From the Max-Planck-Institut für Biochemie,
D-82152 Martinsried, Germany, § Roche Diagnostics GmbH,
D-82372 Penzberg, Germany, and
Ludwig-Maximilians-Universität, Genzentrum,
D-81377 München, Germany
Received for publication, October 19, 2002, and in revised form, November 13, 2002
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ABSTRACT |
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Coagulation factor IXa (fIXa) plays a central
role in the coagulation cascade. Enzymatically, fIXa is characterized
by its very low amidolytic activity that is not improved in the
presence of cofactor, factor VIIIa (fVIIIa), distinguishing fIXa from
all other coagulation factors. Activation of the fIXa-fVIIIa complex requires its macromolecular substrate, factor X (fX). The 99-loop positioned near the active site partly accounts for the poor activity of fIXa because it adopts a conformation that interferes with canonical
substrate binding in S2-S4. Here we show that residues Lys-98 and
Tyr-99 are critically linked to the amidolytic properties of fIXa.
Exchange of Tyr-99 with smaller residues resulted not only in an
overall decreased activity but also in impaired binding in S1.
Replacement of Lys-98 with smaller and uncharged residues increased
activity. Simultaneous mutagenesis of Lys-98, Tyr-177, and Tyr-94
produced an enzyme with 7000-fold increased activity and altered
specificity. This triple mutant probably mimics the conformational
changes that are physiologically induced by cofactor and substrate
binding. It therefore provides a cooperative two-step activation model
for fIXa. Tyr-177 locks the 99-loop in an inactive conformation which,
in the physiologic complex, is released by cofactor fVIIIa. FX is then
able to rearrange the unlocked 99-loop and subsequently binds to the
active site cleft.
The blood coagulation factor IXa is a trypsin-like vitamin
K-dependent serine protease that circulates in the plasma
as a single chain inactive zymogen (1, 2). Its activation requires cleavage of two peptide bonds by either the activated factor VII (fVIIa)-tissue factor complex or activated factor XI (fXIa) (3, 4) to
remove a 35-residue activation peptide. The active enzyme, fIXa The unique behavior of fIXa is related to a set of
surface-exposed structure elements. In particular, the conformation of the fIXa 99-loop deviates considerably from those observed in the
related enzymes of the fIX gene family (10-14). Hopfner et al. (15) show that exchange of the 99-loop (chymotrypsinogen numbering used throughout within the serine protease domain) with the
corresponding sequence of fXa results in a 22-fold increase in activity
toward synthetic substrates. Kolkman and Mertens (16) further
demonstrate that the 99-loop restricts enzymatic activity toward fX and
synthetic substrates in the absence, but not in the presence, of the
cofactor fVIIIa.
The side chain of Tyr-99 adopts different conformations in the two
crystal structures of fIXa reported to date (11, 13). In both
structures Tyr-99 is in steric conflict with canonical substrate
binding in the S2-S4 sites. These observations suggest a critical role
of Tyr-99 and spatially neighboring amino acids for the substrate
dependent activity of fIXa. In particular, Lys-98 is likely to
electrostatically interfere with the basic substrate preference of
fIXa.
Important questions remain open, however, including (i) What are the
distinguishing mechanisms of substrate-assisted activity enhancement of
fIXa?, (ii) What are the mechanisms underlying the cofactor-assisted
activity enhancement of fIXa?, and (iii) Are these mechanisms
independent from each other or coupled on a molecular basis?
To address these questions, we introduced a series of point
mutations in the 99-loop of a truncated version of fIXa, designated rf9a, lacking the N-terminal Gla and epidermal growth factor-1 domains. In detail, residues Lys-98 and Tyr-99 were exchanged with
smaller and/or uncharged residues. In addition, two solvent-exposed alanine residues from the 99-loop were deleted, and we constructed multiple mutants, where several interactions stabilizing the 99-loop were simultaneously abolished. We investigated the influence of the
primary specificity pocket on activity by mutating the fIX-specific residues Glu-219 and Ser-190 positioned at the entrance and within the S1 site, respectively.
