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INTRODUCTION |
Pyrococcus furiosus is a hyperthermophilic member of
the domain Archaea, one of the three major phylogenetic divisions of life. There is a paucity of data on the mechanisms of homologous enzymes from the three domains (Archaea, Bacteria, and Eukarya). Such
data have the potential to enhance our understanding of the evolutionary history of proteins and organisms and will certainly contribute to understanding of enzyme function at high temperatures.
Certain hyperthermostable enzymes share great similarities in catalytic
mechanism with their counterparts from mesophilic organisms. The
-glucosidase of P. furiosus has been compared with its
homologue from the mesophilic bacterium Agrobacterium faecalis (1). These studies suggest that the transition states of
these enzymes are extremely similar, indicating that the mechanism is
highly conserved. Mechanistic and structural studies of the
-glucosidase (2) and glutamate dehydrogenase (3, 4) from P. furiosus have demonstrated the similarities between enzyme homologues from organisms with very different temperature optima, and
structural data obtained for mesophilic proteins can be useful for
predicting the structure of hyperthermostable proteins.
Recently, the gene for the prolyl oligopeptidase of P. furiosus was cloned and overexpressed in Escherichia
coli as a functional protease (5). The recombinant prolyl
oligopeptidase displayed characteristics identical to those of the
enzyme expressed in P. furiosus (5), affording the
opportunity to readily obtain sufficient quantities of this protein for
kinetic studies.
The prolyl oligopeptidase from P. furiosus (EC 3.4.21.26,
formerly termed prolyl endopeptidase) is a serine protease of unusual
specificity, because it cleaves the sequence
X-Pro-Y (where X and Y are
any amino acids) on the carboxyl side of proline. Although it possesses
the catalytic triad composed of serine, histidine, and aspartate that
is characteristic of the serine proteases, its primary structure is
otherwise unrelated to that of the chymotrypsin, trypsin, or elastase
classes of serine proteases (6-8). In fact, the order of the
amino acids in the primary structure is different for prolyl
oligopeptidase (Ser, Asp, His) than it is for either the chymotrypsin
(His, Asp, Ser) or subtilisin (Asp, His, Ser) families (9). Prolyl
oligopeptidases (POPs)1 were
first isolated from human tissue (10) and subsequently from the tissues
of other mammals (11, 12), fungi (13, 14), bacteria,
(15-17), and archaea (5).
Despite its wide distribution, little is known about the physiological
role(s) of POP in any organism. The presence of the enzyme in mammalian
brain tissue and its ability to cleave proline-containing neuroactive
peptides (18, 19) have led to the suggestion that it plays a role in
regulation of these peptides. Evidence is emerging that suggests that
POP influences memory in mammals, because POP inhibitors have
antiamnesiac properties (20).
As a first step in the detailed investigation of P. furiosus (Pfu) POP, its substrate specificity, pH
activity profiles, temperature-dependent activity
profile, and influences by anions were studied. In addition, a
structural model of Pfu POP was constructed based on the
folding patterns from the POP crystal structure from Sus
scrufa (pig) (21). The two enzymes share 32% identity and 57%
similarity at the amino acid level (7). The similarities of these
enzymes from phylogenetically divergent sources are intriguing given
their ~55 °C difference in temperature optimum. Even more
striking, however, are their differences, which may ultimately shed
some light on the evolution of this class of serine proteases.
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EXPERIMENTAL PROCEDURES |
Prolyl Oligopeptidase--
The prpA gene of P. furiosus, which encodes prolyl oligopeptidase, was inserted in the
pET expression plasmid (Novagen). The resulting plasmid was designated
pJS3, and prpA was expressed in E. coli
BL21(plysS) as previously described (5). POP from E. coli BL21(pJS3) was partially purified by first precipitating the
bulk of E. coli protein with a series of 100 °C heating
steps, followed by anion-exchange chromatography (5), which yielded purified POP as assayed by SDS-polyacrylamide gel electrophoresis. Polyl oligopeptidase concentration was determined using the BCA assay
(Pierce Biochemicals). The concentration of POP in the reaction assays was between 0.5 and 0.75 µg/ml (7-10 nM). In all
experiments where rates are compared, the same enzyme preparation with
the same enzyme concentration was used.
