In this study, the pH dependence of the catalytic activity and binding of the low-molecular mass inhibitor N-a-2-naphthylsulfonyl-glycyl-4-amidinophenylalanine-piperidine a-NAPAP to the
Trang 1thrombin A-chain affects the pKa value of catalytic
residues, the overall enzyme’s stability and conformational
Raimondo De Cristofaro1, Andrea Carotti2, Sepideh Akhavan3,*, Roberta Palla3, Flora Peyvandi3, Cosimo Altomare2 and Pier Mannuccio Mannucci3
1 Haemostasis Research Centre, Institute of Internal Medicine and Geriatrics, Catholic University School of Medicine, Rome, Italy
2 Department of Pharmaceutical Chemistry, University of Bari, Italy
3 Angelo Bianchi Bonomi Hemophilia and Thrombosis Center and Fondazione Luigi Villa, IRCCS Maggiore Hospital University of Milan, Italy
Recently, a homozygous deletion mutation of one of the
two contiguous Lys9⁄ Lys10 residues in the A-chain of
a-thrombin (DK9) was identified in patients with severe
prothrombin deficiency and hemorrhagic diathesis [1,2]
Compared with the wild-type (WT) form, the specificity constants of hydrolysis by DK9 of the synthetic substrate d-Phe-Pip-Arg-pNA and fibrinopeptide A were found to be 18- and 60-fold lower, respectively
Keywords
allostery; molecular dynamics; pKavalues;
stability; thrombin
Correspondence
R De Cristofaro, Haemostasis Research
Centre, Institute of Internal Medicine and
Geriatrics, Catholic University School of
Medicine, Largo F Vito 1, 00168 Rome,
Italy
Fax: +39 6 30 155 915
Tel: +39 6 30 154 438
E-mail: rdecristofaro@rm.unicatt.it
C Altomare, Department of Pharmaceutical
Chemistry, University of Bari, Via E.
Orabona 4, 70125 Bari, Italy
Fax: +39 80 544 2230
Tel: +39 80 544 2781
E-mail: altomare@farmchim.uniba.it
*Present address
INSERM E0348, Faculte´ Xavier Bichat,
University Paris 7, France
(Received 2 September 2005, revised 12
October 2005, accepted 7 November 2005)
doi:10.1111/j.1742-4658.2005.05052.x
The catalytic competence of the natural thrombin mutant with deletion of the Lys9 residue in the A-chain (DK9) was found to be severely impaired, most likely due to modification of the 60-loop conformation and catalytic triad geometry, as supported by long molecular dynamics (MD) simula-tions in explicit water solvent In this study, the pH dependence of the catalytic activity and binding of the low-molecular mass inhibitor N-a-(2-naphthylsulfonyl-glycyl)-4-amidinophenylalanine-piperidine (a-NAPAP) to the wild-type (WT) and DK9 thrombin forms were investigated, along with their overall structural stabilities and conformational properties Two ioniz-able groups were found to similarly affect the activity of both thrombins The pKa value of the first ionizable group, assigned to the catalytic His57 residue, was found to be 7.5 and 6.9 in ligand-free DK9 and WT thrombin, respectively Urea-induced denaturation studies showed higher instability
of the DK9 mutant compared with WT thrombin, and disulfide scrambling experiments proved weakening of the interchain interactions, causing faster release of the reduced A-chain in the mutant enzyme The sodium ion bind-ing affinity was not significantly perturbed by Lys9 deletion, although the linked increase in intrinsic fluorescence was lower in the mutant Essential dynamics (ED) analysis highlighted different conformational properties of the two thrombins in agreement with the experimental conformational stability data Globally, these findings enhanced our understanding of the perturbations triggered by Lys9 deletion, which reduces the overall stability
of the molecule, weakens the A–B interchain interactions, and allosterically perturbs the geometry and protonation state of catalytic residues of the enzyme
Abbreviations
a-NAPAP, N-a-(2-naphthylsulfonyl-glycyl)-4-amidinophenylalanine-piperidine; Bis-Tris, (2-hydroxyethyl)iminotris(hydroxymethyl)methane; CHES, 2-(N-cyclohexylamino)ethanesulfonic acid; ED, essential dynamics; DK9, Lys9 deleted mutant; MD, molecular dynamics; Pip, pipecolyl; pNA, para-nitroanilide; SAS, solvent-accessible surface; WT, wild-type.
