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Originally published In Press as doi:10.1074/jbc.M402971200 on April 16, 2004

J. Biol. Chem., Vol. 279, Issue 28, 29485-29492, July 9, 2004
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Structural Role of Gly193 in Serine Proteases

INVESTIGATIONS OF A G555E (GLY193 IN CHYMOTRYPSIN) MUTANT OF BLOOD COAGULATION FACTOR XI*

Amy E. Schmidt{ddagger}§, Taketoshi Ogawa||, David Gailani||, and S. Paul Bajaj{ddagger}§**

From the {ddagger}UCLA/Orthopaedic Hospital, Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, Los Angeles, California 90095, the §Department of Pharmacological & Physiological Sciences, St. Louis University School of Medicine, St. Louis, Missouri 63104, and the ||Department of Pathology, Vanderbilt University, Nashville, Tennessee 37232

Received for publication, March 17, 2004 , and in revised form, April 16, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
In serine proteases, Gly193 is highly conserved with few exceptions. A patient with inherited deficiency of the coagulation serine protease factor XI (FXI) was reported to be homozygous for a Gly555 -> Glu substitution. Gly555 in FXI corresponds to Gly193 in chymotrypsin, which is the numbering system used subsequently. To investigate the abnormality in FXIG193E, we expressed and purified recombinant FXIaG193E, activated it to FXIaG193E, and compared its activity to wild type-activated FXI (FXIaWT). FXIaG193E activated FIX with ~300-fold reduced kcat and similar Km, and hydrolyzed synthetic substrate with ~10-fold reduced Km and modestly reduced kcat. Binding of antithrombin and the amyloid {beta}-precursor protein Kunitz domain inhibitor (APPI) to FXIaG193E was impaired ~8,000- and ~100,000-fold, respectively. FXIaG193E inhibition by diisopropyl fluoro-phosphate was ~30-fold slower and affinity for p-aminobenzamidine (S1 site probe) was 6-fold weaker than for FXIaWT. The rate of carbamylation of NH2-Ile16, which forms a salt bridge with Asp194 in active serine proteases, was 4-fold faster for FXIaG193E. These data indicate that the unoccupied active site of FXIaG193E is incompletely formed, and the amide N of Glu193 may not point toward the oxyanion hole. Inclusion of saturating amounts of p-aminobenzamidine resulted in comparable rates of carbamylation for FXIaWT and FXIaG193E, suggesting that the occupied active site has near normal conformation. Thus, binding of small synthetic substrates or inhibitors provides sufficient energy to allow the amide N of Glu193 to point correctly toward the oxyanion hole. Homology modeling also indicates that the inability of FXIaG193E to bind antithrombin/APPI or activate FIX is caused, in part, by impaired accessibility of the S2' site because of a steric clash with Glu193. Such arguments will apply to other serine proteases with substitutions of Gly193 with a non-glycine residue.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Amino acids Ser195, His57, and Asp102 (chymotrypsin numbering system) form a catalytic triad, which is integral to the catalytic activity of all serine proteases (1, 2). These residues are located at the entrance to the substrate binding pocket, and their geometry is stabilized by hydrogen bonds. Serine proteases hydrolyze peptide bonds via the formation of tetrahedral transition state intermediates. Stabilization of the transition state intermediate occurs through formation of hydrogen bonds between the oxyanion intermediate and the amido groups of residues Gly193 and Ser195. The substrate binding sites in the enzyme involved in precise interactions are referred to as Sn,... S3, S2, S1, S1', S2', S3',... Sn' sites, and the amino acid residues of the substrate or inhibitor that occupy these sites are referred to as Pn,... P3, P2, P1, P1', P2', P3',... Pn', respectively. These complementary sites permit specific alignment of the substrate/inhibitor with the catalytic triad and the oxyanion hole for enzymatic specificity (3). In trypsin-like serine proteases, Asp189 is at the bottom of the primary S1 substrate binding site, and forms a salt bridge with the guanidino group of P1 Arg residues in the substrate/inhibitor.

Gly193, which is part of the oxyanion hole structural unit, is highly conserved in serine proteases with only a few exceptions (see "Discussion"). Several blood coagulation proteins with mutations at Gly193 (chymotrypsin equivalent) in their protease domains have been reported. These include FXI1 (Gly555 -> Glu), FIX (Gly363 -> Ala, Arg, Glu, or Val), and FVII (Gly342 -> Glu or Arg) (4-11). Each patient had normal plasma antigen levels associated with very low coagulant activity. In two cases (FXIG193E [555]2 and FIXG193V [363]) the protein was activated normally (4, 9). These data indicate that the functional abnormalities in these proteins stem from their inability to interact with their biological macromolecular substrate/inhibitors. This has been demonstrated for FIXaG193V (9).

FXI is a disulfide-linked homodimer with a molecular weight of ~160,000 (12). Deficiency of FXI results in a bleeding diathesis sometimes referred to as hemophilia C, and is most common in the Ashkenazi Jewish population (13, 14). FXI can be activated to FXIa by FXIIa, thrombin, or by autoactivation (15, 16). Upon cleavage of the Arg15-Ile16 [Arg369-Ile370] peptide bond, a heavy chain and a light chain are formed that are held together by a disulfide bond (12, 17). Thus, FXIa contains two heavy chains and two light chains. Each heavy chain contains four Apple domains, and each light chain a serine protease domain containing the catalytic triad His57 [413], Asp102 [464], and Ser195 [557] (18). The new NH2 terminus of the light chain contains the sequence Ile16-Val17-Gly18 [370-372]. The NH2-terminal Ile16 [370], which is characteristic of serine proteases, inserts into the protease domain of FXIa, and its NH2 group forms a salt bridge with the COOH group of Asp194 [556]. This salt bridge is a defining feature of active serine protease formation (19). FXIa contributes to coagulation by activating FIX to FIXa in a Ca2+-dependent manner (20, 21). The activation of FIX by FXIa has been shown to involve the Apple 2, Apple 3, and protease domains in FXIa and the activation peptide region and {gamma}-carboxyglutamic acid domains of FIX (22-25).

