Ser214 Is Crucial for Substrate Binding to Serine Proteases*

Highly conserved amino acids that form crucial structural elements of the catalytic apparatus can be used to account for the evolutionary history of serine proteases and the cascades into which they are organized. One such evolutionary marker in chymotrypsin-like proteases is Ser214, located adjacent to the active site and forming part of the primary specificity pocket. Here we report the mutation of Ser214 in thrombin to Ala, Thr, Cys, Asp, Glu, and Lys. None of the mutants seriously compromises active site catalytic function as measured by the kinetic parameter k cat. However, the least conservative mutations result in large increases in K m because of lower rates of substrate diffusion into the active site. Therefore, the role of Ser214 is to promote the productive formation of the enzyme-substrate complex. The S214C mutant is catalytically inactive, which suggests that during evolution the TCN→AGY codon transitions for Ser214 occurred through Thr intermediates.

Serine proteases have been classified into evolutionarily and structurally unrelated clans (1), which nevertheless maintain a strictly conserved active site geometry among their catalytic Ser, His, and Asp residues. This shared catalytic structure suggests that common architectural motifs can be found in the molecular design of active sites utilizing a Ser-His-Asp triad. The identification of discrete evolutionary markers encountered in the sequences contributing to the catalytic apparatus has shed light on both crucial structural motifs and evolutionary events in the history of serine protease families (2) and serine protease cascades (3). One such structural motif common to chymotrypsin-like, subtilisin-like, and ␣/␤-hydrolasefold serine proteases is the presence of a highly conserved fourth active site residue whose backbone atoms form contacts with the substrate and whose polar side chain extends into the catalytic pocket. Among chymotrypsin-like proteases, the backbone of Ser 214 contributes to the S1 binding pocket (4). The side chain of the same residue is hypothesized to generate a polar environment for the catalytic Asp 102 of trypsin (5), and both oxygens of Ser 214 form H-bonds with waters located in the active site cleft of duodenase (6). Ser 125 (subtilisin BPNЈ numbering) in subtilisin-type proteases (4,7) and the unpaired Cys 341 (yeast carboxypeptidase W numbering) in ␣/␤-hydrolase-fold proteases (8,9) play similar roles. Each of these residues (Ser 214 , Ser 125 , and Cys 341 ) serves as an evolutionary marker and appears to contribute to catalytic function in its respective protease clan (2).
The role of Ser 214 in chymotrypsin-like proteases has been addressed in previous studies (5). The trypsin mutants S214E and S214K disrupted the environment of the catalytic Asp 102 as demonstrated kinetically and crystallographically. However, the S214A mutant described in the same report had improved specificity (k cat /K m ) compared with wild type, calling into question the requirement for the Ser 214 side chain in catalysis. More importantly, previous studies on trypsin have not addressed how mutations of Ser 214 affect the individual steps of the catalytic mechanism of substrate hydrolysis and have left the precise origin of the perturbation unanswered.
Here we report the effect of several substitutions (Ala, Asp, Glu, Lys, Thr, and Cys) of Ser 214 in thrombin by dissecting the kinetics of substrate hydrolysis using a novel and powerful approach (10). Considering the TCN3 AGY Ser codon transitions that must have taken place at Ser 214 (2), it has now become relevant to also generate S214T and S214C mutants as the codons for Thr and Cys (ACN and TGY, respectively) represent intermediates in the single nucleotide transitions between TCN and AGY. In addition to trypsin, thrombin is a most relevant system for studying the contribution of Ser 214 to serine protease catalysis. Thrombin is a Na ϩ -activated enzyme, which adds complexity to the mechanism of substrate recognition, and has a more stringent specificity profile than trypsin that may be more sensitive to mutations that alter k cat or K m . Hence, the mutation of Ser 214 in thrombin would reveal whether that evolutionary marker is linked to Na ϩ binding in serine proteases and would test whether previous results on trypsin can be generalized to the majority of chymotrypsin-like proteases.