Enzyme Assays--
The hydrolysis of
benzyloxycarbonyl-glycyl-prolyl-p-nitroanilide
(Z-Gly-Pro-pNA) and several other substrates was monitored with a spectrophotometer (Beckman DU640). The initial rate of hydrolysis of all substrates was obtained by monitoring the release of
the chromophore pNA (p-nitroanaline) at 410 nm
(
= 9600 M
1 cm
1
was found constant under all conditions in this study). All buffers contained 0.1 M NaCl unless otherwise specified. The
concentration of methanol in all assays was adjusted to 0.228 mM to enhance the solubility of the
Z-Gly-Pro-pNA stock solutions. Methanol was found to inhibit
POPs hydrolysis of ZGP non-competitively; therefore, the rate of
Z-Gly-Pro-pNA hydrolysis by POP in 0% methanol was
extrapolated from the linear plot of rate versus methanol concentration (data not shown). The pH of each buffer was adjusted to
the desired value at 85 °C. All assays were carried out in 30 mM buffers, acetate/acetic acid at pH 4.2-4.7, MES at pH
4.8-6.2, HEPES at pH 6.3-7.6, TAPS at pH 7.7-8.6, and CAPS at pH
8.7-9.3) in the presence of 0.1 or 0.8 M NaCl. All aqueous
solutions were prepared from deionized water of >18 M
from a MilliQ
system (Millipore, Bedford, MA). The kinetic parameters
kcat and Km were obtained by
non-linear fitting of the data to the hyperbolic Michaelis-Menten equation.
Deuterium Isotope Effect--
The solvent isotope effect on
kcat and
kcat/Km was measured using
Z-Gly-Pro-pNA or Z-Ala-Pro-pNA as the substrate. Isotope effects for both substrates were measured at 85 °C and at
56 °C at pH 5.2 and 8.0. Substrates were prepared in binary solvent
mixtures of H2O/D2O with 30 mM
buffer containing 0.1 or 0.8 M NaCl. The ratios of
H2O/D2O used to construct the proton inventory
were 100:0, 75:25, 50:50, 75:25, and 0:100. A pH electrode with a
temperature reference was used to measure pH at 85 °C. The pD value
was obtained from the relationship pD = pH (meter reading) + 0.40 (22), with extreme care taken to ensure consistent values.
Molecular Modeling--
A homology model for Pfu POP
was constructed based on the crystal structure of porcine POP (21),
Protein Data Bank entry code 1qfs (Fig. 3A). The alignment
obtained from ClustalX and the homology modeling module of MOE
(Chemical Computing Group Inc., Montreal, Quebec, Canada) was
used to build a three-dimensional model of Pfu POP. The
underlying methodology of MOE-Homology is based on a combination
of the segment-matching procedure of Levitt (23) and an
approach to the modeling of indels based on that of
Fechteler et al. (24). By default, MOE-Homology creates 10 models, each of which is generated by making a series of
Boltzmann-weighted choices of side-chain rotamers and loop
conformations from a set of protein fragments selected from the
built-in library of high resolution protein structures. Each of the
candidate models can be saved in a molecular data base for further
analysis, while an average model is created and then submitted to a
user-controlled level of potential energy minimization. One can pick
any of MOE's standard molecular mechanics force fields that include
two variants of the AMBER force field as well as MMFF94.
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RESULTS AND DISCUSSION |
Substrate Specificity of Pfu POP--
The
kcat values for the hydrolysis of
Z-Gly-Pro-pNA and Z-Ala-Pro-pNA (Table
I) are comparable to those obtained for
lamb brain POP and lamb kidney POP against various proline-containing oligopeptides (12, 25). The amino acid residue at the P2
site affects both kcat and
Km. Pfu POP cleaves
Z-Ala-Pro-pNA approximately four times more efficiently than
it does Z-Gly-Pro-pNA (Table I). The preference for alanine
over glycine at the P2 position was also observed for lamb
kidney POP (25). Like other POPs, Pfu POP cleaves dipeptides
with a free amino terminus, e.g. HAP-pNA,
HRP-pNA and HGP-pNA, very poorly (Table
I). This cleavage shows a large decrease in kcat
for these substrates compared with those with a blocked amino
terminus.