In this report, we describe a series of experiments using physiologic macromolecular and small synthetic substrate/inhibitors to discern the nature of the proteolytic defect in a naturally occurring FXI mutant with a Glu substitution for Gly193 [555]. The data on the rate of carbamylation of the NH2 group of Ile16, which serves as an index of salt bridge formation with Asp194, as well as p-aminobenzamidine (pAB) binding and the rate of diisopropyl fluorophosphate (DFP) incorporation, strongly indicate that the S1 binding site and oxyanion hole are incompletely formed in the mutant enzyme. Binding of small synthetic substrates, however weak, provides sufficient energy to reorient the amido group of Glu193 and restore the proper conformation of the oxyanion hole. Modeling efforts indicate that impairment of the interaction with physiologic macromolecular substrate/inhibitors is, in part, because of inaccessibility of the S2' site, which is attributable to a steric clash with Glu193 [555].


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Reagents—H-D-Ile-Pro-Arg-p-nitroanilide (S-2288) and pyro-Glu-Pro-Arg-p-nitoanilide (S-2366) were purchased from DiaPharma (West Chester, OH). Sodium boro[3H]hydride was obtained from PerkinElmer LAS, Inc. Enhanced chemiluminescence (ECL) detection reagents were purchased from GE Healthcare. DFP was purchased from Calbiochem. Activated partial thromboplastin reagent was purchased from Beckman, and normal plasma was bought from George King (Overland Park, KS). Unfractionated heparin was purchased from Pharmacia Hepar, Inc. (Franklin, OH). Fatty acid-free bovine serum albumin (BSA), pAB, PEG 8000, Polybrene, and all other chemicals of the highest grade available were obtained from Sigma.

Proteins—Corn trypsin inhibitor, human FXIIa, antithrombin (AT), and human FIX were purchased from Enzyme Research Laboratories (South Bend, IN). The Kunitz domain of the protease nexin-2/amyloid {beta} protein precursor Kunitz protein inhibitor (APPI) was a gift from Dr. A. H. Schmaier (University of Michigan, Ann Arbor, MI).

SDS-Gel Electrophoresis—SDS-gel electrophoresis was done using the Laemmli buffer system (26). The acrylamide concentration used was 12% and the gels were stained with GelCode blue stain (Pierce).

Expression and Purification of Recombinant FXI Proteins—A Gly to Ala substitution was introduced into the human FXI cDNA (kindly provided by Dr. Dominic Chung, University of Washington, Seattle, WA) at base pair 1761 by site-directed mutagenesis. This substitution replaces the glycine residue normally found at amino acid position 555 (193 in chymotrypsin numbering) with glutamic acid. The cDNAs for wild type FXI and FXIG193E were ligated into mammalian expression vector pJVCMV, which contains a cytomegalovirus promoter, as previously described (24). 293 fetal kidney fibroblasts (50 x 106, ATCC CRL 1573) were cotransfected by electroporation (Electrocell Manipulator 600 BTX) with 40 µg of pJVCMV-fXI-E555 construct and 2 µg of plasmid RSVneo that contains a gene conferring neomycin resistance. Cells were grown in Dulbecco's modified Eagle's medium with 5% fetal bovine serum and 500 µg/ml G418. G418 resistant clones were transferred to 96-well plates. Culture supernatants were tested for protein expression by enzyme-linked immunosorbent assay using goat anti-human FXI polyclonal antibodies (Affinity Biologicals, Hamilton, Ontario, Canada). Expressing clones were expanded in 175-cm2 flasks using Cellgro Complete media (Mediatech, Herndon, VA). Conditioned media was removed every 48-72 h, supplemented with benzamidine (5 mM), and stored at -20 °C pending purification.

Proteins were purified by monoclonal antibody affinity chromatography using anti-FXI IgG 1G5.1. Conditioned media (1 liter) was applied to the column, followed by washing with 25 mM Tris-HCl, pH 7.5, 100 mM NaCl (Tris/NaCl) with 5 mM benzamidine. Protein was eluted with Tris/NaCl containing 2.0 M sodium thiocyanate. Protein containing fractions were pooled and concentrated in an Amicon concentrator, dialyzed against Tris/NaCl, and stored at -80 °C. Protein concentration was determined by the dye binding assay (Bio-Rad), and purity was assessed by SDS-PAGE. Purified proteins were homogenous on SDS-PAGE and had the correct molecular weight of a disulfide-linked homodimer. To prepare FXIaWT and FXIaG193E recombinant protein (~300 µg/ml) was incubated with 5 µg/ml FXIIa at 37 °C. Complete conversion of the zymogen to the heavy and light chains of FXIa was confirmed by reducing SDS-PAGE. The FXIIa in each activation reaction was inactivated by incubation with a 20-fold molar excess of corn trypsin inhibitor. The reactions were tested for residual FXIIa activity by monitoring the loss in activity following addition of corn trypsin inhibitor using a chromogenic assay.

[sialyl-3H]Factor IX—3H-Labeled FIX was prepared as described previously (28). The specific activity of the [sialyl-3H]FIX was 2.1 x 108 cpm/mg. The labeled preparation has 85% of the biological activity of the nonlabeled control as measured in an activated partial thromboplastin time assay (28). It showed a single band corresponding to FIX in both nonreduced and reduced SDS-PAGE (26).