Chemicals--
N-Methylsulfonyl-D-Phe-Gly-Arg-4-nitroanilide
acetate (MS-D-Phe-Gly-Arg-pNA),
methoxycarbonyl-D-Norleucyl-Gly-Arg-4-nitronilide acetate
(MOC-D-Nle-Gly-Arg-pNA), and human antithrombin III were purchased from Roche Diagnostics GmbH (Mannheim, Germany), and Q-Sepharose ff and Heparin-Sepharose CL 6B were from Amersham Biosciences. para-Aminobenzamidine (PABA), porcine heparin
(grade I-A), and crude Russell's viper snake venom (RVV) were obtained from Sigma Aldrich. The fX activator RVV-X was purified as described elsewhere (17). 4-Nitrophenyl-4-guanidiniumbenzoate and benzamidine were from Merck. Purified human Bacterial Strains and Plasmids--
Cloning and expression of
proteins was done in the Escherichia coli K12 strain UT5600
(18) carrying the lacIq repressor plasmid pUBS520 (19). The
plasmid pSA (20) was used for expression of recombinant proteins.
DNA Manipulation Techniques--
All techniques used for
amplification, mutagenesis, and cloning of DNA followed published
protocols (21). Commercial kits were used according to the
manufacturer's instructions.
Construction of Recombinant Proteins--
The rf9 variant
encoded the second epidermal growth factor domain, the activation
peptide, and the catalytic domain of human fIX (15). The mutant
proteins were produced using the plasmids pf9 and
pf9-99loop(F10) as templates and oligonucleotides carrying the
desired mutation as primer (Table I) as
described earlier (15).
Production of Recombinant Proteins--
The rf9 variants
were expressed, folded from inclusion bodies, and purified as
previously described (15). Activation was achieved using RVV-X, and the
concentration of active sites was determined using thrombin-titrated
antithrombin III and heparin as active site reactants (15, 22).
Amidolytic Assays and Determination of Kinetic
Parameters--
Experiments were performed in 50 mM Tris,
150 mM NaCl, 5 mM CaCl2, 0.1%
polyethylene glycol 8000, pH 8.0, at 25 °C in a
temperature-controlled Kontron-Uvikon 933 spectrophotometer (Kontron
Instruments, Milano, Italy). Substrate concentrations ranged from 30 µM to 5 mM. Enzyme concentrations were
between 2 and 200 nM. Reactions were started by adding the
enzyme to the pre-warmed reaction buffer containing different substrate
concentrations. The change in absorption at 405 nm was monitored for
typically 2 min, and Conformational and Catalytic Properties of Mutant Enzymes--
The
location of the mutations is summarized in Fig.
1. We examined enzymatic activity and
conformational aspects of the mutants. The amidolytic activity and
changes in selectivity in S2/S4 were probed by using the peptidic
substrates MOC-D-Nle-Gly-Arg-pNA (substrate A) and
MS-D-Phe-Gly-Arg-pNA (substrate B). To measure the effects
of the mutations on the conformation of the primary specificity pocket,
we also determined the binding constant Ki of the
reversible S1 site inhibitor PABA in competition with substrate A. The
results for the point mutations of Tyr-99 and Lys-98 as well as
the Ala95a/Ala95b deletion mutant are summarized in Table II.
As summarized in Fig. 1, structure analysis indicated that Tyr-99
hinders the direct substrate access to the S4 binding site (11, 13).
Therefore, we replaced Tyr-99 with several smaller residues. These
substitutions always resulted in a decreased activity, and the
reductions inversely correlated with the size of the introduced residue; the activity of rf9a-Y99L with substrate A was reduced to 41%, whereas rf9a-Y99A retained only 5% of its original
activity. Likewise, the affinity for the inhibitor PABA was decreased
depending on the reduction of the side chain size. Despite these
effects on activity and inhibitor binding, only the mutation Y99T had an influence on the selectivity of the enzyme as measured with two
synthetic substrates.
