by the addition of the methylene carbon to the t nitrogen. The modified species is stabilized by a covalent bond with the active-site serine to form a tetrahedral hemiketal (36, 63). The adduct of the protease with the chloromethyl ketone closely approximates the high energy tetrahedral intermediate that occurs in the hydrolysis of pep- tidy1 or anilide substrates by the enzyme (64). Thus, the covalent modification of factor Xa by DEGRck provides a convenient tool to probe alterations in the catalytic residues which may lead to increases in the kcat for the hydrolysis of peptide bonds by an increased complementarity to the transition state or by an increased reactivity of the catalytic residues.

In the present work, the first-order rate constant for the alkylation of factor Xa by DEGRck (Scheme 11, kz) was experimentally indistinguishable from the rate constants observed in the presence of PCPs or a combination of factor Va and PCPs. Since the concentrations of PCPs and factor Va were sufficient to bind all added factor Xa to form the Xa- PCPs binary complex or the Xa-Va-PCPs ternary complex (prothrombinase), these observations suggest that the macromolecular interactions of factor Xa with other constituents of prothrombinase do not lead to dramatic changes in the catalytic residues of factor Xa. Since the incorporation of factor Xa into prothrombinase results in a 1,000-fold increase in the kcat for prothrombin activation (17), our results suggest that this increase in kcst may not result from cofactor-induced perturbations in the catalytic residues of factor Xa. This conclusion is supported by the lack of an effect of factor Va and PCPs on the kcat for S2222 hydrolysis catalyzed by factor Xa (13).

It is possible that the interaction between factors Xa and Va on the membrane surface results in significant changes in

the catalytic residues of factor Xa which lead to the large increase in kcat for the biological substrate but are inconse- quential for the covalent modification of the protease by the tripeptidyl chloromethyl ketone or for the hydrolysis of synthetic nitroanilide substrates. This possibility would imply that the transition state for the cleavage of prothrombin by factor Xa differs from that for nitroanilide hydrolysis or from the tetrahedral hemiketal formed by the reaction with hal- oketones potentially resulting from additional interactions occurring at extended subsites beyond Ss or at S' sites that are not probed by tripeptidyl reagents.

The incorporation of factor Xa into prothrombinase does yield a small but reproducible decrease in the directly determined association rate constant for the binding of DEGRck to the active site of factor Xa (Scheme 11, k+l). The observed decrease in k,, is sufficient to explain the modest decrease in the overall second-order rate constant for the inhibition reaction measured from steady-state kinetic measurements in the presence of saturating concentrations of factor Va or factor Va and PCPs (Table I). Thus, the interaction between protease and cofactor appears to perturb or alter the accessibility of the primary or extended substrate binding sites on the enzyme. These results are in excellent agreement with a 1.6-fold increase in the K,,, for S2222 hydrolysis by factor Xa observed in the presence of factor Va and PCPs (13). Changes in the K,,, were attributed to hydrodynamic effects of the partitioning of factor Xa to the membrane surface to which the synthetic substrate cannot bind (13). Since identical values of kl,oba and k+l were obtained using solution phase factor Xa or factor Xa bound to PCPs, our results suggest that the decrease in k+l observed upon prothrombinase assembly is not related to the partitioning of the enzyme to the membrane surface.

An alteration in the accessibility or orientation of the active site of factor Xa as a result of its interaction with factor Va on the membrane surface or in solution could decrease the frequency of productive collisions between DEGRck and the enzyme, leading to a decrease in the association rate constant. This possibility cannot be presently excluded and is supported by the results of energy transfer studies which indicate that the spatial relationship between the active site of factor Xa and the membrane surface is altered in the presence of factor Va (21).

Changes in the association rate constant could also reflect altered interactions between DEGRck and one or more extended substrate binding sites on factor Xa and may be responsible for the fluorescence changes observed in DEGR- Xa following the addition of factor Va and PCPs (10, 21). This interpretation would suggest that the interaction between factor Xa and factor Va results in an allosteric pertur- bation in the extended binding sites for the substrate which is manifest as a small decrease in the rate constant and affinity for the binding of DEGRck to the active site. Such changes in the binding of the macromolecular substrate to extended recognition sites on the enzyme could lead to the utilization of binding energies to improve catalysis, thereby resulting in an increased keat (57).

In summary, studies of the reaction of factor Xa with the irreversible transition state inhibitor DEGRck have provided no evidence for a change in the catalytic residues of factor Xa resulting from the incorporation of the protease into the prothrombinase complex. The interaction of factor Xa with factor Va does however cause subtle alterations in the binding of the peptidyl inhibitor to the active site of the enzyme. Thus, the large increase in the kcat for prothrombin activation which accompanies the interaction of factor Va with factor

The Active Site of Factor X u 13929

Xa may not result from allosteric changes in the catalytic residues of the enzyme but rather from an effect of the cofactor on the extended substrate binding sites on the protease and/or by binding the substrate leading to the stabili- zation of a structure about the scissile bond in prothrombin which more closely approximates the transition state.