Kinetics of Factor IX Activation—The rate of FIX activation was measured by quantifying the amount of radioactive peptide released at various times of incubation with FXIa. The procedure used was that described previously (28). Each reaction was carried out in TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.5 mg/ml BSA and 5 mM Ca2+ at 37 °C. The concentration of FIX was varied from 2.0 to 25 µg/ml. The concentrations of FXIaWT and FXIaG193E used were 20 ng/ml and 2.0 µg/ml, respectively. Each reaction was started with the addition of FXIa, and at various times, 100-µl aliquots were removed and added to 100 µl of cold stopping buffer consisting of TBS, 50 mM benzamidine, 50 mM EDTA, and 5 mg/ml BSA. An equal volume (200 µl) of 6% trichloroacetic acid was added and the reaction was centrifuged to precipitate the FIX/FIXa and FXIa proteins. One hundred-µl aliquots of the supernatant were then removed and added to 4 ml of Aquasol 2 and counted for tritium in a Beckman LS 5000CE {beta}-counter. Control experiments were performed in the absence of FXIa and were found to give a maximum of 1% of the total counts in the supernatant. These background counts were subtracted from each sample count prior to calculation of the initial FIXa activation rates. The amount of FIXa formed at a given time was obtained by averaging the results from three experiments and, the rates of activation were determined by least squares fitting of the initial data points to a straight line. Complete activation of FIX resulted in ~35% of the total counts in the trichloroacetic acid supernatant. Rates of activation were then plotted versus FIX concentration. The Km and kcat values were obtained using the Enzyme Kinetics program from Erithacus Software.

Measurement of S-2288 and S-2366 Amidolytic Activity of FXIaWT and FXIaG193E—Each reaction contained TBS with 5 mM Ca2+, 100 µg/ml BSA, 0.5-1 nM FXIaWT, or 2 nM FXIaG193E (the results were normalized to 2 nM FXIa protein), and increasing amounts of either S-2288 or S-2366. The rate of release of pNA was measured using a Beckman DU800 spectrophotometer with a Kinetics module at 405 nm for 15 min. An extinction coefficient of 9.9 mM-1 cm-1 at 405 nM was used in calculating the amount of pNA released (29). The initial rate, which was linear, was then converted to micromolars substrate hydrolyzed per min. The program GraFit was used to determine the Km and Vmax using the Enzyme Kinetics Program from Erthicaus Software.

Inhibition of FXIaWT and FXIaG193E by AT in the Presence of Heparin—All reactions were carried out in 25 mM Tris-HCl, 100 mM NaCl, pH 7.4, plus 0.1% Tween 20 (Tris/NaCl/T) at 37 °C. Recombinant FXIa (6 nM) was incubated with AT (30 nM to 3 µM) in the presence of 1 unit/ml heparin. At various times, 20-µl samples were removed and mixed with 80 µl of Tris/NaCl/T containing 500 µM S-2288 and 5 µg/ml Polybrene (to dissociate the FXIa from heparin) in 96-well microtiter plates. The change in absorbance at 405 nm was measured in a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, CA), and the residual FXIa activity was determined at each time point. The residual activity was then plotted as a percent of initial activity and the first-order rate constants, kobs, for each concentration of AT used were obtained using the following equation,

(Eq. 1)

where At and A0 are the percent of FXIa activity at time t and 0 s, respectively. The values of kobs were then plotted against the AT concentration to obtain second-order rate constants.

Interaction of FXIaG193E and FXIaWT with APPI—These reactions were carried out using microtiter well plates from Dynatech. For experiments with FXIaWT, each reaction (150 µl) contained 0.5 nM FXIaWT, 100 µM S-2288, and increasing amounts of APPI in TBS with 5 mM CaCl2 containing 100 µg/ml BSA. For experiments with FXIaG193E, each reaction (150 µl) contained 1 nM FXIaG193E, 500 µM S-2288, and increasing amounts of APPI in TBS with 5 mM CaCl2 containing 100 µg/ml BSA. Absorbance at 405 nM (pNA release) was measured for up to 4 h in a Bio-Rad model 550 microtiter plate reader operated by Bio-Rad Labs microtiter manager PC software. The data were analyzed based upon the slow tight binding mechanism established for FXIa·APPI as follows,

(Eq. 2)

where FXIa·APPI is the steady state complex, which then isomerizes to XIa·APPI*. The forward and reverse rates for the initial binding are k1 and k2, and k3 and k4 are the forward and reverse rates for the isomerization step. To calculate the binding parameters, the initial (vin) and steady state (vst) velocities were calculated using Equation 3, as described by Morrison and Walsh (30),

(Eq. 3)

where [P] represents the concentration of pNA formed at time t, vin and vst are, respectively, the rates of substrate hydrolysis before and after the steady state is achieved, and kobs is the rate of conversion of vin to vst. For these reactions, APPI was in vast excess to FXIa and the free APPI concentration did not change significantly during the course of the reaction. Substrate depletion also did not contribute to vst, as rates of control experiments done in the absence of APPI did not change significantly over time.

The values of kobs obtained were fitted to a single ligand binding site with a defined background using the following equation to obtain Kd(app),

(Eq. 4)

where the y intercept yields k4, the plateau value of kobs yields (k3 + k4), and the midpoint of the curve yields Ki(app), which represents k2/k1 (31).

The value of Ki (k2/k1) was obtained from Ki(app) using,

(Eq. 5)

where [S] is the S-2288 concentration. The Km values obtained with S-2288 were used to obtain Ki. The overall Ki, designated Ki*, was derived using the following equation (30).

(Eq. 6)

Determination of KdpAB of Binding of pAB to FXIaWT and FXIaG193E—The Kd(app) of binding of pAB to FXIaG193E or FXIaWT was determined by its ability to competitively inhibit S-2288 hydrolysis. Details are provided in the legend to Fig. 2. The IC50 (concentration of pAB required for 50% inhibition) was determined by fitting the data to the IC50 four-parameter logistic equation of Halfman (32) given below.