Trang 2Interaction with antithrombin was also reduced in the
mutant, the association rate being 20-fold lower than
in WT thrombin Moreover, DK9 showed very weak
platelet-activating capacity, whereas binding to the
platelet glycoprotein Iba and thrombomodulin was
unaffected At variance with these findings, inhibitors
showed better binding to DK9 than to the WT form A
long-term molecular dynamics (MD) simulation of DK9
thrombin in explicit water solvent supports the role of
the A-chain in affecting the conformation and catalytic
properties of the B-chain, particularly in some insertion
loops of the enzyme, such as the 60-loop, as well as in
the geometry of the catalytic triad residues Our MD
analysis highlighted relevant modifications within the
so-called ‘aryl-binding site’, in particular, expulsion⁄
rearrangement of the W60d side chain (S2 site) and
shifting of W215 (S3) Functional and computational
data show that the catalytic cycle and efficient
interac-tion with substrates and natural inhibitors by DK9
undergo a severe impairment, likely due to propagation
to the active site residues of structural and
conforma-tional perturbations caused by Lys9 deletion in the
A-chain [2]
These findings prompted us to further investigate
the pH dependence of the catalytic activity and
stabil-ity of the DK9 mutant in comparison with WT
throm-bin, using experimental techniques in conjunction with
computational approaches to prove the effects of the
K9 deletion on the ionization of catalytic residues and
the overall stability of the enzyme This investigation
contributes to the unraveling of the mechanisms
responsible for both the impaired catalytic activity of
the K9-deleted natural mutant of thrombin in vitro and its hemorrhagic phenotype in vivo
Results and Discussion
Effect of pH on thrombin catalytic activity and inhibition
The pH-dependent steady-state amidase activities of
WT and K9-deleted mutant thrombins were studied in the pH range 5.5–10, using previously reported experi-mental and theoretical approaches [3,4] Kinetic schemes and equations (Eqns 2–4), allowing the effects
of pH on the Michaelis–Menten parameters kcatand Km
to be assessed, are reported in Experimental Procedures Although protons globally affected the amidase activity of both WT and DK9 thrombin forms in a similar way (Fig 1A–C), the pKa values of the ioniza-ble groups involved in the catalytic cycle were found
to be significantly different As reported in Table 1, we found an appreciable increase in the pKa value of the first ionizable group, which in the free enzyme showed
a pKa value of 7.53 in the DK9 mutant and 6.86 in
WT thrombin Because previous studies have assigned this group to the active site His57 side chain [3–5], this finding suggests that Lys9 deletion allosterically affects the protonation equilibrium of the active site His57, enhancing its affinity for protons both in free and sub-strate-bound forms of the enzymes The second pKa value was assigned to the N-terminal group of Ile16 (NTIle), which holds Asp194 in a salt bridge as a result of zymogen activation [3,5,6] The NTIle pKa
Fig 1 Analysis of pH dependence of Michaelis–Menten constants of D -Phe-Pip-Arg-pNA hydrolysis (A–C) by WT (d) and DK9 thrombin (s), along with the K i values
of NAPAP binding (D) at 25 C and 0.15 M
NaCl Continuous lines were drawn accord-ing to the best-fit parameters values of Eqns (2–4) and listed in Table 1 The vertical bars are the standard errors of the determi-nations.
Trang 3value in the mutant thrombin undergoes a moderate
increase from 8.45 to 8.87 in the free enzyme and from
9.04 to 9.36 in the substrate-bound form, for WT
thrombin and DK9 mutant, respectively
Table 1B reports changes in the kcat and kcat⁄ Km
values at the three protonation levels of WT and DK9
thrombins The kcatand kcat⁄ Kmvalues show a drastic
decrease, mostly due to a net fall in the kcat value,
which expresses the acylation rate By contrast, the
decrease in the DK9 Kmvalue is consistent with better
accommodation of the substrate into the catalytic
pocket of the unprotonated form of the mutant, as
shown recently [2]
Interestingly, the log kcat⁄ Km values pertaining to
WT and DK9 forms with protonated His57 (r1 of
Eqn 4) in the presence of 0.15 m NaCl were inversely
related to the respective His57 pKa values Based on a
Brønsted mechanism [7], this observed relation is
consis-tent with a transition state for breakdown of the
tetra-hedral intermediate involving partial carbon–nitrogen
(C–N) bond cleavage, which is stabilized by H-bonding
to the His57 imidazolium Nenitrogen (see Experimental
procedures Scheme 2) The imidazolium form would act
as a general acid to facilitate amine expulsion (kA) from
the tetrahedral intermediate (TI) Such a conclusion is
in agreement with recent findings obtained with proton
inventory studies of a-thrombin-catalyzed hydrolysis of
amide substrates [6] Thus, in human thrombin the
rate-limiting step in the acylation reaction is the breakdown
of tetrahedral intermediate, being the different affinity
for protons of His57 imidazole Ne nitrogen inversely
related to the specificity constant of the amidase activity
of the two thrombin forms
According to our recent report, DK9 compared with
WT thrombin achieves a better interaction with the low-molecular mass inhibitor N-a-(2-naphthylsulfonyl-glycyl)-4-amidinophenylalanine-piperidine (a-NAPAP) [2,8] As shown in Fig 1D, the pH dependence of a-NAPAP binding was characterized by a bell-shaped curve The best-fit pKavalues calculated from this data set (Table 1C) were very close to those calculated from the pH-dependent enzyme activity profiles (Table 1A) The pKa values of the DK9 residues obtained in the two data sets showed almost the same experimental error This may be because the synthetic substrate used
in the experiments is not a ‘sticky’ substrate for DK9 thrombin, as shown recently [2] In fact, according to the known following relation [3,5]
pKðobsÞ¼ pK logð1 þ k2=k1Þ ð1Þ
in case of the interaction between thrombin and a nonsticky substrate, where k2<< k1, pK(obs) should be equal to the true pK value, as found experimentally Slightly higher pKa values were obtained in a-NAPAP than in the substrate data set for WT thrombin (6.97
vs 6.86 and 8.63 vs 8.45, for the first and second ionizable group, respectively) This is likely because
d-Phe-Pip-Arg-pNA is a ‘sticky’ substrate for WT thrombin [2] and thus, according to Eqn (1), the observed pKa value from steady-state kinetic experi-ments should be lower than the true pKa value by a factor equal to log(1) k2⁄ k1), as seen experimentally
In analogy with the values calculated in enzymatic experiments, an increase of 0.5 pK units of the His57 in DK9 mutant was also calculated analyzing the NAPAP data set (Table 1C) In fact, His57 undergoes
Table 1 Best-fit pKavalues of the ionizable groups of both WT and DK9 mutant thrombins (A), along with the best-fit kinetic parameters contained in Eqns (2–4) involved in the hydrolysis of the synthetic substrate D -Phe-Pip-Arg-pNA (B) at 25 C in the presence of 0.15 M NaCl The best-fit pK a values of the ionizable groups of both WT and DK9 mutant thrombins, calculated using Eqn (2), are reported in C.