Fig. 1 also suggests that the positively charged side chain of Lys-98
might interfere with the access of basic residues to the S1 pocket
(11). Therefore we set out to test the role of both size and charge of
the residue at position 98. Replacement of Lys-98 with the larger but
equally charged arginine had only a minor effect on the amidolytic
activity. In contrast, rf9a-K98M resulted in a 2.8-fold, and
rf9a-K98T resulted in a 6.7-fold increase in activity with
substrate A. Although PABA binding was hardly affected in
rf9a-K98R, it was improved in the other 98 mutants, leading to a
Ki reduced to ~60% that of the original value
(see Table II). In addition to the impact on activity and inhibitor
binding, the mutations also influenced the selectivity for the two
tested substrates. The stronger the activity increased, the more the
preference was altered toward the substrate A, which was less preferred
in the wild-type enzyme.
Ala95a-Ala95b is a factor IXa-specific two-residue insertion as
compared with trypsin-like serine proteases. This distinct sequential
motif prompted us to probe the role of this insertion on rf9a
amidolytic activity. Deletion of Ala95a/Ala95b, thus, aimed to
reconstitute the 99-loop in a conformation resembling that of related
coagulation factors. Nevertheless, this deletion had only little
influence on all parameters examined.
Multiple Mutants Probing the Conformation of the 99-Loop--
The
conformation of the 99-loop in fIXa relates to a series of more subtle
structural characteristics in addition to a two-residue insertion
(Ala95a-Ala95b). Its conformation is stabilized by a hydrogen bond
between the carbonyl of Lys-98 and the hydroxyl of Tyr-94, which is
specific to fIXa. Also, Tyr-177 and Lys-98 would collide if the
fIXa-99-loop were to adopt an fXa-like conformation. Moreover, the
structure analysis revealed that Tyr-177 stabilizes and locks the
99-loop in its inactive conformation by interactions with Asn-97 and
Asn-100 (cf. Fig. 1). Given the impact of mutants in the
99-loop on the activity, in particular K98T, we constructed multiple
mutants to mimic the cooperative mode of fIXa activation in the
physiological context. The kinetic parameters (activity, selectivity,
and inhibitor binding) for these enzymes together with the
corresponding wild-type data are summarized in Table III.
The triple mutant rf9-Y94F/K98T/Y177T had a 7000-fold increased
activity when compared with rf9a and a 1000-fold increased activity compared with the single mutant rf9a-K98T, resulting in
an amidolytic activity comparable with that of rf10a.
rf9-99loop(F10)-Y94F/Y177T had a 3-fold higher activity than
rf9-99loop(F10) and a 51-fold increased activity compared with
rf9a. The affinity for the S1-directed inhibitor PABA in the
triple mutant was essentially the same as in rf9a, whereas in
rf9-99loop(F10)-Y94F/Y177T it resembled that of
rf9-99loop(F10) and increased ~2-fold compared with
rf9a. The selectivity of the mutants for the two substrates was
in between those of rf9a and rf10a.
Influence of S1 Site Mutations on the Activity of
fIXa--
Glu-219 is positioned at the entrance frame to the S1 site
of fIXa. It is particularly interesting because Gly-219 is conserved in
almost all trypsin-like serine proteases, including trypsin itself and
the coagulation factors. Because Glu-219 has an apparently unchanged
backbone conformation compared with trypsin (11), we exchanged Glu-219
against Gln, thereby removing the salt bridge with Lys-224 and Ser-190
against Ala. The kinetic parameters for these mutations and the wild
type as well as our earlier data are shown in Table
IV.
rf9-E219Q had a 30% decreased activity with substrate A,
paralleled by a 20% decreased affinity for PABA. The selectivity for
the two substrates was unchanged. rf9-S190A had a rather
unaltered activity, whereas Ki was increased by
82%. The selectivity was altered toward a preference for substrate A.