(Eq. 7)



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  		FIG. 2.
Binding of pAB to FXIaWT and FXIaG193E. Each reaction was carried out in TBS with 5 mM Ca2+, 100 µg/ml BSA, and either 100 µM S-2288 (for FXIaWT) or 500 µM S-2288 (for FXIaG193E). Increasing amounts of pAB were added to each mixture, and the reactions were initiated by addition of either 1 nM FXIaWT or 2 nM FXIaG193E. The initial rates of pNA release were measured, converted to micromolar substrate hydrolyzed per min, and the percent activity plotted as a function of pAB concentration. Open circles, FXIaWT; closed circles, FXIaG193E.

 
Where y is the rate of pNA release in the presence of a given concentration of pAB represented by x, a is the maximum rate of pNA release in the absence of pAB, and s is the slope factor. Each point was weighted equally and the data were fitted to Equation 7 using the nonlinear regression analysis program GraFit from Erithcus Software. To obtain Kd(pAB) values for the interaction of pAB with FXIaG193E and FXIaWT, we used the following equation described by Cheng and Prusoff (33) and further elaborated by Craig (34)

(Eq. 8)

where [S] is the S-2288 concentration. The Km values obtained for each protein using S-2288 (see Table I) were used to obtain Kd(ppAB).


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  		TABLE I
Kinetic parameters of FXIaWT and FXIaG193E synthetic substrate hydrolysis

Increasing amounts of the substrates S-2366 or S-2288 were added to reactions containing 0.5-2 nM FXIaG193E or FXIaWT in TBS, pH 7.5, containing 100 µg/ml BSA and 5 mM CaCl2. The rates of pNA release were measured as described under "Experimental Procedures." The data presented are the averages of three experiments.

 
Inhibition of FXIaWT and FXIaG193E by DFP—Each reaction mixture contained 250 nM FXIaWT or FXIaG193E in TBS with 5 mM CaCl2 and 100 µg/ml BSA. Increasing amounts of DFP (2 µM to 4 mM) were added to each reaction and incubated at room temperature for various times. At each time point, 5-µl aliquots of each reaction were removed and added to 155 µl of TBS/BSA containing 625 µM S-2288. The change in absorbance at 405 nm was measured in a SpectraMax 190 microplate reader (Molecular Devices) and the residual FXIa activity was determined at each time point. The residual activity was then plotted as a percent of initial activity and the first-order rate constants, kobs, for each concentration of DFP used were obtained using Equation 1, where At and A0 are the percent FXIa activity at time t and 0 s, respectively. The values of kobs were then plotted against the DFP concentration to obtain second-order rate constants.

Carbamylation of Ile16 in FXIaG193E and FXIaWT by Reaction with NaNCO—These experiments were performed as described by Camire (34). Briefly, each reaction mixture contained 1 µM FXIa mutant or normal protein in 20 mM Hepes, 0.15 M NaCl, 0.1% PEG 8000, 2 mM CaCl2, pH 7.5 (HBSP). Each experiment was performed in the absence and presence of pAB (10x the KD pAB) and each reaction was started by the addition of 0.2 M NaNCO. The final pH after the addition of NaNCO was 7.5. Every 30 min, a 5-µl aliquot was removed and added to 145 µl of HBSP containing 500 µM S-2288. The residual activity was determined from the initial linear rates of hydrolysis using a Beckman DU 800 spectrophotometer. The residual activity was plotted as a percent of initial activity and kobs for carbamylation were determined using Equation 1.

Molecular Modeling—The three-dimensional structure information of the zymogen and activated serine protease domains of FXIWT and FXIG193E as well as the complexes of APPI and the serine protease domain of FXIa were derived using software from Biosym/MSI (San Diego, CA) and the Swiss-Model server using the optimize mode (36-38). The crystallographically determined structures of chymotrypsinogen (Ref. 39, Protein Data Bank code 2cga [PDB] , and Ref. 40, Protein Data Bank code 1ex3 [PDB] ) and trypsinogen (Ref. 41, Protein Data Bank codes 2tga [PDB] , 1tgc [PDB] , and 1tgt [PDB] ) were used to model the serine protease domain of zymogen FXIWT and FXIG193E. Although FXI resembles trypsinogen, trypsinogen displays considerable disorder in several parts of the polypeptide chain including the region that contains residue 193 [555]. Instead, this region was modeled based upon chymotrypsin where this region around Gly193 is well ordered. The crystallographically determined structures of enteropeptidase (Ref. 42, Protein Data Bank code 1ekb [PDB] ), {alpha}1-tryptase (Ref. 43, Protein Data Bank code 1lto [PDB] ), {alpha}-chymotrypsin (Ref. 44, Protein Data Bank code 6cha [PDB] ), and {beta}-trypsin (Ref. 45, Protein Data Bank code 3bth [PDB] ) were used to model the serine protease domains of FXIaWT and FXIaG193E. APPI alone (Ref. 46, Protein Data Bank code 1aap [PDB] ), bovine chymotrypsin inhibited with APPI (Ref. 47; Protein Data Bank code 1ca0 [PDB] ), and bovine trypsin inhibited with APPI (Ref. 47; Protein Data Bank code 1taw [PDB] ) served as templates in building models of the serine protease domain of FXIaWT and FXIaG193E with APPI. Bulk solvent is excluded from the protease-inhibitor complex, thus, it is anticipated that hydrogen bonds and ionic interactions can be accurately evaluated and play an important role in specificity. The relative positions of the inhibitor and protease domains were maintained, and adjustments were only made to the side chains. Hydrophobic/van der Waals, hydrogen bonds, and ionic interactions were observed between each protease-inhibitor complex. These interactions were taken into consideration in evaluating each protease-inhibitor complex, and it was assumed that all potential hydrogen bond donors and acceptors would participate in these interactions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
FXIaWT and FXIaG193E Amidolytic Activity toward Small Synthetic Substrates—We initially studied the effects of the Glu193 substitution on protease catalytic activity using the tripeptide chromogenic substrates S-2366 and S-2288. These data are presented in Table I. The catalytic efficiency of FXIaG193E was ~18.5-fold lower (~12-fold increase in Km and ~1.5-fold decrease in kcat) for S-2366 and ~11.5-fold lower (~7-fold increase in Km and ~1.7-fold decrease in kcat) for S-2288. These data indicate that the active site is impaired in FXIaG193E.