(A)
Enzymes
WT 6.86 ± 0.06 6.30 ± 0.06 8.45 ± 0.06 9.04 ± 0.05 DK9 7.53 ± 0.12 7.13 ± 0.09 8.87 ± 0.09 9.36 ± 0.12 (B) Enzymes 0 k cat s)1 1 k cat s)1 2 k cat s)1 K m l M r 0 · 10 6 ( M )1Æs)1) r1 · 10 6 ( M )1Æs)1) r2 · 10 6 ( M )1Æs)1)
WT 31.9 ± 4 76.3 ± 3 32 ± 4 6.6 ± 0.3 5 ± 0.6 42 ± 3 5.3 ± 0.6 DK9 1.02 ± 0.4 3.82 ± 0.3 2.01 ± 0.2 3.6 ± 0.3 0.31 ± 0.04 3.1 ± 0.3 0.69 ± 0.07 (C)
Enzymes
WTa 6.97 ± 0.05 6.52 ± 0.05 8.63 ± 0.05 9.00 ± 0.05 DK9b 7.53 ± 0.05 6.59 ± 0.05 8.99 ± 0.06 9.85 ± 0.07
a Ki ¼ 3.9 ± 0.10 n M b Ki ¼ 2.7 ± 0.13 n M
Trang 4a decrease in pKa value upon NAPAP binding in the
reversible complex formation, both in WT and DK9
thrombins
The change in pKa value of the catalytic His residue
in the mutant thrombin implies that the deletion of
Lys9 allosterically affects the conformational state of
relevant domains of the catalytic B-chain Thus, in an
attempt to unravel the mechanisms responsible for the
observed effects, we investigated the sodium-binding
properties and conformational stability of the mutant
enzyme in comparison with the WT thrombin form
Binding of Na+to thrombin
Sodium ion binding to WT thrombin was
character-ized by a saturable increase in fluorescence at 342 nm
(Fig 2) The apparent equilibrium dissociation
con-stant of Na+ binding was calculated using a single
site binding equation and was 22.0 ± 1.5 and
24 ± 2.6 mm for WT and DK9 thrombins,
respect-ively, in reasonable agreement with previous results [9]
However, the intrinsic fluorescence of the mutant was
20% lower than that pertaining to WT thrombin
and the magnitudes of the fluorescence change differed
significantly, being equal to approximately +18 and
+9% for WT and DK9 mutant thrombin, respectively
These results suggest that the Na+-binding loop in
the K9-deleted mutant retains the intrinsic affinity for
the cation, although the conformational transitions
linked to its binding are of more limited extension
compared with those of the WT form In other words,
in the mutant thrombin the binding of sodium is not intrinsically perturbed, but should be uncoupled from the specific conformational transitions occurring in the
WT form [9] This implies that the conformational transitions induced in the B-chain by Lys9 deletion are unique, being different from that of either the ‘fast’ or
‘slow’ form of WT thrombin [10]
Essential dynamics (ED) analysis [11] was applied to key regions of WT and DK9 thrombin forms, with the aim of identifying motions relevant for their folding, separating them from those describing irrelevant local fluctuations ED analysis has proven to be a valid method allowing the correlation between motions of different parts of the protein to be assessed, overcom-ing possible artifacts which could derive from a simple rmsd analysis As shown in Fig 3, the A-chain, as well
as the Na+-binding site, proved more flexible in WT than in the K9-deleted mutant The Na+-binding site undergoes a significant conformational transition after
10 ns in DK9 thrombin, whereas this phenomenon occurs after 5 ns in WT thrombin The motions of the Na+-binding site were found to be correlated with those of regions of particular importance, such as the S3 specificity site (Trp215–Ile174), the Trp60 insertion loop, the Cys168–Cys182 disulfide bond, and the fibrinogen secondary binding exosite, all occurring after 10 ns (results not shown) A previous X-ray diffraction study demonstrated that the Cys168– Cys182 disulfide bond undergoes a re-registration upon sodium binding [10] In particular, the distance between the sulfur atom of Cys182 and the Cb of Tyr225 was reduced by 1 A˚ in WT thrombin by
