From the Department of Biological Sciences,
University of Warwick, Gibbet Hill Road, Coventry CV4 7AL,
United Kingdom and the ¶ Institute of Enzymology, Biological
Research Center, Hungarian Academy of Sciences, P. O. Box 7, H-1518 Budapest 112, Hungary
Received for publication, August 3, 2000, and in revised form, September 29, 2000
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ABSTRACT |
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Structure determination of the inactive S554A
variant of prolyl oligopeptidase complexed with an octapeptide has
shown that substrate binding is restricted to the P4-P2' region. In
addition, it has revealed a hydrogen bond network of potential
catalytic importance not detected in other serine peptidases. This
involves a unique intramolecular hydrogen bond between the P1' amide
and P2 carbonyl groups and another between the P2' amide and N Prolyl oligopeptidase (EC 3.4.21.26), previously called prolyl
endopeptidase or post-proline cleaving enzyme, is a large intracellular
enzyme (molecular mass 80 kDa) that preferentially hydrolyzes
proline-containing peptides at the carboxyl end of proline
residues (1-3). It is presumably involved in the maturation and
degradation of peptide hormones and neuropeptides (1). Prolyl
oligopeptidase has recently gained pharmaceutical interest, because
specific inhibitors reverse scopolamine-induced amnesia in rats (4-6).
Its activity in plasma correlates with different stages of depression
(7). The enzyme also has a role in the regulation of blood pressure by
participating in the renin-angiotensin system through metabolism of
bradykinin and angiotensin I and II (8).
Prolyl oligopeptidase is unrelated to the well known trypsin and
subtilisin families and belongs to a new class of serine peptidases
(clan SC, family S9), which also includes dipeptidyl peptidase IV,
acylaminoacyl peptidase, and oligopeptidase B (9, 10). These enzymes
display distinct specificities, and each contains a peptidase domain at
the carboxyl-terminal region of the single polypeptide chain. In
the case of prolyl oligopeptidase, the active site serine and histidine
have been identified as Ser554 and His680,
respectively (11, 12). A structural relationship between lipases and
the peptidase domain of oligopeptidases has been indicated by the
similar topology of the catalytic groups and by the homologous amino
acid sequences around these residues (13). The 1.4-Å resolution crystal structures of the enzyme and its complex with
Z1-Pro-prolinal (Protein Data
Bank codes 1qfm and 1qfs) have recently been determined (14). The
results show that the enzyme contains a peptidase domain with an
The mechanism of action of serine peptidases involves an acyl enzyme
intermediate. Both the formation and the decomposition of the acyl
enzyme proceed through the formation of a negatively charged
tetrahedral intermediate that is stabilized by the oxyanion binding
site providing two hydrogen bonds to the oxyanion (16, 17). In the
chymotrypsin-type enzymes the hydrogen bonds are contributed by the
main chain NH groups of the catalytic Ser195 and the nearby
Gly193. In the subtilisin-type enzymes the side chain amide
of an asparagine replaces the main chain NH of Gly193. In
prolyl oligopeptidase one of the hydrogen bonds is formed between the
oxyanion and the main chain NH group of Asn555, adjacent to
the catalytic serine, Ser554. The second hydrogen bond is
unique among serine peptidases and provided by the OH group of
Tyr473 (14). Experiments with the Y473F variant of prolyl
oligopeptidase have shown that the Tyr473 OH indeed
markedly contributes to the transition state stabilization, but the
effects are greatly dependent upon the substrate and pH (18).
Structure determination of the hemiacetal group of prolyl
oligopeptidase formed with an aldehyde inhibitor, Z-Pro-prolinal, has
revealed a limited region of the substrate binding site, involving the
S1-S3 subsites (14). To identify the ligand binding mode of a longer
substrate, which possesses amino acid residues on both sides of the
scissile bond, we prepared the inactive S554A variant. We also examined
the binding of inhibitors to this variant, which allowed us to estimate
the pKa of the catalytic histidine
(His680) and of tyrosine (Tyr473) at the
oxyanion binding site.
Enzyme Preparation--
Prolyl oligopeptidase of porcine brain
was expressed in Escherichia coli and purified as described
(18), and its concentration was determined at 280 nm (4). The S554A
mutation was performed with the two-step polymerase chain reaction
procedure as described for the Y473F mutant (18). The following
primers were used for mutagenesis: 5'-AACGGAGGTgCAAATGGAGG-3' and
3'-TTGCCTCCAcGTTTACCTCC-5'.
Activity Measurements--
The activity of prolyl oligopeptidase
was determined fluorometrically with Z-Gly-Pro-Nap (Bachem, Ltd.),
using a Jasco FP 777 spectrofluorometer. The excitation and
emission wavelengths were 340 nm (1.5 nm bandwidth) and 410 nm (5 nm
bandwidth), respectively. The substrate with internally quenched
fluorescence,
Abz-Gly-Phe-Gly-Pro-Phe-Gly-Phe(NO2)-Ala-NH2, was prepared with solid phase synthesis, and its hydrolysis was followed similarly as in the case of Z-Gly-Pro-Nap, except that the excitation and emission wavelengths were 337 and 420 nm, respectively.