FIX Activation by FXIaWT and FXIaG193E—During physiologic coagulation, the macromolecular substrate for FXIa is FIX. We examined activation of FIX by FXIWT and FXIG193E. These data are presented in Fig. 1. The values for Km were similar for the two proteases (FXIaWT, 145 nM and FXIaG193E, 170 nM); however, kcat was ~300-fold slower for activation by FXIaG193E. The Km data are consistent with previous observations indicating that exosites distant from the FXIa protease domain, such as the Apple 2 and Apple 3 domains, are important in binding to FIX (22-25). One should note that the kcat value in this situation is influenced by the interaction of the activation peptide cleavage sites of FIX with the active site of FXIa, as well as by catalysis of the peptide bond by FXIa. Thus, the ~300-fold difference in kcat for WT and mutant FXIa represents a cumulative effect of restricted binding and catalysis at the active site. The ~15-fold average reduction in catalytic efficiency for synthetic substrates (Table I) versus ~300-fold reduction for FIX (Fig. 1) could be accounted for by misalignment at the S2'/P2' binding sites in FIX and FXIaG193E (see "Discussion").



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  		FIG. 1.
FIX activation by FXIaWT and FXIaG193E. A, FXIaWT; B, FXIaG193E. The rate of FIX activation was determined by measuring the amount of radioactive peptide released at various times of incubation with FXIa. Each reaction was carried out in TBS containing 0.5 mg/ml BSA and 5 mM Ca2+ at 37 °C. The concentration of FIX was varied from 2.0 to 25 µg/ml. The concentrations of FXIaWT and FXIaG193E used were 20 ng/ml and 2.0 µg/ml, respectively.

 
FXIaG193E and FXIaWT Inhibition by AT and APPI—AT and APPI are two known physiologic inhibitors of FXIa. AT belongs to the serpin family of inhibitors and APPI to the Kunitz family of inhibitors (similar to bovine pancreatic trypsin inhibitor). In contrast to small synthetic substrates and the activation peptide cleavage sites in FIX, AT and APPI are likely to make extensive contacts with the protease domain of FXIa. For this reason, we hypothesized that binding of these inhibitors to FXIaG193V may be severely impaired in comparison to synthetic substrate hydrolysis and FIX activation. The second-order rate constant, k, for binding of AT to FXIaWT was 6.1 µM-1 s-1 and to FXIaG193V was 8 x 10-4 µM-1 s-1. These data indicate that binding of AT to FXIaG193V is ~8,000-fold weaker than binding to FXIaWT.

Binding data for APPI inhibition of FXIaWT and FXIaG193E are presented in Table II. The initial rapid equilibrium binding (Ki) of APPI to the mutant enzyme was impaired ~6000-fold. Furthermore, the isomerization step (k3) that leads to the tight binding complex was also impaired ~20-fold. However, dissociation of the tightly bound complex was equivalent for both enzymes. Thus, from the AT and APPI binding data, one may conclude that FXIaG193V may not be locked into an active enzyme conformation. Instead it may exist in an equilibrium state that fluctuates between the active and zymogen forms of the enzyme. The experiments described in the following sections were designed to test this concept.


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  		TABLE II
APPI Inhibition of FIXaWT and FXIaG193E

For experiments with FXIaWT, each reaction (150 µl) contained 0.5 nM FXIaWT, 100 µM S-2288, and increasing amounts of APPI in TBS with 5 mM CaCl2 containing 100 µg/ml BSA. For experiments with FXIaG193E, each reaction (150 µl) contained 1 nM FXIaG193E, 500 µM S-2288, and increasing amounts of APPI in TBS with 5 mM CaCl2 containing 100 µg/ml BSA. The pNA release was measured for up to 4 h, and the data were analyzed as described under "Experimental Procedures."

 
Binding of pAB to FXIaG193E and FXIaWT—The inhibitor pAB is known to bind to the S1 site of serine proteases, and was employed to investigate the integrity of the S1 site in FXIaG193E. The data are presented in Fig. 2. Notably, FXIaG193E bound to pAB with 6-fold weaker affinity (Ki = 126 µM versus 21 µM) than to FXIaWT. Thus, the S1 site is impaired in FXIaG193E, in agreement with the synthetic substrate hydrolysis data presented in Table I.

Inhibition of FXIaG193E and FXIaWT by DFP—One of the underlying features of serine proteases is the presence of the oxyanion hole, which develops upon conversion of the zymogen to the enzyme form. DFP specifically reacts with Ser195 and contains an oxyanion that enables it to be used as a probe to test the integrity of the oxyanion hole (48). As shown in Fig. 3, FXIaG193E inhibition by DFP was ~30-fold slower when compared with inhibition of FXIaWT (20 M-1 min-1 versus 610 M-1 min-1, respectively). This demonstrates that the oxyanion hole in the mutant enzyme is not properly formed, and that the amide N of Glu193 [555] in FXIaG193E is not pointing precisely toward the oxyanion hole.



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  		FIG. 3.
Inhibition of FXIaWT and FXIaG193E by DFP. A, FXIaWT; B, FXIaG193E. Each reaction was carried out in TBS/BSA, 5 mM CaCl2 at room temperature. The enzyme concentration for FXIaWT or FXIaG193E was 250 nM, and the concentration of DFP was varied from 2 µM to 4 mM. At various times, 5-µl aliquots were removed and added to 155 µl of TBS/BSA containing 625 µM S-2288. The percent residual FXIa activity at each point was plotted as a function of time to obtain the values of kobs. The first-order rate constants, kobs, are plotted against the DFP concentration.