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Table I
Kinetic parameters for the hydrolysis of various substrates by Pfu POP
at 85 °C in the presence of 0.1 M NaCl at pH 7.5
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Measurements of kcat for P. furiosus
POP toward Z-Ala-Ala-pNA were approximately 7-fold slower
than kcat toward the proline-containing, N-blocked substrates Z-Ala-Pro-pNA and
Z-Gly-Pro-pNA (Table I). The Km for
Z-Ala-Ala-pNA is very similar to that of
Z-Ala-Pro-pNA. The POPs from lamb kidney and
Flavobacterium cleaved Ala-X 100 to 1000 times
slower than Pro-X (25, 26). The relatively high rate of
cleavage of Z-Ala-Ala-pNA by Pfu POP suggests
that the specific recognition and active site conformation of
Pfu POP differs from those of the mammalian and bacterial POPs.
Solvent Isotope Effect--
The solvent isotope effect was
measured in mixtures of H2O and D2O at 85 °C (Fig.
1) and 56 °C (data not shown). When
either Z-Gly-Pro-pNA or Z-Ala-Pro-pNA was used as
the substrate, the graph of kn
(kcat at deuterium mole fraction of
n) versus n was linear (Fig. 1),
indicating that the rate-limiting step of the reaction is dependent
upon exchange of a single protonic site (27). The overall solvent
isotope effect (k0/k1)
ranged from 2.0 to 2.2 under all conditions tested in both 0.1 and 0.8 M NaCl. The solvent isotope effect suggests that proton
transfer is the rate-limiting step in the mechanism of Pfu
POP, as it is in the case of chymotrypsin (28).

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Fig. 1.
Proton inventory at 85 °C for the
hydrolysis of ZGP, catalyzed by Pfu POP at pH/pD 8.0 ( ) and pH/pD 5.2 ( ) in the presence of 0.1 M
NaCl. The linear plot suggests that the transfer of one proton is
the rate-limiting step of the reaction.
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These results contrast with those of the POP from pig muscle (29),
where a deuterium isotope effect of ~2 was observed only under low pH
conditions (
6), but not under higher pH conditions (~8). The
explanation offered for the pH-dependent difference in
isotope effect for the porcine enzyme is that the enzyme undergoes a
conformation change between low and high pH, and only in the low pH
form is a proton exchange event rate-limiting (29). Further studies on
the porcine POP have suggested that the rate-limiting step of the
reaction at physiological pH (~7) is a conformation change at the
-propeller domain (30-32), which may be induced by the substrate.
It was proposed that the conformation change enlarges the 4-Å wide
entrance to the
-propeller structure, which otherwise prevents
substrate entry into the active site (21). Evidently, such a
conformational change is not the rate-limiting step for catalysis by
Pfu POP.
Model of Pfu POP--
Although Pfu POP functions
optimally at temperatures some 55 °C higher than the porcine enzyme,
numerous shared characteristics exist. At the amino acid level, the
POPs of P. furiosus and S. scrufa display 32%
sequence identity and 57% similarity (7). There is also a very high
degree of sequence identity around the active site residues as
highlighted in Fig. 2. The molecular mass of the enzyme from porcine muscle is ~73 kDa (33) and 70.0 kDa for Pfu POP (5). The enzymes from pig and archaeon share the double-sigmoidal pH-rate profiles and response to solvent ionic strength (discussed later).

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Fig. 2.
Alignment of porcine POP with Pfu
POP. The residues are colored based on
calculated conservation values that range from 0 to 10 where 10 is
identity (residues in red). The green residues
have values above 5, which show increasing similarities in
physico-chemical properties. The yellow regions are the
residues composing the active site. The catalytic triad is
boxed. Numbers on the top correspond
to the primary amino acid sequence of porcine POP; those on the
bottom are for Pfu POP.
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A structural model of Pfu POP was constructed based on its
amino acid sequence similarity with porcine POP (Fig. 2) and on the
crystal structure with 1.4-Å resolution (21) of porcine POP. Fig.
3A illustrates the results
from the homology modeling by superimposing ribbon structures of
porcine POP with Pfu POP. Fig. 3B focuses on the
active site of the enzymes. The model predicts that there is a high
degree of structural identity between the mammalian and archaeal
enzymes. The transition-state analogue Z-Pro-proline inhibitor
is illustrated to show its position with respect to the catalytic
residues. The position of the substrate was taken as that found in the
x-ray crystal structure for the porcine POP (21). The position of the
substrate in the Pfu POP active site was not adjusted. The
stereochemical quality of the Pfu model structure can be
addressed using a variety of stereochemical parameters (34). Using the
Protein Report of MOE we have analyzed the porcine POP structure (1qfs)
and the Pfu homology structure. The stereochemical analysis
results for the porcine POP indicates that 88% of the residues are in
the core regions of the Ramachandran plot (34) The catalytic residue
Ser-554 is located in the outside region with
/
values of
61.9°/
110.2°. Only three other residues (Thr-311, Ser-346, and
Leu-520) were reported with stereochemical dihedral angle violations.