MATERIALS AND METHODS
Site-directed mutagenesis of human ␣-thrombin was carried out in a HPC4-pNUT expression vector using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA). Expression of thrombin mutants was carried out in baby hamster kidney cells as described previously (11). Mutants were activated with the prothrombinase complex between 40 and 60 min at 37°C. Enzymes used in the activation were supplied by Enzyme Research (South Bend, IN). Mutants were purified to homogeneity by fast protein liquid chromatography using Resource Q and S columns with a linear gradient from 0.05 to 0.5 M choline chloride (ChCl), 5 mM MES, 1 pH 6.0, at room temperature. Active site concentrations were determined by titration with hirudin.
The substrate H-D-Phe-Pro-Arg-p-nitroanilide (FPR) was synthesized by Midwest Biotech (Carmel, IN). Individual rate constants defining the mechanism of substrate hydrolysis by serine proteases were extracted from the values of k cat and k cat /K m obtained as a function of temperature (10) from 5 to 45°C under solution conditions of 5 mM Tris, 0.1% PEG-8000, 200 mM NaCl, pH 8.0. The values for the Na ϩ -free slow form of wild type were obtained by replacing NaCl with ChCl in the * This work was supported in part by National Institutes of Health Research Grants HL49413 and HL58141. 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 The acylation step is typically rate-determining for amide substrates. The Michaelis-Menten parameters s ϭ k cat /K m and k cat accessible to direct experimental measurements are composite functions of the individual kinetic rates in Scheme 1. The explicit expressions as a function of temperature are shown in Equations 1 and 2 (10), where E j is the activation energy associated with the rate constant k j , R is the gas constant, T is the absolute temperature, and the k values refer to T 0 ϭ 298.15 K. The measurements of s and k cat as a function of temperature can resolve all the parameters in Equations 1 and 2 provided the plots show curvature. In the event of k 3 Ͼ Ͼk 2 (␤Ͼ Ͼ1) as typically observed for thrombin, k 3 and E 3 cannot be resolved and the plot of logk cat versus 1/T is a straight line. When k cat values cannot be determined because of high K m values, a plot of logs versus 1/T yields k 1 , E 1 , the ratio ␣ ϭ k 2 /k Ϫ1 , and the difference E ␣ ϭ E Ϫ1 Ϫ E 2 , again provided the plot shows curvature. Physiologic substrates and inhibitors were studied under the conditions of 5 mM Tris, 0.1% PEG-8000, 200 mM NaCl, pH 8.0 at 25°C. The cleavage of fibrinogen leading to the release of fibrinopeptides A and B and cleavage of the protease-activated receptor (PAR) peptides PAR1, PAR3, and PAR4 were carried out by HPLC as described previously (12). The activation of protein C in the presence of 5 mM CaCl 2 and 10 nM rabbit thrombomodulin was measured and analyzed as reported elsewhere (11). Antithrombin inhibition was studied by changing the concentration of unfractionated heparin to determine the optimal inhibition of each mutant (13), monitored by following progress curves of FPR hydrolysis in which the antithrombin concentration was varied. The observed rate of inhibition, k on , was calculated as described previously (11).
Equilibrium dissociation constants for Na ϩ binding were determined by fluorescence titration using a FluoroMax-3 SPEX spectrophotometer. Fluorescence titrations took place under experimental conditions of 5 mM Tris, 0.1% PEG-8000, 800 mM ionic strength, pH 8.0 at 10°C. The temperature was chosen to maximize both the intrinsic fluorescence of the enzyme and the fluorescence increase induced by Na ϩ binding. Titrations were carried out by adding aliquots of thrombin in 800 mM NaCl to a solution containing the enzyme in 800 mM ChCl. Ionic strength (800 mM) and enzyme concentration (200 nM) were held constant, whereas [Na ϩ ] was varied. Excitation was at 295 nm, and emission was measured at 333 nm. The value of thrombin intrinsic fluorescence, F, as a function of [Na ϩ ] was fit according to the Equation 3, where F 0 and F 1 are the values of F in the absence and under saturating [Na ϩ ] and K d is the equilibrium dissociation constant for Na ϩ binding. Na ϩ dissociation constants were alternately determined by following the linkage between the k cat /K m for FPR hydrolysis and [Na ϩ ] under conditions of 50 mM Tris, 0.1% PEG-8000, pH 8.0 at 25°C. The value of s ϭ k cat /K m was fit according to the Equation 4 (11), where s 0 and s 1 are the values of k cat /K m in the absence and under saturating [Na ϩ ], pertaining, respectively, to the Na ϩ -free slow and Na ϩ -bound fast forms and K d is the equilibrium dissociation constant for Na ϩ binding. Temperature melting curves were measured under conditions of 200 mM NaCl or ChCl, 5 mM Tris, pH 8.0, 0.1% PEG-8000 over the temperature range of 15-80°C. Denaturation was monitored by following absorbance at 280 nm. Experiments were conducted using sufficient enzyme to generate an absorbance of ϳ0.05 absorbance units at 15°C. Temperature was then gradually increased to 80°C at a rate of 1.5°C/ min. The melting temperature was calculated from the midpoint of the melting transition.