Na+ binding and mediated the conformational transi-tions in the catalytic pocket of the enzyme responsible for the enhanced catalytic activity [10] The results of our calculations were in qualitative agreement with this behavior of the WT form, whereas the reduction of that distance in DK9 thrombin upon Na+ binding, along the whole productive MD, was found about half
of that of WT (data not shown), highlighting reduced conformational mobility of the Cys168–Cys182 disul-fide bond linked to Na+binding
It had been shown by others that there is an inverse relation between fluorescence intensity and exposure to solvent in a number of Trp residues (60d, 96, 148, 207, and 215) [12] In particular, Trp207 and Trp29 residues, located at the boundary between the A- and B-chains and interacting with Arg137 via three water structural molecules having low mobility (w321, w325, and w454) [12], have been shown to contribute about 35 and 9%, respectively, to the total intrinsic fluorescence of WT thrombin [12] In an attempt to understand the
Fig 2 Titration by steady-state fluorescence of Na + binding to
75 n M WT (d) and DK9 thrombin (s) Na + binding was investigated
at 25 C at ionic strength of 0.2, pH 8.00 Excitation wavelength
was 280 nm Continuous lines were drawn according to single-site
binding isotherms with best-fit Kd values of 22 ± 1.5 and
24 ± 2.6 m M for WT and DK9 thrombin, respectively The vertical
bars are the standard errors of the measurements.
Trang 5observed difference in the fluorescence properties of the
WT and DK9 thrombins, we calculated the average
sol-vent (water)-accessible surface areas (SAS) of the above
residues along the whole MD simulations and found
that Trp207, whose predominant contribution to the
total fluorescence is well established [12], exposes its
surface to solvent about three times more in the DK9
mutant (average SAS¼ 24.9 A˚2) than in the WT form
(average SAS¼ 9.3 A˚2) A similar trend was observed
for Trp29 (average SAS¼ 9.6 and 14.1 A˚2 in WT and
DK9, respectively), whereas the other Trp residues
(60d, 96, 148 and 215), contributing < 11% to the total
fluorescence [12], vary in their average SASs by < 25%
in DK9 compared with WT thrombin Furthermore, it
is likely that as a consequence of Lys14 deletion, the
hydrogen bond between Glu8 and Trp207 is lost, as
our MD calculations proved, with a consequent
decrease in tryptophan fluorescence, in agreement with
the known inverse relationship between the intrinsic
fluorescence of a molecule and its conformational
mobility [13] These computational results suggest that
perturbation in the polarity and⁄ or flexibility of the
environment of Trp207 and 29 may significantly affect
the intrinsic fluorescence of the DK9 variant, the higher
the surface exposure and flexibility of their side-chains
to the solvent the smaller the intrinsic fluorescence of
the enzyme
The smaller gain in the intrinsic fluorescence of DK9
upon Na+concentration (9 vs 18% in WT) may reflect
not only the lower flexibility of the Na+-binding loop,
but also a different conformational rearrangement of
Trp215 (as indicated by the ED results), which contri-butes solely to the gain in fluorescence observed in the
Na+-bound conformer of thrombin [10,14]
Thrombin stability studies Urea at 6 m concentration induced complete denatura-tion of both WT and DK9 thrombin (Fig 4) The denaturating process at pH 6.80 was monitored by the
A
B
Fig 3 Essential dynamics analysis of (A)
A-chain and (B) Na+-binding site of WT ( )
and DK9 thrombin (—) Motions along the
first eigenvector of the selected protein
regions in the time frame of the MD
simula-tion are reported.
Fig 4 Urea-induced denaturation of WT (d) and DK9 thrombin (s) Measurements were performed at 25 C in 10 m M Bis ⁄ Tris, 0.15 M
NaCl, pH 6.80 Continuous lines were drawn according to the best-fit EC 50 values of urea-induced denaturation: 2.94 ± 0.05 and 2.49 ± 0.04 M for WT and DK9 thrombin, respectively.