 
Carbamylation of Ile16 Using NaNCO—The data in the previous two sections indicate that the S1 site and the oxyanion hole in the mutant enzyme are not properly formed. The development of the S1 site and oxyanion hole in serine proteases requires formation of a salt bridge between the amino group of Ile16 [370] and the carboxylate group of Asp194 [556]. Covalent modification of the amino group of Ile16 [370] by carbamylation results in an inactive enzyme, and a faster rate of carbamylation is indicative of impaired and destabilized salt bridge formation. As shown in Table III, the rate of carbamylation of FXIaG193E was ~4-fold faster than for FXIaWT. From these data, one can conclude that the main chain conformation involving residues 189-194 [551-556], which lines one side of the S1 pocket, is altered in the mutant enzyme. Because residue 193 [555] is a part of this peptide backbone, the amide N of Glu193 [555] may not be pointing toward the oxyanion hole in the mutant enzyme.


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  		TABLE III
Kinetic parameters for carbamylation of Ile16 [370] in FXIaWT and FXIaG193E

The rates of carbamylation of the free amino group of Ile16 [370] of FXIaG193E and FXIaWT were measured over a period of 5 h. Upon carbamylation, FXIa becomes inactive and cannot hydrolyze a synthetic substrate. Aliquots of each reaction were removed every 30 min and assayed for residual FXIa activity. Saturating amounts of pAB were added to each reaction to examine the effects of S1 site occupancy on carbamylation rates. The results presented are the averages of two experiments.

 
Occupancy of the S1 site is known to order the binding pocket and stabilize the salt bridge between Ile16 [370] and Asp194 [556] in serine proteases (35). We therefore examined the effect of pAB of the S1 site on the stability of the salt bridge between Ile16 [370] and Asp194 [556] in FXIaG193E. Notably, pAB occupancy of the S1 site in FXIaG193E led to an ~8-fold decrease in the rate of carbamylation (Table III), indicating substantial stabilization of the salt bridge. Interestingly, occupancy of the S1 site of FXIaWT by pAB also resulted in an ~2-fold slower rate of carbamylation (Table III), suggesting that pAB further stabilizes the wild type enzyme in a more active conformation. Remarkably, the presence of pAB corrected the rate of carbamylation of FXIaG193E to that of FXIaWT. Thus, the presence of pAB in the S1 site appears to largely correct the abnormality in the main chain conformation involving residues 189-194 [551-556] in the mutant enzyme.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
We have investigated the structural role of Gly193 [555] in the proper conformation and activity of the mutant coagulation serine protease FXIaG193E. The substitution of Glu for Gly193 [555] was based upon a naturally occurring mutation found in a patient with a history of excessive bleeding (4). In preliminary work reported in abstract form (4), zymogen FXIG193E could be cleaved normally between Arg15 [369] and Ile16 [370] to generate the protease FXIaG193E. Thus, bleeding associated with FXIG193E is likely because of the inability of FXIaG193E to function as an enzyme. FXIa was chosen for the present study because it has significant amidolytic activity and a well characterized physiologic macromolecular substrate/inhibitor profile.

Replacement of Gly193 [555] by Glu in FXIa may have two consequences: 1) it may change the conformation around the latent active site in the zymogen, and 2) it may not allow the specific conformational changes that accompany conversion of the zymogen to the enzyme to occur. Analysis of a model for the human FXI protease domain suggests that Glu at position 193 [555] can be easily accommodated in the zymogen form of the protein (Fig. 4A). When Gly193 [555] is replaced with Glu, close contacts with the side chains of Val138 [498] and Thr213 [575] are observed. This steric conflict is easily relieved by a slight rotation around the N-C{alpha} (13°) and C{alpha}-C' (32°) bonds resulting in movement of the Glu193 [555] side chain away from these residues. The Glu193 [555] side chain would then point into a cavity that is filled with solvent in most serine proteases. The Glu193 side chain may also be able to make a hydrogen bond with the hydroxyl group of Tyr228 [590]. Thus, it is expected that Glu substitution at position 193 [555] can be easily accommodated in FXI without significant structural consequences.