The Ethylene Glycol Effect Depends on the 99-Loop--
The
reactivity of some mutants in the presence of ethylene glycol was
examined with two different substrates. The results are summarized in
Fig. 2. All tested rf9a variants
had a maximum stimulation at ~35% ethylene glycol. The extent of
stimulation correlates inversely with the catalytic turnover, which
depends on both the mutant enzyme and the used substrate. The maximum 20-fold activity enhancement was observed with the wild-type
rf9a and the worse substrate A (see Fig. 2). Analogously, the
more active mutants showed less stimulation. The fXa mutant
rf10a-99loop(F9), containing the fIXa-derived loop, was stimulated to a
maximum of 1.5-fold at 15-20% ethylene glycol and was inhibited at
concentrations above 27 and 37%, depending on the substrate. Wild type
fXa is inhibited by ethylene glycol already at low concentrations.
Proteolytic Activation of fIX Occurs in Two Steps--
Even at
millimolar concentrations, all zymogens were unable to auto-activate.
This behavior contrasts that of related enzymes such as factor VII
where the zymogen autoactivates spontaneously (23). Instead, the
wild-type and mutant fIX enzymes required an exogenous protease (RVV-X)
to cleave the peptide bond Arg-180-Val-181 (residues 15 and 16, chymotrypsinogen numbering) and to initially produce fIXa FIX Lacks Any Zymogen Activity--
In
vitro the activation of fIX requires two distinct enzymatic
events. RVV-X performs the initial cleavage, at Arg-180-Val-181, of
single chain fIX to produce fIXa
The active site mutant rf9a-S195A was cleaved only once in the
presence of RVV-X, thereby converting single-chain rf9-S195A into the active conformation. Two-chain rf9a-S195A was not
further processed. This contrasts the situation in related serine
proteases, which display residual activity when carrying the
corresponding mutation of the catalytic residue Ser-195 (26-28).
Tyr-99 Stabilizes the 99-Loop--
Structure analysis indicated
that Tyr-99 hinders the direct substrate access to the S4 binding site.
Therefore, we replaced Tyr-99 with several smaller residues.
Counterintuitively, the activity of these mutants decreased. The
reactivity was lowest with the smallest side chain substitution, Y99A.
Moreover, binding of the S1 inhibitor PABA was also impaired. This
indicates that the removal of Tyr-99 results in complex changes,
affecting not only the S2-S4 but also the S1 site by either blocking
access to the primary specificity site or by even destabilizing its
conformation. The side chain of Tyr-99 is the part of the 99-loop
closest to the S1 pocket. Removal of the bulky side chain of Tyr-99
probably causes the loop to slide further into the S2-S4 substrate
binding cleft. As a consequence, the positively charged side chain of Lys-98 comes closer to the entrance to S1 and repels basic substrate residues from binding into the primary specificity pocket.
Charge and Size of Lys-98 Disturb Substrate Binding--
To
elucidate the influence of charge and size of Lys-98 on substrate
binding, the activity of mutants with uncharged and shorter side chains
in position 98 was examined. rf9a-K98M has an increased activity, confirming the working hypothesis of an electrostatic repulsion of basic substrate residues by Lys-98. The activity of
rf9a-K98T is even higher, in line with the assumption that in
the wild-type enzyme both charge- and size-hinder substrate binding.
The binding affinity of the S1 inhibitor PABA is doubled by the charge
removal but independent of the size of the side chain at position 98. This indicates that, whereas the binding of positively charged P1
residues is electrostatically hindered by Lys-98, the size of the side
chain at position 98 presumably only affects the accessibility to the
S2/S4 site but not to the S1 site.
The Ala95a/Ala95b Insertion in the 99-Loop Does Not
Affect the Amidolytic Activity of fIXa--
The Ala95a/Ala95b
insertion is specific to fIXa and, based on sequence comparison, is the
most prominent difference to the related enzymes. Therefore, the
deletion of these residues represents a straightforward approach to
examine whether the low catalytic activity originates from these amino
acids. However, the deletion of Ala95a/Ala95b did not have a
significant impact on either activity or selectivity of rf9a.