When the same analysis was performed on the Pfu POP model,
41 residues had stereochemical dihedral angle violations, with only
eight being
/
violations. The catalytic residue Ser-477 was one
of these having
and
values of 72.5° and
87.5°,
respectively. None of the other active site residues appeared in the
violation list. The percentage of residues residing in the core region
of the Ramachandran map for Pfu POP was 69%. MOE reports
the percentage of residues in the core region as defined by
Morris et al. (34). Using this percentage along with the conclusions from the study by Morris et al. (34), we believe that the stereochemical quality of our Pfu POP model is that
of a protein x-ray structure that has a resolution of less than 3.0 Å and an R-factor of 0.28 and that it is of sufficient quality to be used to rationalize our experimental data.

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Fig. 3.
Top, structure of Pfu POP
(green ribbon representation) superimposed on the porcine
POP (1qfs.pdb; red string representation). The porcine POP
inhibitor (Z-Pro-prolinal) is represented as a multicolored thick
bond representation. The proposed "hinge" region is located at
the arrows. For clarity the amino-terminal residues 1-23
are omitted from the porcine structure. Bottom,
ball-and-stick stereoview of the Pfu POP
catalytic triad plus Tyr-401. The residues represented as solid
purple are the superimposed, equivalent porcine POP residues. The
multicolored thick bond structure is the porcine POP
inhibitor. The cyan dashed lines illustrate possible
hydrogen bond network between the active residues.
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Both the crystal structure of porcine POP and the structural model of
Pfu POP have an
/
hydrolase fold and share a unique feature in that both active sites are covered by a
-propeller structure (Fig. 3A). The Pfu POP model structure
has a cylindrical shape with a length of ~65 Å and a diameter of
~50 Å. The active site domain and the
-propeller domain are
connected through a "hinge" region composed of amino acids Arg-361
to Gly-365 and Ser-47 to Ile-51. This region is located at the top
center of the model (Fig. 3A). The active site triad
residues are Ser-477, His-592, and Asp-560 (corresponding to Ser-554,
His-680, and Asp-641 of porcine POP) and are located in the center of
the enzyme at the interface of the two domains (Fig. 3B).
The side chains of Tyr-473 and Arg-643 of porcine POP are catalytically
important. They are H-bonded to the substrate as suggested by the
crystallographic study of the inhibitor complex with the porcine
enzyme. The corresponding Tyr-401 and Arg-562 of Pfu POP
also reside at the same locations, and thus may be catalytically important.
It has been suggested that the
-propeller domain of porcine POP
blocks the active site to entry of large substrates, and, when flexed,
provides a pathway for substrate entry to the active site (21).
However, because Pfu POP has been shown to be
autoproteolytic and can cleave large substrates (5), it would seem
unlikely that a dramatic conformation change would occur at the
-propeller domain. On the other hand, enlarging the crevice between
the two domains with respect to the "hinge" may be a low energy
route for the substrate to gain access to the active site.
Effect of pH on Hydrolytic Rate--
The effects of pH on
kcat and
kcat/Km were measured
throughout a pH range of 4.0-9.5 (Fig.
4). The kinetic parameters were obtained
by nonlinear regression fitting of the data to the hyperbolic
Michaelis-Menten equation. Profiles of pH versus
kcat and
kcat/Km exhibited a doubly
sigmoidal pattern with respect to the activation of the enzyme at low
pH (Fig. 4), which was also observed in both profiles for porcine POP
(29). Although pH-rate profiles have only been published for a few
POPs, a bell-shaped curve was obtained for a prolyl oligopeptidase
isolated from a Xanthomonas sp. when
Z-Gly-Pro-pNA was used as the substrate (17), suggesting
that not all POPs share the same response to pH.

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Fig. 4.
pH dependence of (A)
kcat, (B)
kcat/Km at 0.1 M NaCl and at 0.8 M NaCl for the hydrolysis of
Z-Gly-Pro-pNA. The data were fitted by non-linear
regression to Eq. 1 for a three-ionization process. C, pH
dependence of the inhibitor constant Ki of AEBSF.