Acknowledgments-We acknowledge gratefully the expert assistance provided by Drs. Robert Kelm and Xavier Villareal of the Protein Microchemical Facility, Department of Biochemistry, Uni- versity of Vermont and by Dr. Jan Pohl, Microchemical Facility, Emory University for amino acid analyses and peptide sequencing. We are grateful to Dr. John Lee, Department of Biochemistry, University of Georgia, for assistance in the lifetime measurements and for the gracious use of his fluorescence lifetime instrumentation. We thank Dr. Pete Lollar for useful discussions and critical com- ments.

REFERENCES 1. Walker, R. K., and Krishnaswamy, S. (1992) Circulation 86,1-466 (abstr.) 2. Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27,

3. Jackson, C. M., and Nemerson, Y. (1980) Annu. Rev. Biochem. 49, 765-

4. Mann, K. G., Jenny, R. J., and Krishnaswamy, S. (1988) Annu. Reu.

5. Mann, K. G., Nesheim, M. E., Church, W. R., Haley, P., and Krishnaswamy,

6. Nesheim, M. E., Taswell, J. B., and Mann, K. G. (1979) J. Biol. Chem.

7. Mann, K. G. (1987) Trends Biochem. Sci. 12,229-233 8. Nesheim. M. E.. Kettner. C.. Shaw. E.. and Mann. K. G. (1981) J. Biol.

157-162

811

Biochem. 67,915-956

S. (1990) Blood 76, l -16

254,10952-10962

10. 9.

11.

12. 13.

14.

15.

16. Carlisle, T. L., Bock, P. E., and Jackson, C. M. (1990) J. Biol. Chem. 265,

17. Rosing, J., Tans, G., Govers Riemslag, J. W., Zwaal, R. F., and Hemker, H.

18. van Rijn, J. L., Govers Riemslag, J. W., Zwaal, R. F., and Rosing, J. (1984)

19. Esmon, C. T., Owen, W. G., and Jackson, C. M. (1974) J. Biol. Chem. 249,

20. Higgins, D. L., Callahan, P. J., Prendergast, F. G., Nesheim, M. E., and

21. Husten, E. J.. Esmon, C. T., and Johnson, A. E. (1987) J. Biol. Chem. 262,

22. 23.

266; 1379-1382

22044-22055

C. (1980) J. Biol. Chem. 256,274-283

Biochemistry 23,4557-4564

1190-1807

Mann, K. G. (1985) J. Biol. Chem. 260,3604-3612

Chem. '256,65374540' '

Krishnaswamy, S. (1990) J. Biol. Chem. 266,3708-3718 Krishnaswamy, S., Jones, K. C., and Mann, K. G. (1988) J. Biol. Chem.

Boskovic, D. S., Giles, A. R., and Nesheim, M. E. (1990) J. Biol. Chem.

Pryzdial, E. L. G., and Mann, K. G. (1991) J. Biol. Chem. 266,8969-8977 Nesheim, M. E., Eid, S., and Mann, K. G. (1981) J. Biol. Chem. 266,9874-

Nesheim, M. E., Tracy, R. P., and Mann, K. G. (1984) J. Biol. Chem. 259,

Giesen, P. L. A.. Willems. G. M., and Hermens, W. T. (1991) J. Biol. Chem.

263,3823-3834

266,10497-10505

9882

1447-1453

12953-12962 Esmon, C. T., and Jackson, C. M. (1974) J. Biol. Chem. 249,7791-7797 Luckow, E. A,, Lyons, D. A., Ridgeway, T. M., Esmon, C. T., and Laue, T. M. (1989) Biochemistry 28,2348-2354

24. Armstrong, S. A., Husten, E. J., Esmon, C. T., and Johnson, A. E. (1990)

25. Nesheim, M. E., and Mann, K. G. (1983) J. Biol. Chem. 268,5386-5391 26. Krishnaswamy, S., Church, W. R., Nesheim, M. E., and Mann, K. G. (1987)

27. Ye. J.. Llu. L. W.. Esmon. C. T.. and Johnson. A. E. (1992) J. Biol. Chem.

J. Biol. Chem. 266,6210-6218

J. Biol. Chem. 262,3291-3299 ~I

267, 11023-11028 . .