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  		FIG. 4.
Model of the zymogen and activated serine protease domain of FXIG193E. FXIWT, FXIG193E, FXIaWT, and FXIaG193E were modeled as described under "Experimental Procedures." In all structures, oxygen atoms are red, nitrogens are blue, carbons are green, and sulfurs are yellow. The chymotrypsin numbering system is used to identify the residues. A, zymogen FXIWT and FXIG193E. In this figure, the backbone of residues 192-194 [554-556] from FXIWT is shown in cyan superimposed on FXIG193E. Glu193 [555] is easily accommodated in FXIG193E, and may make a H-bond with the hydroxyl of Tyr228 [590]. Glu217 [579] in both FXIWT and FXIG193E makes a H-bond with the backbone amide of residue 191 [553]. Notably, Asp194 [556], which in the activated serine proteases makes a salt bridge with Ile16 [370], makes a H-bond with His40 [396] in the zymogen and Ile16 [370] is located distant from the active site. Such is the case in other zymogens in this family as well. B, active serine protease FXIaG193E. In the activated enzyme, Asp194 [556] makes H-bonds with the amide N of Ile16 [370] instead of with His40 [396] as shown in A for the zymogen. The backbone of residues 16-18 [370-372] is shown as a magenta ribbon. Glu193 [555] is shown stabilized by making H-bonds with Arg39 [395]. C{beta} of Glu193 [555] sterically conflicts with the carbonyl O of residue 192 [554], which will not be the case for FXIaWT. To relieve the steric conflict in FXIaG193E, the 192-193 peptide bond may flip or reorient. The backbone of residues 192-194 [554-556] with this peptide bond flipped is shown in purple. Note that the amide N of 193 [555] is now pointing away from the oxyanion hole. The catalytic triad residues consisting of His57 [413], Asp102 [464], and Ser195 [557] and residues 214-217 [576-579], which line one side of the S1 binding pocket are also shown. C, model of FXIaG193E in complex with APPI. The protease domain backbone of FXIaG193E is shown in magenta and that of APPI in cyan. Helices are depicted as cylinders, {beta}-sheets as long thick arrows, and turns in the loops with short thin arrows. The NH2 and COOH termini are labeled N and C, respectively. The interacting residues in FXIaG193E and APPI are colored by atom type. Residues from FXIa are labeled in white and residues from APPI are labeled in cyan. As in A and B, the protease domain numbering is based upon chymotrypsin and the APPI numbering is based upon that of bovine pancreatic trypsin inhibitor. Specifically, Tyr60 [416] from FXIa makes H-bonds with Tyr35 and Arg20 from APPI. Arg39 [395] from FXIa makes a H-bond with Ser19 from APPI. This is important as Arg39 [395] in the absence of APPI may help to stabilize Glu193 [555] through a hydrogen bond (see B), which will not exist in the complex because of the role of Arg39 [395] in binding to APPI. Tyr143 [503] and Ile151 [510] of FXIa are expected to have van der Waals interactions with Phe34 of APPI. Arg15 of APPI interacts with the S1 site residue Asp189 [551] in FXIa, and the carbonyl of Pro13 in APPI forms a H-bond with the N of Gly216 [578] in FXIa. These last two interactions are present between all bovine pancreatic trypsin-like inhibitors and their respective associated enzymes. Note that Glu193 [555] protrudes into the S2' site and sterically conflicts with Met17, the P2' residue of APPI. This steric conflict is highlighted with a yellow circle. This conflict will not exist in the FXIaWT-APPI complex where residue 193 [555] is Gly.

 
In contrast, in the active form of the enzyme, the polypeptide backbone at Gly193 [555] assumes a conformation compatible only with a Gly residue. Gly193 [555] is located in position 3 of a type II hairpin loop, and has {varphi}-{psi} conformation parameters of (105, -10) in the modeled FXIaWT structure. These parameters are compatible with those reported in crystal structures of many serine proteases, and places this residue in a region of the Ramachandran plot that is compatible only with a Gly residue. Replacement of Gly193 [555] with any other residue will introduce a {beta}-carbon that will have a steric conflict with the carbonyl O of residue 192 [554] (Fig. 4B). Relief of this conflict can only be achieved by a change in the conformation around the 192-193 [554-555] peptide bond. One possible way to achieve this is to flip the bond, thereby converting the type II turn into a type I turn. In this process, rotation ({varphi}) around the N-C{alpha}i+1 bond and rotation ({psi}) around the C{alpha}-C'i changes the orientation of the peptide bond without affecting side chain positions or the main chain conformation (49). The maximum activation barrier for a concerted flip of this type is <3 kcal/mol (49). In the case of FXIaG193E, this results in {varphi}-{psi} conformation parameters of (-61, -45) in the modeled structure (Fig. 4B). Support for the premise that the 192-193 [554-555] bond can be readily flipped in FXIaG193E comes from structures of the serine protease factor VIIa (bound to tissue factor) in complex with a certain class of inhibitor (50). Thus, it would appear that the facile interconversion of the 192-193 peptide bond occurs with a relatively low energy barrier. In both the type II and type I turns, the Glu193 [555] side chain points into the solvent and may make a hydrogen bond with Arg39 [395] (Fig. 4B).

The experimental evidence supports the argument that the conformation of the 192-193 [554-555] bond is altered when Glu occupies position 193 [555] instead of Gly. An altered conformation in the mutant would be expected to perturb the structure of the S1 binding site, oxyanion hole, and salt bridge formation between Ile16 [370] and Asp194 [556]. The data clearly demonstrate that the S1 site (Fig. 2), oxyanion hole (Fig. 3), and Ile16-Asp194 salt bridge formation (Table III) are disrupted in FXIaG193E. Thus, a concerted flip of the 192-193 [554-555] peptide bond is supported by three independent sets of experimental data, and suggests that the amide N of Glu193 [555] may point away from the oxyanion hole in the absence of active site occupancy by a substrate/inhibitor.

Interestingly, FXIaG193E hydrolyzes synthetic substrates quite effectively. This could occur only if the oxyanion hole is restructured by correctly orienting the amide N of residue 193 [555] so that it points toward the oxyanion hole. This implies that occupancy of the S1 site and the oxyanion of the substrate provide enough energy to flip the 192-193 [554-555] peptide bond to the proper conformation. This allows the amide N of 193 [555] to make a hydrogen bond with the oxyanion, stabilizing the tetrahedral transition state intermediate. This also implies that formation of the salt bridge between Ile16 [370] and Asp194 [556] is favored upon occupancy of the S1 site by the substrate. Data presented in Table III show that occupancy of the S1 site alone can correct the impairment in formation of the salt bridge in FXIaG193E. Similarly, binding of DFP (Fig. 3) should also reorient the amide N of residue 193 [555] into the proper conformation for formation of a hydrogen bond with its oxyanion. The observations that both pAB and DFP binding are impaired in FXIaG193E is strong evidence that the S1 site and oxyanion hole are not preformed in the mutant enzyme, but can be reordered upon binding of a substrate that occupies the S1 site and/or oxyanion hole.