Trang 6decrease in fluorescence of the intrinsic enzyme and
was almost perfectly reversible, because a 10-fold
dilu-tion of the thrombin samples in the same buffer
with-out urea resulted in recovery (after correction for the
dilution factor) of the fluorescence in the absence of
urea The process was highly cooperative for both
thrombin species (slope factor 8 in both cases), and
the concentration of urea inducing the 50% effect on
the fluorescence signal, [urea]1⁄ 2, was determined as
2.94 ± 0.05 m for WT and 2.49 ± 0.04 m for DK9
thrombin These values suggested that, in the presence
of a saturating Na+concentration, the mutant species
was less stable than WT thrombin, which in turn
showed a behavior similar to that reported previously
[15] The higher sensitivity to urea denaturation shown
by the mutant may explain why DK9 thrombin is
clin-ically characterized by a much lower phenotypic
expression in vivo, likely as a consequence of
intracellu-lar precipitation or enhanced degradation [2]
The stability of the two thrombin variants was
fur-ther investigated using a more complex denaturation
procedure, referred to as disulfide scrambling [16] One
disulfide bond connects covalently the A- and B-chain
(Cys1–Cys122) in the thrombin molecule, whereas the
B-chain is stabilized by three intrachain disulfide bonds
(Cys42–Cys58, Cys168–Cys182 and Cys191–Cys220)
[17] In disulfide scrambling, urea breaks noncovalent
interactions between the two chains, subsequently
enhancing the susceptibility of the connecting disulfide
bonds to the reductive action of b-mercaptoethanol
However, the low reducing agent concentration allows
the disulfide bonds to scramble and rearrange
accord-ing to conformational changes induced by the
denatu-rant This allowed us to assess, better than by the
simple urea-induced denaturation, whether the deletion
of the K9 residue in the A-chain could affect the
con-formation stability of the whole thrombin molecule,
as a consequence of perturbed intra- and interchain
bonds
After 180 min denaturation with 6 m urea and
0.2 mm b-mercaptoethanol, the A-chain was released
and the the native enzyme form disappeared for both
WT and mutant thrombin (Fig 5) The kinetic rate
constant of free A-chain release was 1.69 ± 0.03·
10)2Æmin)1 in the WT form and 2.96 ± 0.05·
10)2Æmin)1 in DK9 thrombin (Fig 6) The
disappear-ance of the intact enzyme (A- + B-chains) was
charac-terized by a first-order rate constant equal to 1.69 ±
0.06 and 3.01 ± 0.02· 10)2Æmin)1 for WT and DK9
thrombins, respectively Furthermore, the lag time for
the early appearance of the stable isomer ‘3’ shown in
Fig 6 is shorter in DK9 (20 min) than in the WT form
(33 min)
Characterization of stabilizing interactions between A- and B-chains
In WT thrombin, the A-chain assumes an overall boomerang-like shape interacting with the B-chain sur-face opposite to the active site [17] Stabilization within the A-chain and between the A- and B-chains occurs mainly through salt bridges and H bonds involving charged side chains The A-chain is intramolecularly cross-linked by five side-chain electrostatic interactions grouped into three separate clusters (D1a–K9, K14a– D14–R4–E8 and E13–R14d) Besides the covalent disulfide connection between Cys1 and Cys122, seven salt bridges, grouped into five clusters (D1a–R206, E8–K202–E14c, D14–R137, K135–E14e–K186d and K14a–E23), interconnect A- with B-chain; almost 90%
Fig 5 HPLC chromatograms of disulfide scrambling of WT and DK9 thrombin Disulfide scrambling was obtained under 6 M urea and 0.2 m M b-mercaptoethanol at 0.5 min (upper) and 180 min (remaining chromatograms) The HPLC chromatogram pertaining to
WT thrombin after 180 min of treatment is reported in the middle
of the figure, whereas the chromatogram of DK9 form is given at the bottom The primed numbers refer to the stable B-chain iso-mers of DK9 thrombin.
Trang 7of the total electrostatic energy of the A–B-chain
inter-action has been calculated to be due to these salt
clus-ters [17] We calculated the total number of stabilizing
A–B interchain electrostatic⁄ H-bonding interactions
along the whole MD simulations, by using gromacs
routines (g_saltbr and g_hbond), and found that in the
WT form they are 10% more than in the DK9
mutant (25969 and 23471 in WT and DK9,
respect-ively) These computational results suggest that in this
case the higher the flexibility of the A-chain the higher
the number of electrostatic⁄ H-bonding contacts
between the A- and B-chain in the WT thrombin, in
agreement with slower release of the light chain under
mild reducing conditions, as shown by the disulfide
scrambling experiments
Conclusions
The results obtained in this study provide knowledge
about the perturbations triggered by deleting the Lys9
residue in the A-chain of thrombin [1,2]
Measure-ments of the pH dependence of both steady-state
ami-dase activity and binding of the high-affinity inhibitor
a-NAPAP showed pKa values of the catalytic His57
higher in DK9 mutant than in WT thrombin
Applica-tion of the Brønsted theory on acid⁄ base-catalyzed
reactions indicated that in the thrombin amidase cycle the His57 imidazolium form acts as a general acid to facilitate amine expulsion from the tetrahedral interme-diate, and this process is the rate-limiting step for the overall acylation reaction The increased basicity of the His57 N (nitrogen in the DK9 mutant would oppose this function, resulting in a decrease in its catalytic competence, as shown experimentally by both in vitro and in vivo data)