RESULTS
Ser 214 thrombin mutants have reduced catalytic activity as measured by FPR hydrolysis with the most significant reductions corresponding to the mutants carrying the largest side chains at position 214 (Table I). Although the Ala, Thr, and Lys mutants have reduced k cat compared with wild type, the Asp a Values were calculated from the k cat at 25°C. b n.d., not determined because k cat and K m could not be measured individually over the entire temperature range.
and Glu mutants have increased k cat . Therefore, changes in k cat do not correlate with the size of the side chain placed at position 214. However, the presence of a residue other than Ser at position 214 dramatically increases K m . The least conservative mutant, S214K, shows a 1000-fold increase in K m with only a 3-fold drop in k cat . Only limited kinetic data could be collected for the S214C mutant (see below). The impact of Ser 214 on K m rather than k cat suggests a major role in substrate binding, which is confirmed by the individual rate constants gleaned from temperature dependence studies of k cat /K m and k cat for FPR hydrolysis ( Fig. 1 and Table I). The increases in K m are primarily attributable to decreases in the rate of association k 1 , which are most significant for the least conservative mutations. The values of k 1 for the Ala and Thr mutants approach that of the slow form of wild type, whereas those of the Asp, Glu, and Lys mutants deviate more to the downside and underline more drastic structural perturbations of the accessibility of the active site. The reductions in k 1 tend to be linked to significant decreases in the activation energy, E 1 , for this step. This suggests that wild-type thrombin under-goes an induced fit rearrangement on substrate binding that cannot be duplicated fully in the Ser 214 mutants. The integrity of the Ser side chain ensures this structural change and optimizes the productive collision of substrate with the active site. When the catalytic rate constants are considered, all mutants are like wild type in that k 3 Ͼ Ͼk 2 and the logk cat versus 1/T plot is linear. Interestingly, none of the mutants compromises k cat significantly, and the presence of a negatively charged side chain at position 214 actually increases the acylation rate and k cat relative to wild type.
There is an apparent dichotomy in the functional behavior of the mutants of Ser 214 , because they tend to behave like the Na ϩ -free slow form of wild type when the rate of substrate diffusion into the active site is considered but mimic more closely the Na ϩ -bound fast form in their catalytic rate constants. This finding suggests that Ser 214 is energetically linked to Na ϩ binding especially in the steps that control substrate binding and formation of the enzyme-substrate complex. This expectation is confirmed by the inspection of the Na ϩ binding affinities of the Ser 214 mutants as measured directly by intrinsic fluorescence titration (Fig. 2) that are compromised to a significant extent (up to 50-fold). Consistent with these findings, Na ϩ binding partially rescues the Ser 214 mutants (Table  II) because the value of the specificity constant s 0 in the absence of Na ϩ (slow form) is more compromised relative to wild type than the value s 1 obtained under saturating [Na ϩ ] (fast form). This finding demonstrates that the deleterious effect of replacing Ser 214 is felt more on the Na ϩ -free slow form.