The data were fitted by non-linear regression for a one-deprotonation
process.
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The rate constants can be derived with respect to the pH to contain
three ionization constants pK1,
pK2, and pK3 by means of
steady-state approximation and the rate law for two active forms,
EH2 and EH, rate = k[EH2·S] + k'[EH·S].
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(Eq. 1)
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In this equation k(lim)1 and
k(lim)2 are the pH-independent rate constants
for the maximum rates associated with pK1 and pK2. The pH-dependent rate constants
kcat and
kcat/Km have been fitted to
Eq. 1 using non-linear regression, yielding three
pKa values. The results are shown in Table
II for rate constants obtained in the
presence of 0.1 and 0.8 M NaCl. These
pKa values are similar to those of porcine POP, which displayed pKa values of 3.3, 6.2, and 9.6 in
the presence of 0.1 M NaCl and 4.7, 6.9, and 9.6 in the
presence of 0.8 M NaCl (29). Porcine POP denatures at pH
values below 5.0, making it difficult to obtain accurate data in acidic
pH conditions, whereas Pfu POP incubated at low and high pH
values showed no loss of activity when assayed at physiological pH. The
application of constant ionic strength ensured the accuracy of rates
determined throughout the pH range. There was virtually no difference
in rates obtained in regions where buffers overlapped, ensuring the precision and accuracy of the experiments.
Measurements of pH-dependent inhibition of
Pfu POP by the serine-specific inhibitor
4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) were obtained to
provide more information for assignments of the
pKes2 values to specific amino acid residues.
The inhibition of Pfu POP by AEBSF was measured over a pH
range of 3.7 to 9.9 (Fig. 4C). The data were fitted to a
single-deprotonation process by non-linear regression. The resultant
pKa of 6.76 ± 0.03 indicates that the
nucleophilic, reactive site serine is activated by the deprotonation of
an ionizable group with pKa of 6.76. The
pH-independent Ki value was 5.00 ± 0.07 mM
1.
The assignments for the pKa values were also
corroborated by monitoring the change in pKa values
at different temperatures, allowing calculation of the heat of
ionization (
Hioniz) of the
ionizable groups according to the van't Hoff plot
(pKa versus 1/T, data not
shown).
Hioniz for
pKes2 from the pH-rate profile was calculated to
be 22 kJ/mol. This value is comparable to that of an imidazole (25-35
kJ/mol), although it is too low for an amine (46-54 kJ/mol), too high
for a carboxylic acid (
4-8 kJ/mol), and much lower than values
expected for a pH-dependent conformational change (60-105
kJ/mol) (35). These results suggest that Ser-477 is activated by the
deprotonation of the imidazole ring on His-592, which displays a
pKa of ~7.0 in Pfu POP (Table II). The
deprotonation of the imidazole with pKa ~ 7.0 agrees with the pKa assignment for the well studied serine proteases trypsin and chymotrypsin.
The pKa2 of porcine POP was much lower
than the value for Pfu POP (~4.5), but it was also
ascribed to ionization of an imidazole (His-680). It was proposed that
the pKa of His-680 in porcine POP is perturbed by
the ionization of a second functional group (associated with
pK2) (29, 31) and that this ionization may
contribute to the conformation change that renders the enzyme fully
active. This argument is supported by the difference in the deuterium
isotope effect at low and physiological pH for the porcine enzyme (29).
The deuterium isotopic effect observed in Pfu POP is
markedly different. The isotope effects at acidic and physiological pH
values are very similar, indicating that a general base/acid catalysis
is the rate-limiting step at neutral pH. It is therefore unlikely that
a conformation change is involved with the ionization of
pK2 in Pfu POP. This contention is
also supported by the AEBSF inhibition study and heat of ionization, which both suggest that Ser-477 is activated by the ionization of the
imidazole from His-592.
The ionization of pK1 for Pfu POP has
some unusual characteristics compared with most serine proteases;
however, similar observations have been made for thermophilic proteases
(36, 37) and another POP (38). Fig. 4 shows that a high percentage of
the enzyme activity is dependent upon the ionization of the first
ionizable group with a pKa at ~4.0-4.7 depending
on the ionic strength. The van't Hoff plot for
pKes1 (data not shown) displays a
Hioniz of 4.7 kJ/mol, which
suggests that pK1 is due to the deprotonation of
a carboxyl group from either an aspartic acid or a glutamic acid.