Based on disulfide scrambling denaturation experi-ments, we inferred that in the DK9 mutant a refolded A-chain should reduce the structural stability of the whole a-thrombin molecule, weakening A–B interchain contacts Previously reported MD simulations showed
a transition of the A-chain from a boomerang-like shape (WT) to a handle-like shape (DK9) [2] Computa-tional studies highlighted lower conformaComputa-tional flexibil-ity in the A-chain resulting in fewer A–B interchain electrostatic⁄ H-bonding contacts in the DK9 mutant These A-chain folding effects should be allosterically transmitted to the active site cleft: (a) altering the geometry and protonation state of the residues involved
in catalysis and inhibitor binding, and (b) limiting the allosteric effects triggered by sodium binding
X-ray structures of human thrombin show that the A-chain closely follows the contour of the catalytic B-chain, hinging the two interacting six-stranded bar-rel-like domains of the B-chain [17] A well-structured network of ionic and H-bond interactions stabilize the correct orientation of the two barrels in the catalytic B-chain Deletion of Lys9 may cause a re-registration
of this ionic network In particular, in the WT forms Asp14 makes a very strong salt bridge with Arg137 In the DK9 mutant this salt bridge should be severely per-turbed, because Asp14, as a consequence of Lys9 dele-tion, preferentially interacts with Lys202, which is electrostatically linked to Glu14C in WT thrombin [17] Destruction of the salt bridge Asp14–Arg137 could alter the environment in which two Trp residues, namely Trp207 (at vdW distance from Arg137) and Trp29, are located, modifying its polarity and inducing conformational changes in the two Trp side chains which would be in DK9 more exposed to the solvent,
as our calculations on MD conformations proved It is known that the higher the conformational flexibility of
a fluorescent molecule the lower its fluorescence quan-tum yield [13], and Trp207 predominantly contributes
to the global fluorescence of thrombin [12] Taking these reports and our data into account, it is reason-able to hypothesize that even a subtle perturbation in the polarity and⁄ or flexibility of the environment of the Trp residues could significantly affect the fluores-cence of the DK9 thrombin Moreover, the
hydropho-Fig 6 Kinetics of disulfide scrambling of WT and DK9 thrombin.
Kinetics of reduced A-chain release from WT (s) and DK9 thrombin
(h) Continuous lines were drawn according to a single exponential
equation with the best-fit first order rate constant equal to
1.69 ± 0.03 · 10)2Æmin)1 for the WT form, and 2.96 ± 0.05 ·
10)2Æmin)1for DK9 thrombin The kinetics of disappearance of the
intact adduct of A with B chain for WT (d) and DK9 thrombin (n) is
also shown The single exponential decay rate constant was equal
to 1.70 ± 0.06 and 3.01 ± 0.02 · 10)2Æmin)1 for WT and DK9
thrombin, respectively The vertical bars are the standard errors of
the experimental measurements.
Trang 8bic cluster below Arg137, comprising the side chains of
Phe181, Phe199, Phe227, and Tyr228, close to the
act-ive site, may be destabilized, with repercussions for the
catalytic pocket, as shown in crystal structure studies
[10] In particular, a perturbed conformational change
of Trp215, linked to Na+ binding and likely
respon-sible for the reduced fractional change in fluorescence
in the Na+-bound DK9 conformer, may be another
evidence that the conformational transition caused by
Lys9 deletion in the A-chain are sensed by catalytic
subsites of the mutant enzyme These findings lead us
to propose that a significant uncoupling between Na+
binding and conformational changes sensed by the
fluorescence change of Trp215 takes place in DK9
thrombin
Globally taken, the experimental and computational
studies reported herein provide mechanistic support to
the phenomenological evidence that the A-chain would
affect both the conformation and the catalytic activity
of the thrombin B-chain, thus corroborating the belief
of an extraordinary conformational plasticity of this
enzyme
Experimental procedures
Site-directed mutagenesis and construction
of expression vectors
Site-directed mutagenesis, expression, activation and
purifi-cation of WT and DK9 thrombin form were obtained as
recently detailed [1,2] The active-site titration of thrombin
forms, obtained by using p-nitro-phenyl guanidinobenzoate
gave a concentration of 95 ± 5% with respect to that
measured spectrophotometrically at 280 nm using E¼
1.83 mgÆmL)1 SDS⁄ PAGE showed a single band of
~ 36 kDa for all the thrombin forms
Effect of pH on thrombin amidase activity and
binding of the inhibitor a-NAPAP to the thrombin
active site
The effects of pH (5.5–10) on the hydrolysis of
d-Phe-Pip-Arg-pNA substrate by both WT and DK9 mutant thrombin
and binding of the inhibitor a-NAPAP to the enzyme active
site were analyzed The experiments were carried out in an
appropriate triple buffer (25 mm Bis⁄ Tris, 25 mm Tris,
50 mm CHES, 0.15 m NaCl, 0.1% PEG 6000) This buffer
system allowed us to keep the ionic strength of the solution
nearly constant over the entire pH range [18] Calculations