Tyr-177 Locks the 99-Loop in an Inactive Conformation, Which Is
Released by Cofactor fVIIIa and Rearranged by fX--
The unique
conformation of the 99-loop in fIXa relates to several elements,
including a two-residue insertion (Ala95a-Ala95b) and a series of more
subtle structural characteristics. Its conformation is stabilized by a
hydrogen bond between the carbonyl of Lys-98 and the hydroxyl of
Tyr-94. Because fXa has a Phe-94, this hydrogen bond is not existent in
fXa. Also, Tyr-177 and Lys-98, both of which are threonines (or
serines) in fXa, fVIIa, and protein C, would collide if the
fIXa-99-loop were to adopt an fXa-like conformation. Moreover, the
structure analysis revealed that Tyr-177 stabilizes and locks the
99-loop in its inactive conformation by interactions with Asn-97 and
Asn-100.
This rationale was examined by constructing a triple mutant
rf9a-Y94F/K98T/Y177T as well as a chimerical rf9a
containing the 99-loop of fXa and the fXa-like environment of the loop
(Y94F/Y177T). Both mutants had a largely increased amidolytic activity.
rf9a-99loop(F10)-Y94F/Y177T did not achieve the same activity as
the triple mutant. Although the access to the substrate binding cleft
is facilitated, and the steric clash between Lys-98 and Tyr-177 has
been removed, the heterologous loop can obviously not adopt the optimal conformation.
In the highly active triple mutant rf9a-Y94F/K98T/Y177T, the
99-loop and the 177-segment probably approach the physiologically active conformation and open the active site cleft for substrate binding. These data suggest a mechanism of the physiological fIXa activation by cofactor and substrate binding. In this model, cofactor binding close to the 177-segment (5, 10) will release the lock of the
inactive 99-loop imposed by Tyr-177. Only the physiological substrate
fX is able to rearrange the released 99-loop, paving itself its way
into the active site cleft of fIXa. Both steps of the fIXa activation
are combined in the triple mutant, explaining its high activity toward
peptidic substrates.
Notably, the conformation of the S1 site remains mainly unaltered in
both multiple mutants as shown by the binding affinity of the S1 site
inhibitor PABA (Table III). Thus, the introduced multiple mutations
mainly affect the S2-S4 recognition sites.
S1 Site Mutations Decrease the Activity of fIXa--
In serine
proteases, the S1 site usually contributes most to substrate
recognition and catalysis. Therefore, we examined the impact of
mutations localized at the S1 site on the fIXa activity. None of the
S1-directed mutations presented in this work and in earlier work (15)
resulted in an improved activity.
Glu-219 is particularly intriguing because Gly-219 is conserved in
almost all trypsin-like serine proteases, including trypsin itself and
the coagulation factors. Although energetically unfavorable, Glu-219
has an apparently unchanged backbone conformation compared with
trypsin. We attempted to clarify the importance of the charge of
Glu-219 and its salt bridge with Lys-224 for fIXa activity by
isosterically replacing it with Gln. Both activity and inhibitor binding were slightly impaired, indicating a moderate disturbance of
the S1 site geometry. In the S190A mutant, the absence of a hydroxyl in
the S1 site explains the weakening of PABA binding.
If the S1 site is affected during physiological activation, these
changes are very subtle and are difficult to mimic by mutagenesis. Alternatively, the primary specificity pocket may be only of minor relevance for cofactor activation. The latter hypothesis is supported by only small differences in PABA binding between fIXa and fXa (Table
IV).
Ethylene Glycol Binding at the 60-Loop Allosterically Stimulates
the Activity of fIXa by Reorganizing the
99-Loop--
Stürzebecher and co-workers (29) speculate that
the stimulation of fIXa activity by ethylene glycol and other alcohols is mediated by interaction of these alcohols with the 99-loop. Kolkman
and Mertens (16) report that a fIXa variant with mutations in the
99-loop and increased amidolytic activity was stimulated by ethylene
glycol to a lesser extent than the wild type but resulted in the same
maximum activity. We recently showed that fIXa and fVIIa have a common
ethylene glycol binding site between residues 60 and 90 that is not
accessible in fXa (23). Occupation of this site by ethylene glycol
appears to mimic the effects of macromolecular substrate binding on the
99-loop. Thereby it stimulates catalysis of synthetic substrates in a
way that is usually only observed for macromolecular substrates that
are able to occupy both sites (60-loop region and active site cleft)
simultaneously in the presence of fVIIIa. The decreased stimulation of
the more active 99-loop mutants by ethylene glycol underlines this
interpretation; the 99-loop is already reorganized in these mutants,
thus precluding a further activity enhancement.