Similar observations exist in the pH-rate profile for the thermophilic
serine protease from Sulfolobus solfataricus, which displays
double-sigmoidal activation and suggests that ~80% of the activity
is associated with the first ionizable group (36). The prolyl
oligopeptidase from Sphingomonas capsulata has an activity maximum at 43 °C and displays double-sigmoidal activation for the
hydrolysis of ZGP. Approximately 35% of the activity is attributable to pK1 (38). The pH-rate profile from the
thermophilic protease caldolysin of Thermus aquaticus
suggests that an ionization with pKa ~ 4.5 may
play an important role in the enzyme mechanism (37). These similarities
suggest that the double-sigmoidal character associated with the pH
activation of thermophilic prolyl oligopeptidases may result from an
ionization of a functional group (possibility a carboxyl group in
Pfu POP) with a pKa of ~4.5. It cannot be determined from the model of Pfu POP how the
deprotonation of a carboxyl group can result in the emergence of a
double-sigmoidal pH-rate profile, but forthcoming site-directed
mutagenesis of Asp-560 and other residues in the active site may
provide insight into the activation of this enzyme at low pH.
Effect of Ionic Strength on Catalytic Rate--
The rate of
Pfu POP is ionic-strength-dependent and is
activated by salts such as NaF, NaCl, and NaBr (Fig.
5). Although the activity of the
well-studied serine proteases such as trypsin, chymotrypsin, and
subtilisin are affected little by ionic strength, the porcine (29) and
Pfu POPs have similar responses to ionic strength. Ionic
strength does not affect kcat of Pfu
POP (Fig. 5, inset). Ionic strength does affect the second
order rate constant for the hydrolysis of ZGP by changes in
Km, because the substrate binds more efficiently at
higher salt concentrations. Activation by halides and other simple
anions is not uncommon in enzymes. For example, thermolysin (39) and
angiotensin-converting enzymes (40) are activated by chloride via
changes in Km to different degrees depending on the
substrate. The data indicate that the activation pattern is not a
"specific activation" pattern (41) as the enzyme still exhibits
activity without the anion, and neither is it a "hyperbolic
activation" (characterized by the equilibrium ES + A
ESA (41)), as the plots of 1/V
against reciprocal anion concentration are not linear.

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Fig. 5.
Plot of
kcat/Km
versus [NaF] ( ), [NaCl] ( ), and [NaBr]
( ). The fittings were obtained by non-linear regression
analysis to Eq. 4. The inset is a plot of
kcat versus [NaCl], showing that
kcat remains unchanged with increasing
[NaCl].
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Although both the porcine and Pfu POPs are activated by
ionic strength via a change in Km, the nature of
activation is different for the two enzymes. In the case of porcine POP
the plot of kcat/Km
versus [NaCl] is linear at low [NaCl] and levels off at
higher salt concentrations around 1.0 M; whereas Pfu POP exhibits a sigmoidal curve for the binding of NaF,
NaCl, and NaBr (Fig. 5). The sigmoidal pattern of halide binding
suggests that there are multiple binding sites for the halide ions,
reminiscent of oxygen binding in hemoglobin. However, the binding of
the first oxygen molecule to one subunit of hemoglobin increases the
affinity of O2 binding for the remaining binding sites
(i.e. a cooperative binding pattern), whereas the binding of
the first or first few halides to Pfu POP does not seem to
affect the activity of the enzyme. The binding of halide ions to
Pfu POP is illustrated in Reaction 1,
where K1, K2,
K3, K4, and
Ki are the stepwise formation constants for halide
(X) binding. In this sequential binding pattern, the binding
of the first or first few X
ions can be
"non-productive" and do not affect the activity. If
EXi is considered the step that results in
increased activity, i.e. k = ko + klim
([EXi]/ET), Eq. 2 is
obtained,
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(Eq. 2)
|
where k is the rate constant, ET
is the total enzyme concentration (= E + EX + . . . . . EXi), ko is
the rate constant without anions, and klim is a
constant that represents the limiting rate above
ko. A good fit for Cl
activation
can be obtained when four binding constants are chosen for the fitting
(Fig. 4). The binding pattern in Reaction 1 can be further simplified
to Reaction 2,
where n represents the number of non-productive halide
ions that do not affect the activity,
Xn+1 represents the (n+1)th
halide ion responsible for activation,
n represents the overall formation constant for the non-productive binding site(s), and Kn is the stepwise formation
constant for the (n+1)th halide ion responsible for
activation. Eq. 2 can be now simplified to Eq. 3 below.