performed with a program written in basic showed that
using the triple-buffer system in presence of 0.15 m
mono-valent salts, the ionic strength falls to a value 2.5% lower
than that at extreme pH values The Michaelis–Menten
constants of d-Phe-Pip-Arg-pNA (Instrumentation Labor-atory, Milan, Italy) hydrolysis, as well as the equilibrium dissociation constant of a-NAPAP (Sigma-Aldrich, St Louis, MO) binding to thrombin, were calculated as detailed previously [8] The kinetic scheme for the catalytic cycle of thrombin in the steady-state analysis is given in Scheme 1
E, ES and EP are the free, Michaelis–Menten and
acylat-ed enzyme forms, whereas k1, k-1, k2and k3are the kinetic constants for substrate binding, dissociation, acylation and deacylation, respectively Within the overall acylation step
of the canonical Scheme 1 (k2), tetrahedral intermediate (TI) formation (kB) or breakdown (kA) may be rate limiting [7,19–21] (Scheme 2)
Recently, a kinetic study showed that DK9 thrombin catalyses the hydrolysis of a synthetic amide substrate with
a k2<< k3[2] Under these conditions, k2 kcat Further-more, for both WT and DK9 thrombin Km Kd, that is the equilibrium dissociation constant of substrate’s binding
to thrombin The effect of protons on this kinetic scheme was analyzed by an appropriate extension of Scheme 1, whereby binding and dissociation of protons were consid-ered much more rapid than all binding and catalytic steps
of the substrate [3,4] Thus the kinetic Scheme 1 was expan-ded, assuming the existence of two ionizable thrombin groups involved in catalysis, as emerged by a best-fit mini-mization procedure of the experimental data taken over a 5.5–10 pH range Accordingly, Scheme 1 was expanded as shown in Scheme 3
In Scheme 31K and 2K are the equilibrium association constant for proton binding to the unprotonated and mono-protonated thrombin form, respectively, whereas the
‘s’ and ‘p’ subscript refer to ES and EP thrombin species, and ‘1’ and ‘2’ superscript refer to the mono- and diproto-nated thrombin species, respectively Using the linkage scheme analysis detailed previously [3,4], the pH effects on the Michaelis–Menten parameters kcat, Km, and kcat⁄ Km of
WT and DK9 mutant thrombin hydrolysis of d-Phe-Pip-Arg-pNA were analyzed as follows:
obs
Km¼0Km
ð1 þ1KHÞð1 þ2KHÞ ð1 þ1KsHÞð1 þ2KsHÞ ð2Þ where Kmvalues were approximated to the equilibrium dis-sociation constant of the substrate binding to thrombin [2],
0
Kmis the asymptotic Kmvalue in absence of protons, and
1
K and2K are the equilibrium association constant of pro-ton (H) binding to the first and second ionizable group for free, and ES thrombin species (the latter denoted by the
Scheme 1.
Trang 9‘s’ subscript), respectively Likewise, the observed kcat
val-ues were analyzed as a function of pH as follows [3,4]:
obs
kcat¼
0kcatþ ð1kcatð1Ksþ 2KsÞH þ2kcatð1Ks 2KsÞH2
ð1 þ1KsHÞð1 þ2KsHÞ ð3Þ where0kcat,1kcat, and2kcatrefer to the kcatvalue pertaining
to unprotonated, mono- and diprotonated thrombin form,
respectively Hence, the pH dependence of the observed
kcat⁄ Kmvalue (referred to as ‘r’) is:
obs
r¼ ½r0þ ðr1 1K HÞ þ r2ð1K2K H2Þ=Z ð4Þ
where the superscript 0, 1, and 2 refer to the unprotonated,
mono- and diprotonated thrombin form, respectively, and
Z¼ (1 +1KH) (1 +2KH)
In the data sets of NAPAP inhibition of
d-Phe-Pip-Arg-pNA hydrolysis at each pH value, the steady-state velocity
of cleavage of seven substrate concentrations (0.5–32 lm)
was studied in the presence of seven fixed NAPAP concen-trations (1–64 nm) and were simultaneously analyzed using
a simple scheme, whereby both binding of NAPAP and
d-Phe-Pip-Arg-pNA to the thrombin active site were mutu-ally exclusive [8] In the analysis of pH effects on NAPAP binding, the Km values were replaced in Eqn (2) by the Ki
values, calculated by the above competitive scheme [8] Fitting of catalytic parameters was constrained to an internally consistent picture, such that, at the three proto-nation levels, values of kcat, Km, and kcat⁄ Km must be closely related according to Eqns (2–4) Global and simul-taneous analysis of the experimental data by grafit soft-ware allowed computation of the pKa values of the two groups both in the free and substrate-bound species of thrombin forms, along with the kinetic parameters’ values pertaining to the three protonated thrombin species The values of standard errors (± SD) were also obtained in the fitting procedure
Denaturation by urea Urea-induced denaturation curves of both WT and DK9 thrombin were obtained in 10 mm Bis⁄ Tris, 0.15 m NaCl,
pH 6.80, by monitoring the fluorescence emission at
342 nm (excitation at 280 nm) in a Varian Eclipse spectro-fluorometer (Leini, Italy) Spectra were taken with an exci-tation⁄ emission slit of 5 nm The results were expressed as the percentage of the measure fluorescence at any urea con-centration compared with that obtained in the absence of the denaturating agent
Denaturation by disulfide scrambling Denaturation of both WT and DK9 thrombin was per-formed by disulfide scrambling, that is the unfolding of thrombin by urea in the presence of low concentrations of the reducing agent b-mercaptoethanol, as reported
(50 lgÆmL)1 in 0.1 m Bis⁄ Tris buffer, pH 6.8) were treated with 6 m urea in the presence of 0.2 mm
b-mercaptoetha-Scheme 2.