FXa is not stimulated by ethylene glycol. An fXa variant with the
99-loop of fIXa was stimulated but only to less than 10% compared with
fIXa. The inferior activity modulation of this mutant can be explained
by the lack of a proper ethylene glycol binding site in fXa despite the
presence of a 99-loop that is in principle susceptible to ethylene
glycol stimulation in this mutant.
The Closed Conformation of the 99-Loop Is a Major Insurance against
Premature Onset of Coagulation--
fIXa has evolved as an enzyme with
a strictly regulated activity. It is virtually inactive in the absence
of cofactor and physiologic substrate. This can be attributed mainly to
the 99-loop. The side chains of Tyr-99 and Lys-98 impair substrate
binding and are only removed by major reorganizations of several parts of the enzyme after binding of both the cofactor and the substrate. This "closed conformation" of the active site cleft is guaranteed by several mechanisms. Lys-98, Tyr-94, and Tyr-177 prevent a
spontaneous reorganization of the 99-loop. Tyr-99 blocks the canonical
substrate binding but also stabilizes the whole loop. A spontaneous
point mutation of Tyr-99 will, therefore, result in an even less active enzyme rather than open the substrate binding cleft. The cofactor and
substrate-assisted activation of fIXa specified by our results together
with other control mechanisms like Ca2+ and membrane
dependence of fIXa activity emphasize the exceptionally important role
of fIXa as a central enzyme of the coagulation cascade at the
intersection between extrinsic and intrinsic coagulation pathway as
well as between initiation and amplification of coagulation. Dysfunction of this enzyme is extremely dangerous in either direction and, therefore, underlies multiple control mechanisms.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
consists of two disulfide-linked chains. The light chain comprises the
N-terminal Gla domain with
-carboxylated glutamic acid residues and
two epidermal growth factor-like domains. The heavy chain forms the
serine protease domain. Activated factor IX
(fIXa)1 then activates fX in
a reaction that is dependent on the presence of calcium ions, a
membrane surface, and a nonenzymatic protein cofactor, activated factor
VIII (fVIIIa) (2). The importance of fIXa in hemostasis is reflected by
the occurrence of the bleeding disorder hemophilia B in individuals
carrying mutations in the fIX gene (5). fIXa displays only very little
proteolytic activity against natural or synthetic substrates in the
absence of its cofactor. Binding of fVIIIa results in a
106-fold increase in proteolytic activity for fX, whereas
the activity with peptidic substrates remains unaltered (6, 7). The
latter dramatic substrate-dependent activity modulation
distinguishes fIXa from the related coagulation enzymes fXa and fVIIa,
which in the presence of their cofactors, achieve a significant
activity enhancement with synthetic substrates (8, 9).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-thrombin was a generous gift from
Dr. Wirthensohn (Roche Diagnostics GmbH). Restriction enzymes and
enzymes used for PCR and cloning were from Roche Diagnostics GmbH and
New England Biolabs (Schwalbach, Germany). All other materials were of
the highest grade commercially available.
Oligonucleotides for cloning and mutagenesis of rf9a variants
E/min was calculated from the linear
part of the curve. Kinetic parameters were calculated by nonlinear
curve-fitting of the initial rates to the Michaelis-Menten equation
using the SigmaPlot software (Jandel GmbH, Erkrath, Germany). Inhibition constants of PABA were measured by competition with MOC-D-Nle-Gly-Arg-pNA. A 7 × 6 matrix was created by
varying the concentration of MOC-D-Nle-Gly-Arg-pNA (45 µM to 3 mM) and PABA (0-2 mM) by
repeated dilution by a factor of 2 and 3, respectively. The highest
concentration was usually 5-10-fold the Km of
MOC-D-Nle-Gly-Arg-pNA or Ki of PABA.