Two crystal structures of serine proteases with bound inhibitors support our argument that occupancy by the substrate/inhibitor can correct the defects in the FXIaG193E S1 site and oxyanion hole. These are human brain trypsin with Arg at position 193 and the S1 site inhibitor (Ref. 51, Protein Data Bank code 1h4w [PDB] ) and Trimeresurus stejnejeri plasminogen activator with Phe at position 193 and the chloromethylketone inhibitor (Ref. 52, Protein Data Bank code 1bqy [PDB] ). Furthermore, a flip of the 192-193 peptide bond upon substrate binding is also supported by crystal structure and biochemical data for Staphylococcus aureus exfoliative toxins A (53-55) and B (55). The crystal structure of mouse glandular kallikrein-13 with Asp at position 193 and no inhibitor is also known (Ref. 56, Protein Data Bank code 1ao5 [PDB] ). Some residues lining both sides of the S1 site have very high B factors, suggesting that this region is somewhat mobile in the absence of inhibitor occupancy. In this structure, although the amide N of residue 193 is pointing toward the oxyanion hole to some extent, this region appears to be quite mobile. Thus, the S1 site may not be completely formed in the absence of occupancy of the S1 site.

The hydrolysis of the macromolecular substrate, FIX, is significantly more impaired than is hydrolysis of synthetic substrates by FXIaG193E. This could be because of the side chain of Glu193 occupying part of the S2' site where the P2' residue of FIX is expected to reside (see below), in addition to the abnormal conformation of the S1 site and oxyanion hole. The sequence in FIX at its NH2-terminal most cleavage site (Arg145-Ala146) from P2 to P2' is Thr-Arg-Ala-Glu, and at the COOH-terminal cleavage site is (Arg180-Val181) Thr-Arg-Val-Val. Considering the P2' residues, it appears that the steric clash would be larger at the Arg145-Ala146 cleavage site for two reasons: 1) the Glu at this site is larger than Val, and 2) Glu at the P2' position would produce a charge repulsion. Thus, we expect that the Arg145-Ala146 peptide bond in FIX would be cleaved very slowly in comparison to cleavage of the Arg180-Ala181 peptide bond by mutant FXIaG193E.

The inhibition of FXIaG193E by AT and APPI was even more impaired than was activation of FIX. This may be due primarily to the larger contact regions between the protease domain of FXIa and the two inhibitors when compared with the limited contact region with the FIX activation peptide. A model of APPI in complex with FXIaG193E is shown in Fig. 4C. The electrostatic and hydrophobic interactions are described in the figure legend. The indicated interactions are in agreement with data presented in abstract form by Navaneetham et al. (27). Clearly, the presence of Glu at position 193 [555] will change the electrostatic potential around the active site and interfere with the binary collision with AT or APPI. Moreover, Glu193 [555] will have a steric clash with the P2' Met residue of APPI as shown in Fig. 4C. Data presented in Table II indicate that the isomerization step that leads to the formation of the tight complex after the initial binary collision with FXIaG193E is also severely affected. This is expected because minor rearrangements of Glu193 [555] in FXIaG193E and the P2' residue Met17 in APPI must take place for a stable complex to form. However, once a stable complex is formed, dissociation of the complex does not appear to be affected.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
The Gly193 [555] residue of FXIa, a serine protease involved in blood coagulation, was changed to Glu to study the role of Gly193 in serine proteases. The experimental and modeling data presented in this paper indicate that the S1 site, oxyanion hole, and salt bridge formation between Ile16 [370] and Asp194 [556] are impaired in the mutant enzyme. However, occupancy of the active site by substrate/inhibitors can correct these defects. The hydrolysis of macromolecular substrates and binding of macromolecular inhibitors can be further impaired by a steric clash between the S2' site of the enzyme and P2' residue of the substrate/inhibitor. Enzymes with residues other than glycine at position 193, such as human brain trypsin (Arg193) (51), T. stejnejeri plasminogen activator (Phe193) (52), and mouse glandular kallikrein-13 (Asp193) (56) would be compatible with substrate/inhibitors that have small side chains at the P2' position. This will introduce a new level of structural plasticity and selectivity in serine proteases. Thus, it would appear that enzymes with Gly at position 193 will be more active but not have selectivity at the S2' site; whereas, enzymes with non-Gly residues at position 193 will be an order of magnitude less active but have a restricted S2' site and selectivity for their substrate/inhibitors. Bode and co-workers (52) have previously elaborated such plasticity and substrate/inhibitor selectivity in this class of proteases.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants HL36365 and HL70369 (to S. P. B.) and HL02917 and HL58837 (to D. G.). 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. Back

Supported, in part, by Predoctoral Fellowship 0315211Z from the American Heart Association Heartland Affiliate. Back

** To whom correspondence should be addressed: UCLA/Orthopaedic Hospital, Dept. of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, 1000 Veterans Ave., Rehab Bldg., Rm. 21-31, Los Angeles, CA 90095. Tel.: 310-825-5622/7603; Fax: 310-825-5972; E-mail: pbajaj{at}mednet.ucla.edu.

1 The abbreviations used are: FXI, factor XI; FIX, factor IX; FXIWT, wild-type factor XI; FXIG193E, factor XI with Gly193 -> Glu mutation; FXIIa, factor XIIa; APPI, protease nexin-2/amyloid {beta} protein precursor Kunitz domain inhibitor; TBS, Tris-buffered saline; pNA, p-nitro-aniline; S-2288, H-D-Ile-Pro-Arg-p-nitroanilide; S-2366, pyro-Glu-Pro-Arg-p-nitroanilide; pAB, p-aminobenzamidine; AT, antithrombin; BSA, bovine serum albumin; PEG, polyethylene glycol 8000; DFP, diisopropyl fluorophosphate; WT, wild type. Back

2 For comparison, the chymotrypsin amino acid numbering system is used throughout. Residue 555 in FXIa, 363 in FIXa, and 342 in FVIIa each correspond to residue 193 in chymotrypsin. Where necessary, the amino acid corresponding to a given protease is given in brackets (e.g. [555]). Back


   ACKNOWLEDGMENTS
 
We thank Dr. Alvin Schmaier for providing the APPI protein and Dr. Jens Birktoft for useful discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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