Fitting the data to Eq. 3 affords n = 4.9 for
Cl
binding, n = 2.1 for F
,
and n = 2.2 for Br
.
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(Eq. 3)
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This result indicates that there are five non-productive binding
sites for Cl
, and two for F
and
Br
, suggesting that Cl
must bind
differently from F
and Br
. Furthermore, the
data cannot be fitted for only one halide binding site, suggesting that
an activation mechanism with more than one halide binding site must be
applied to the explanation of the halide activation.
Thermolysin is similarly activated by halide ions (39). Recent studies
on thermolysin have shown that saturated concentrations of
Cl
and Br
lower Km
values ~10-fold for the hydrolysis of dansyl substrates. Furthermore,
chloride ions show a greater effect on substrate binding than bromide
ions. Chloride may activate thermolysin by competing with a salt bridge
that is believed to have an influence in substrate binding (39). The
thermolysin crystal structure illustrates that the salt bridge (Arg-203
to Asp-170) distance is lengthened upon binding with transition-state
analogue inhibitors (39). This observation suggests that substrate
binding to thermolysin would disrupt the salt bridge between Arg-203
and Asp-170. If chloride were to compete with Asp-170 for binding to
Arg-203 by replacement of an adjacent water molecule, which exists in
the thermolysin crystal structure, then the ESX (S = substrate) complex would be more stable then the ES complex,
thus accounting for the reduction in Km. The porcine
POP crystal structure with a bound transitional state analogue,
Z-Pro-prolinal, supports this halide activation mechanism. The
Z-Pro-prolinal molecule is hydrogen-bonded to Arg-643 in the active
site of this enzyme with the hydrogen bond formed between the
main-chain carbonyl group of the prolinal molecule and the NH hydrogen
of Arg-643 side chain. The guanidinium group on Arg-643 is involved in
an extensive hydrogen bond network, which includes a salt bridge with
Asp-149. The Pfu POP model shows a similar arrangement in that Arg-562 forms a salt bridge with Asp-119 and is in position to
hydrogen bond to the substrate. These results suggest that the halide
ions may compete with Asp-149 in porcine POP and Asp-119 in
Pfu POP for the salt bridges to the arginine residues. This interaction would place the arginine residue in a more suitable position to enhance substrate binding, which could result in lower Km values.
Enhancement of substrate binding by salts could, alternatively, result
from improving the access of the substrate to the active site. The
active site is hidden in a large cavity at the interface of the two
domains for both the porcine and Pfu POPs (Fig.
3A). The
-propeller domain in the porcine POP is the
proposed entrance for the substrate to the active site (21). The
-propeller has a small opening that is ~4 Å wide, which is too
small to permit the entrance of the peptide substrates without some
conformational change taking place. Studies of the activity of porcine
POP on different substrates suggested that the opening of the
-propeller domain is induced by the substrate and that this
conformational change may be the rate-limiting step of catalysis at
physiological pH (21, 28-31). The crystal structure of
porcine POP illustrates that there are three lysine residues involved
in narrowing the entrance to the tunnel of the propeller domain.
Because anions have been shown to bind to positively charged residues
such as lysine and arginine (42) and only influence the activity of porcine POP by changes in
Km,2 it is
possible that the substrate or the anions could independently induce a
conformational change at the
-propeller domain for porcine POP.
Although the model of the POP from P. furiosus shows that
Lys-330 and Lys-255 could partially block the entrance to the tunnel of
the
-propeller structure and that halide binding at these residues
could conceivably cause a conformational change at the
-propeller,
it is very unlikely that halides activate Pfu POP via such a
conformation change. The autoproteolytic nature of Pfu POP
as well as this enzyme's ability to hydrolyze azocasein (5) argue
against substrate entry via the
-propeller, because this opening is
too small (
3 Å wide). Alternatively, the substrate may be afforded
entry to the active site through the crevice of the two domains via a
conformational change at the hinge region, which separates the
and
domains and is located near the top and center of the model (Fig.