Scheme 3.
Trang 10nol Denaturation was performed at 25C for 3 h to allow
the reaction to reach equilibrium To monitor the kinetics
of denaturation, 50 lL of the samples were removed at
dif-ferent time intervals, mixed with an equal volume of 4%
TFA, and analyzed by RP-HPLC, using a Bio-Rad C18
Hi-Pore RP-318, 250· 4.6 mm column The eluant was
composed of solvent A (0.1%) trifluoroacetic acid, and
solvent B was acetonitrile⁄ water (9 : 1 v ⁄ v) containing
0.08% trifluoroacetic acid The gradient was 20–40%
sol-vent B for 10 min, linearly increased from 40 to 55% for
40 min, kept at 55% for 10 min, and reduced to 20%
sol-vent B in 20 min, while the flow rate was 0.5 mLÆmin)1
The HPLC instrument was a PU-2080 instrument
connec-ted to a UV-2075 spectrophotometer (Jasco Europe s.r.l.,
Cremella, Italy) The chromatographic peaks were analyzed
and quantified using borwin-1 software (Jasco Europe)
The results were expressed as a percentage with respect to
the peak area measured at the start The kinetic data
refer-ring to the disappearance of the native enzyme, [A + B%],
were fitted to the single exponential decay equation:
where [A + B%]t is the percentage of the native enzyme
present at time t, and k is the first-order rate constant of
this process The appearance of the free A-chain at time t,
referred to as [A%]t, was fitted to single exponential
rela-tion:
½A%t¼ 100 ð1 expðktÞÞ ð6Þ
where k is the first-order rate constant of the A chain
release
At the end of the denaturation process three stable B
chain isomers were produced, in agreement with previous
studies, although obtained at different pH [16]
The kinetic Eqns (5–6) were fitted to the experimental
data using the grafit program (Erithacus Software Ltd,
Staines, UK)
Binding of sodium ion
Steady-state fluorescence titration measurements were
car-ried out using 500 nm WT and mutant thrombin, as
des-cribed previously [9] Fluorescence emission spectra (kex¼
280 nm) were recorded at 25C in a 1 cm quartz cell, using
a Varian Eclipse spectrofluorometer (Leini, Italy), in
5 mmolÆL)1 Tris, pH 8.0 and increasing amount of NaCl
Emission spectra between 300 and 400 nm did not show
any significant peak shift Titrations were performed by
acquiring the changes in fluorescence intensity at the peak
of the emission spectrum at 342 nm The concentration
of Na+ was increased by aspirating a defined volume of
thrombin solution dissolved in the cuvette in the above
buf-fer containing 0.2 m tetramethylammonium chloride and by
adding the same volume of a thrombin solution (at the
same concentration) dissolved in the same buffer but
taining 0.2 m NaCl This procedure allowed us to keep con-stant both the concentration of thrombin and the ionic strength of the solution The decrease in fluorescence signal, usually defined as ‘bleaching effect’, due to the iterative exposure of the sample to high intensity light beam, was restricted to < 3% of the initial intensity and was always taken into consideration in the analysis of the titration data
Computational methods gromacs 3.2.1 software [22], running on a Linux PC clus-ter, was used for the MD simulations and analysis of the trajectories
Using the gromacs utilities, the ED analysis was carried out in to separate the motions of the examined protein models into an essential subspace, describing most of the functional motions, and into a physically constrained sub-space, describing irrelevant local fluctuations Eigenvectors defining the direction of higher displacement are extracted
in decreasing order of the corresponding eigenvalue, and the first few eigenvectors capture most of the essential motions of the proteins The cross-correlation function between the projection of protein segments pairs onto the first eigenvector obtained from the ED analysis of both seg-ments was also calculated to estimate correlation of the essential motions
Acknowledgements
Financial support from Italian Ministry of Education, Universities and Research (MIUR) is gratefully acknowledged by CA and RDC (PRIN 2003, Grant
no 2003064812) We thank Dr Vincenzo De Filippis (University of Padova, Italy) for helpful comments and critical reading of the manuscript
References
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2 De Cristofaro R, Akhavan S, Altomare C, Carotti A, Peyvandi F & Mannucci PM (2004) A natural pro-thrombin mutant reveals an unexpected influence of A-chain structure on the activity of human alpha-thrombin J Biol Chem 279, 13035–13043
3 Di Cera E, De Cristofaro R, Albright DJ & Fenton JW
II (1991) Linkage between proton binding and amidase activity in human a-thrombin: effect of ions and tem-perature Biochemistry 30, 7913–7924