28. Hofsteenge, J., Taguchi, H., and Stone, S. R. (1986) Biochem. J. 237,243-

29. Jakubowski, H. V., Kline, M. D., and Owen, W. G. (1986) J. Biol. Chem.

30. Ye J Esmon N. L., Esmon, C. T., and Johnson, A. E. (1991) J. Biol. 261,3876-3882

31. Lu, R., Esmon, N. L., Esmon, C. T., and Johnson, A. E. (1989) J. Biol. b$m. 266,'23016-23021

32. Pedersen, A. H. Nordfan , 0 , Norris, F., Wiber F. C., Christensen, P. Chem. 264,12956-12962

U., and Kisiel, W. (1990) J. Biol. Chem. 266, 16786-16793 M., Moeller, K. B., Mei&hl Pedersen, J., Beck, 8 C., Norris, K., Hedner,

33. Ruf, W., Rehemtulla, A., Edgington, T. S., and Morrissey, J. H. (1991) J. Biol. Chem. 266,2158-2166

34. Lawson, J. H., Butenas, S., and Mann, K. G. (1992) J. Biol. Chem. 267, 4834-4843

35. Krishnaswamy S. (1992) J. Biol. Chem. 267,23696-23706 36. Poulos, T. L., Alden, R. A., Freer, S. T., Birktoft, J. J., and Kraut, J. (1976)

37. Higgins, D. L., and Mann, K. G. (1983) J. Biol. Chem. 268,6503-6608 38. Gomori, G. (1942) J. Lab. Clin. Med. 27,955-960 39. Krishnaswamv. S.. Mann. K. G.. and Nesheim. M. E. (1986) J. Biol. Chem.

LO1

J. Biol. Chem. 261,1097-1103

261,8977-8984 . ,

40. Jesty, J., and Nemerson, Y. (1976) Methods Enzymol. 46,95-107 41. Krishnaswamy, S. and Mann K. G. (1988) J. Biol. Chem. 263,5714-5723

43. Laue. T. M.. Johnson. A. E.. Esmon. C. T.. and Yuhantis. D. A. (1984) 42. Laemmli, U. K. (y970) Naturi 227,680-685

Biichemistry 23,1339-1348 . ~I

44. Jackson, C. M., Johnson, T. F., and Hanahan, D. J. (1968) Biochemistry 7 , AAO9-AFin5

45. Fujikawa, K., Coan, M. H., Legaz, M. E., and Davie, E. W. (1974) Biochem-

46. Chase, T., Jr., and Shaw, E. (1967) Methods Enzymol. 19,20-27 47. Lackowicz, J. R. (1983) Principles of Fluorescence Spectroscopy, Plenum

48. Lee, J., O'Kane, D. J., and Gibson, B. G. (1989) Biophys. Chem. 33, 99-

49. Lee, J., O'Kane, D. J., and Gibson, B. G. (1989) Biochemistry 28, 4263-

50. Liu, W. and Tsou C. L. (1986) Biochim. Biophys. Acta 870,185-190 51. Tsou, d. L. (1988)'Adu. Enzymol. 61, 381-436 52. Leytus, S. P., Toledo, D. L., and Mangel, W. F. (1984) Biochim. Biophys.

53. Bevington, P. R. (1969) Data Reduction and Error Analysis in the Physical

54. Straume M., and Johnson, M. L. (1992) Methods Enzymol. 210,87-105 55. Moore. j. W.. and Pearson, R. G. (1981) Kinetics and Mechanism:A Studv

.."- """ lstry 13,5290-5299

Press, New York

111

4271

Acta 788, 74-86

Sciences, McGraw-Hill, New York

of Eiomogei~ow Chemical Reactions, pp. 290-300, John Wiley and Sons, New York

56. Cornish-Bowden. A. (1979) Fundamentals of Enzvme Kinetics. DD. 177-179. ButterworthsyLondon '

, " 1.1

57. Jencks, W. P. (1987) Catalysis in Chemistry and Enzymology, pp. 571-576,

58. Odegaard, B., and Mann, K. G. (1987) J. Biol. Chem. 262,11233-11238 59. Seeel. I. H. (1975) Enzvme Kznetm: Behavzor and Analvsls of Raoid

Dover Publications Inc., New York

Equilibriumand Sted$-state Enzyme Systems, pp. 113-lf8, Jo6n Wiley &Sons, New York

"

60. Fersht, A. (1977) Enzyme Structure and Mechanism, pp. 111-112, W. H. Freeman and Co San Francisco

61. Kettner C., and Sdaw E. (1981) Thromb. Res. 22,645-652 62. Kettner: C., and Shawl E. (1981) Methods Enzymol. 80,826-842 63. Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S. R., and Hofsteenge,

64. Powers, J. C. (1977) Methods Enzymol. 46, 197-208 J. (1989) EMBO J. 8,3467-3475

Date post:	04-Feb-2017
Category:	Documents
Upload:	trannguyet
View:	218 times
Download:	0 times

The Influence of Factor Va on the Active Site of Factor Xa*

Documents