Ser 214 thrombin mutants also demonstrate impaired cleavage of physiologic substrates (Table III). Specificity constants for the release of fibrinopeptides A and B, which measure the ability of thrombin to cleave fibrinogen to form a fibrin clot, are compromised to a similar degree. Protein C activation, an anticoagulant activity, is slightly less compromised with the exception of S214K. The cleavage of PAR peptides is reduced to a similar extent for PAR1, PAR3, and PAR4. Because of the severely impacted catalytic efficiency of the S214K mutant, specificity constants toward PAR3 and PAR4 could not be measured. The least conservative mutants show the greatest reduction in antithrombin inhibition as measured by the rate of inactivation k on . The mutants require slightly higher concen- trations of heparin relative to wild type to ensure inhibition. The optimal heparin concentration for wild type is 0.5 USP units/ml, whereas the majority of the mutants require 1-2 USP units/ml heparin. Selected S2 and S3 specificity subsite mutants of thrombin also demonstrate increased requirements for heparin (13).
The S214C mutant could not be characterized extensively. During the first attempt at activation of S214C prethrombin-1, the mutant thrombin appeared to aggregate in solution. Despite this result, it was possible to purify a fraction of the mutant whose absorbance indicated a concentration of 700 nM. However, titration with hirudin indicated an active site concentration of ϳ30 nM. Thus, 96% of the activated material was functionally inactive. A subsequent activation using a lower prothrombin concentration prevented aggregation, but once again the titration of the purified fraction revealed that nearly all of the activated material was catalytically inactive. The loss of activity seen for S214C is puzzling. Polyacrylamide gel electrophoresis of aliquots from the activation reactions and purified fractions does not indicate proteolysis. As the unpaired Cys points into the active site and away from the surface of the of the enzyme, intermolecular disulfide bonding appears improbable. It is possible that the presence of the extra Cys at position 214 causes improper intramolecular disulfide bonds to form, resulting in an incorrectly folded enzyme.
Temperature denaturation experiments were used to assess the relative stabilities of wild type, S214T, and S214C. The melting temperatures for the S214C and S214T mutants are 10 and 5°C, respectively, lower than that for wild type (62°C). The instability of S214C does not result from impaired Na ϩ binding as wild type has the same melting temperature in both NaCl (fast form) and ChCl (slow form) solutions. Ser 214 appears to be an important residue for the folding of thrombin. The mutation to Thr slightly destabilizes the fold, but providing a free disulfide at this position destabilizes the fold enough to produce an inactive and presumably misfolded enzyme. DISCUSSION The results presented here on the role of Ser 214 in serine proteases extend those reported previously in the case of trypsin (5) that have left several basic questions unanswered. In particular, previous studies have characterized the kinetic origin of the perturbations only in terms of k cat and K m . Our study has examined the effect of Ser 214 mutations on each step of the catalytic mechanism of substrate hydrolysis and has identified the precise events in which Ser 214 is involved, namely the optimization of substrate diffusion in the active site, the induced fit rearrangement of the enzyme-substrate complex, and the unanticipated energetic linkage with Na ϩ binding.
Ser 214 is the only residue among the four with side chains in the active site that is not required for proper function of the Ser-His-Asp charge relay system. As such, it presents an opportunity to probe the environment of the active site around Asp 102 . The active site is extremely tolerant to substitution at position 214 as the presence of a buried Lys produces only a 3-fold reduction in k cat . However, it should be noted that in trypsin, the reduction in k cat was more dramatic. In ␣-lytic protease, the mutation of Ser 214 to Ala brought about a 4900fold reduction in specificity, but Epstein and Abeles (14) surmise that the catalytic function remained intact.
Ser 214 seems to play a minimal role in governing the catalytic rate reflected by the minor effects on k cat . Crystal structures of trypsin (5) and duodenase (6) have led to the hypothesis that Ser 214 helps position Asp 102 of the charge relay system for optimal catalysis. Slight reductions in k cat for S214A, S214T, and S214K lend support to this idea but prove that Ser 214 is not essential for effective catalysis. Catalytically, the most interesting mutants are S214D and S214E, which display increases in k cat despite the potential introduction of an additional charge into the active site. The Asp and Glu side chains may repel Asp 102 , pushing it closer to His 57 and therefore increasing the potency of the charge relay system.