3A). The model of Pfu POP places four lysine
residues (159, 161, 511, and 589) and two arginine residues (158 and
602) at the interface of the
/
hinge region, which could interact
with anions to promote a conformational change that allows the
substrate to enter to the active site.
The sequential binding observed for halide ions suggests that a
conformational change might occur at the hinge region between the two
domains. The binding of the first few halide ions may cause the
conformational change, which enhances the binding of the later halide
ion, allowing the substrate entry to the active site. If halides
activate the enzyme by interacting with a salt bridge near the active
site, it should require only one halide ion to disrupt the salt Arg/Asp
salt bridge as was suggested for thermolysin. The last halide to bind
may serve this role. There is no evidence for this mechanism of
activation in porcine POP, because sequential halide binding was not
observed (29).
Temperature Dependence of Pfu POP Catalysis--
It has been shown
that Pfu POP is extremely stable when incubated at
temperatures of
95 °C for several hours at concentrations of less
than 1 µg/ml (5). This thermal stability provides a unique
opportunity to probe the thermodynamic properties for POP catalysis.
The first order rate constant (kcat) for ZGP
hydrolysis catalyzed by this enzyme was obtained at temperatures
between 60 °C and 90 °C. The data were fitted to the Arrhenius
equation, k = A
exp(
Ea/RT) to obtain the
activation energy (Ea), where k is
the rate constant, A is the Arrhenius constant, R
is the gas constant, and T is the temperature. Fig.
6 shows two Arrhenius plots at pH 6.0 and
7.6 for both the acidic/neutral and basic active enzyme forms. These
two pH values are within the regions in the pH profile where
kcat reaches a plateau (Fig. 4), which allows
the thermodynamic parameters to be accurately studied. The linear plots
in Fig. 6 imply that the rate-limiting step does not change as the
temperature is increased (43). The other thermodynamic parameters can
be calculated from the following equations:
G
=
RT
ln(kcath/kBT),
H
= Ea
RT, and
S
= (
H
G
)/T, where
kB and h are the Boltzmann and Planck
constants, respectively (Table III). The
large positive enthalpy obtained from Pfu POP suggests that
a conformational change occurs upon substrate binding, possibly
attributable to hydrogen bond formation during the transition state
(43). The large negative entropy suggests that molecular motion is lost
during the ES
transition state, which could
be due to hydrogen bond formation with residues near the active site.
The interactions may include Arg-562 of the active site, which, based
on the model structure, is positioned to form a hydrogen bond with the
substrate.

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Fig. 6.
Arrhenius plot of
kcat versus
1/T at pH 6.0 ( ) and 7.6 ( ). The data
are fitted by non-linear regression to the Arrhenius equation to give
the activation energy Ea, which is displayed as
linear plots for better visualization.
|
|
The large free energy of activation is a composite of the enthalpy and
entropy, which literally prevents the Pfu POP-catalyzed hydrolysis from occurring at room temperature. Interestingly,
H
differs for the two turnover
numbers at two different pHs, with 28.7 kJ/mol in the acidic/neutral
region compared with 37.2 kJ/mol in the basic region; whereas
G
remains nearly unchanged.
Based on the estimates of hydrogen-bond energies (44), one or two extra
hydrogen bonds could be broken during the transition state of the
acidic/neutral active enzyme form to account for the difference in the
enthalpy of activation of the two active forms.
Concluding Remarks--
Although P. furiosus and
S. scrufa, the pig, are from different phylogenetic domains
and live at vastly different temperatures, their POPs share several
characteristics that are unusual among serine proteases. Both enzymes
display doubly sigmoidal pH-activity profiles. Anions have been shown
to activate both POPs by changes in Km. However,
Pfu POP displays sequential halide binding, and several
non-productive binding sites are suggested by the effect of chloride on
enzyme activity. Halide binding could result in a conformational change
at the hinge region between the
and
domains, allowing substrate
access to the active site. The solvent isotope effect studies coupled
with the heat of ionization studies and the ability of Pfu
POP to hydrolyze large proteins (5) offer evidence that substrate
access to the active site is not regulated by a conformational change
at the
-propeller, as has been proposed for the mammalian enzyme.
The model constructed for Pfu POP suggests a significant
structural similarity to porcine POP, however, the
propeller domain
does not appear to carry out the same function, i.e.
exclusion of large peptides from the active site. The solution to the
differences between these two enzymes awaits further structural and
mechanistic studies, including site-directed mutagenesis.