Kinetic data were calculated by global nonlinear least square fitting
of the reaction rate v to v = eTkcat[S]/(Km(1 + [I]/Ki) + [S]), where eT,
[S], and [I] denote the total concentration of enzyme, substrate,
and the inhibitor PABA, respectively. The influence of ethylene glycol
on the reactivity of the enzymes was examined by measuring their
activity in the presence of different ethylene glycol concentrations
and 1 mM substrate.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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View larger version (24K):
[in a new window]
Fig. 1.
Superposition of the 99-loops of fXa
(white) and fIXa (orange). The
main chain atoms of the loop are shown as well as the side chains of
residues 94, 98, 99, and 177. The red dotted line indicates
the steric clash between Tyr-177 and Thr/Lys-98, and the green
dotted line indicates the stabilizing hydrogen bond between Tyr-94
and Lys-98.
Specificity constants (kcat/Km) for the
hydrolysis of the synthetic substrates
MOC-D-Nle-Gly-Arg-pNA (A) and
MS-D-Phe-Gly-Arg-pNA (B) and binding constants (Ki)
for PABA for rf9a-99-loop single mutants
A95a/A95b denotes the two-residue deletion mutant where
the fIXa-specific insertions Ala95a and Ala95b are removed.
Specificity constants (kcat/Km) for the
hydrolysis of the synthetic substrates
MOC-D-Nle-Gly-Arg-pNA (A) and
MS-D-Phe-Gly-Arg-pNA (B) and binding constants (Ki)
for PABA for rf9a-99-loop multiple mutants
Specificity constants (kcat/Km) for the
hydrolysis of the synthetic substrates
MOC-D-Nle-Gly-Arg-pNA (A) and
MS-D-Phe-Gly-Arg-pNA (B) and binding constants (Ki)
for PABA for rf9a-S1 site mutants
View larger version (24K):
[in a new window]
Fig. 2.
Influence of the ethylene glycol
concentration on the hydrolysis of the synthetic substrates
MOC-D-Nle-Gly-Arg-pNA (A) and
MS-D-Phe-Gly-Arg-pNA (B) by several
rf9a (a) and rf10a
(b) variants.
. Notably,
we observed significant differences in the rate of the subsequent
cleavage at Arg-145-Ala-146 to generate fIXa
. Although not
quantified, the rate of fIXa
to fIXa
conversion qualitatively
correlated with the amidolytic activity of the mutants. Consistent
herewith, the active site mutant rf9a-S195A was cleaved only
once in the presence of RVV-X, thereby converting single chain
rf9-S195A into the active conformation. Therefore, fIXa, but not
RVV-X, performs the second cleavage at Arg-145-Ala-146, as proposed
earlier (4).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
but is unable to complete activation to fIXa
. Instead, the second cleavage, at
Arg-145-Ala-146, is performed auto-catalytically by fIXa. This
observation implies that proteolytic activation of fIX critically
depends on the presence of an already active enzyme, contrasting the
situation of, for example, fVII, which is able to auto-activate without
a prior "jump start" (23, 24). Given the sequence similarity of
both activation peptide cleavage sites, we expect active fIXa to be able to cleave at both sites. The need for an exogenous activator indicates that single-chain fIX is unable to undergo the conformational switch necessary for proteolytic activity (23, 25).
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence may be addressed. Tel.: 49-8856-60-7672; Fax: 0049-8856-60-3573; E-mail: Katrin.Sichler@roche.com.
** To whom correspondence may be addressed. Tel.: 49-89-8578-2828; Fax: 49-89-8578-3516; E-mail: hbs@biochem.mpg.de.
Published, JBC Papers in Press, November 19, 2002, DOI 10.1074/jbc.M210722200
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ABBREVIATIONS |
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The abbreviations used are: fIXa, activated factor IX; MS-D-Phe-Gly-Arg-pNA, N-methylsulfonyl-D-Phe-Gly-Arg-4-nitroanilide acetate; Nle, norleucine; MOC-D-Nle-Gly-Arg-pNA, methoxycarbonyl-D-Norleucyl-Gly-Arg-4-nitronilide acetate; PABA, para-aminobenzamidine; RVV, Russell's viper venom.
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