The mutation of Ser 214 primarily disturbs the S1 pocket as active site function (measured by k cat ) does not appear to suffer from the energetic perturbation associated with Ser 214 mutation. The decrease in k 1 and the increase in K m would then be explained by displacement of the backbone atoms of residue 214, the carbonyl oxygen of which makes contact with substrate (4). Ser is the appropriate connector at position 214 that ties the very stable active site to the S1 pocket to maintain optimal contact with substrate and correct alignment of the catalytic register. For trypsin substitutions involving large side chains (S214E and S214K), steric clashes with the side chain of Trp 215 occur. This results in the Trp side chain "flipping" and blocking access to the S2 and S3 specificity pockets (5) and may explain the extremely poor specificity of S214K mutants in both thrombin and trypsin.
In thrombin, Na ϩ binds in a water channel extending from the surface of the enzyme through the S1 specificity pocket into the active site and containing Ͼ20 water molecules intercon-TABLE II Specificity constants of wild type and mutant thrombins in the slow (Na ϩ -free) and fast (Na ϩ -bound) forms The parameters s 0 and s 1 are the values of k cat /K m for the slow and fast forms, respectively, whereas K d is the dissociation constant for Na ϩ binding (see also Equation 4 under "Materials and Methods"). nected by a hydrogen-bonding network that includes the protein (15). The positions of these internal water molecules are highly conserved and well ordered. Several of those water molecules are involved in Na ϩ coordination, and the hydrogen bond connectivity of the S1 pocket water channel is altered when Na ϩ binds, accounting for the change in specificity associated with the Na ϩ -triggered slow3fast transition (16). Residues that are linked to this water channel are thus energetically linked to Na ϩ , and the perturbation of such residues may affect Na ϩ binding as well as specificity. As a result, Na ϩ binding is extremely sensitive to the architecture of the S1 pocket (16). Ser 214 is not involved in Na ϩ coordination, and its side chain extends away from the Na ϩ site and into the active site. In fact, the Na ϩ binding site of thrombin resides 15 Å from Ser 214 at the opposite end of the S1 specificity pocket. However, residues that are thermodynamically linked to Na ϩ binding need not be located near the Na ϩ binding site. For example, the mutations of Trp 60d and Trp 215 located 17 and 10 Å away from the Na ϩ site practically abolish Na ϩ binding (17,18). The backbone atoms of Trp 215 adjacent to Ser 214 make contact with two water molecules of the S1 channel (19), and perturbations of Ser 214 may be transmitted via Trp 215 to the four water ligands coordinating the Na ϩ ion (15,16). The significantly reduced Na ϩ binding of Ser 214 mutants indicates the long range influence of that residue on the S1 pocket structure. Also, the structural perturbation caused by the replacement of Ser 214 affects predominantly the Na ϩ -free slow form (Table II) and is corrected by Na ϩ binding by virtue of the reciprocity of the linkage between this residue and Na ϩ as seen for mutants of residues Trp 60d and Trp 215 (17,18).
The S214C mutant is 95% inactive. The residue presumably engages in an improper disulfide bond with another Cys residue; however, there is no outstanding candidate for this other residue because the nearest Cys to Ser 214 is 9 Å away. Temperature denaturation studies also suggest that Ser 214 plays a role in thrombin stability. Among ␣/␤-hydrolase-fold serine proteases, the residue analogous to Ser 214 is frequently the unpaired Cys 341 . A subtilisin-like protease with Cys at the analogous position 125 also exists (2). Therefore, unpaired Cys residues are easily tolerated adjacent to serine protease active sites except in the chymotrypsin-like clan. There are several instances of Thr usage at position 214 including human complement factor D. In factor D, Thr and Ser are interchangeable (20), indicating the viability of Thr at position 214. The unsuitability of Cys and the feasibility of Thr at position 214 of thrombin suggest the codon switch for Ser 214 from TCN to AGY if occurring by single-nucleotide transitions utilized a Thr intermediate.
The case for a true catalytic tetrad, at least in clan PA, now seems weaker than originally suggested by sequence data (2). Among chymotrypsin-like proteases, Ser 214 appears to have been conserved because of its role in substrate recognition, ensuring optimal diffusion into the active site and proper induced fit rearrangement of the enzyme-substrate complex. Upon first glance, this function may not seem as crucial as that performed by the catalytic nucleophile Ser 195 , but Ser 214 is conserved almost as strongly as Ser 195 , highlighting the evolutionary and physiologic importance of tight substrate binding (21) in the vast majority of serine proteases.