Kinetics--
The specificity rate constants
(kcat/Km) were determined
under first-order conditions, i.e. at substrate
concentrations lower than Km. The first-order rate
constant, calculated by nonlinear regression analysis, was divided by
the total enzyme concentration to provide
kcat/Km. The pH dependence of catalysis was measured in a four-component buffer composed of 25 mM glycine, 25 mM acetic acid, 25 mM Mes, and 75 mM Tris and which contained 1 mM EDTA and 1 mM 1,4-dithiothreitol
(standard buffer). The buffer was titrated to the desired pH with HCl
or NaOH, while the ionic strength remained fairly constant over a wide
pH range. Small changes in the conductivity were adjusted by the
addition of NaCl. After the reaction had been completed, the pH of each
sample was determined and found to be practically identical with the
starting value.
Theoretical curves for bell-shaped pH rate profiles were calculated by
nonlinear regression analysis, using the following equation
The Ki values, the dissociation constant of
the enzyme-inhibitor complex, were calculated from the following
equation
Crystallization, X-ray Data Collection, and Structure
Refinement--
The peptides with the S554A variant of prolyl
oligopeptidase were co-crystallized using the conditions established
for the wild type enzyme (14). Crystals belong to the orthorhombic
space group P212121 with cell
dimensions a = 70.7 Å, b = 99.7 Å,
c = 110.7 Å for the octapeptide complex and
a = 71.1 Å, b = 100.1 Å,
c = 111.3 Å for the Z-Gly-Pro-OH complex. X-ray
diffraction data were collected at 100 K on a MAR345 image plate
detector at the beam line X11 (EMBL, Hamburg, Germany) using a
0.909-Å wavelength. Data were processed using the HKL suite of
programs (20). Refinement of the structures were carried out by
alternate cycles of X-PLOR (21) and manual refitting using O (22),
based on the 1.4-Å resolution model of wild type enzyme (14) (Protein Data Bank code 1qfm). A bulk solvent correction allowed all measured
data to be used. Water molecules were added to the atomic model at the
positions of large positive peaks (>3.0 Binding of an Octapeptide--
We have synthesized an internally
quenched fluorogenic peptide,
Abz-Gly-Phe-Gly-Pro-Phe-Gly-Phe(NO2)-Ala-NH2,
which is to be cleaved between the Pro-Phe bond. Fig.
1A illustrates its binding to
the S554A variant. The binding mode of P3-P1 residues (Phe-Gly-Pro) is
very similar to that of Z-Pro-prolinal (Fig. 1C) (14). The P1 proline ring is stacked against the aromatic Trp595,
whose indole nitrogen is hydrogen-bonded to the carbonyl oxygen of the
Phe residue at the P3 position. The carbonyl oxygen of the scissile
bond is in the oxyanion binding site, forming hydrogen bonds to the
main chain NH of Asn555 and the OH of Tyr473.
Both carbonyl oxygens of glycines at the P2 and P4 positions are
hydrogen-bonded to the same N
An additional important feature of substrate binding concerns the P2
carbonyl oxygen, which forms two hydrogen bonds, one with
Arg643 as already pointed out (15), and the other with the
leaving group (NH of the P1' residue). These hydrogen bonds appear to stabilize the substrate in the proper position for catalysis and explain an earlier observation that a sulfur atom in place of the P2
carbonyl oxygen makes the substrate practically unsuitable for
hydrolysis (23). The sulfur substitution of the carbonyl group in the
P2 position seems to induce a greater effect on catalysis than does
such a substitution in the P1 position, where the carbonyl oxygen is directly involved in the peptide bond cleavage (23). An
extensive network of hydrogen bonds
(P2'-NH···His680-N Binding of a Product-like Inhibitor--
It has previously been
shown that the acyl product of Z-Gly-Pro-Nap, the classic substrate of
prolyl oligopeptidase, is an inhibitor to the enzyme (18). Fig.
1B illustrates that the binding mode of Z-Gly-Pro-OH greatly
resembles the complex formed between prolyl oligopeptidase and the
aldehyde inhibitor Z-Pro-prolinal (Fig. 1C) (14). Similarity
to the P1-P3 residues of the octapeptide binding (Fig. 1A)
is also apparent, and the interactions are listed in Table II. One of
the oxygen atoms of the carboxylate ion of Z-Gly-Pro-OH is located in
the oxyanion binding site, forming hydrogen bonds with the OH group of
Tyr473 and the main chain NH of Asn555. The
other oxygen atom is linked to the N The pH Rate Profile for the Octapeptide Reaction--
The simplest
way of estimating the pKa values of catalytically
competent groups utilizes
pH-kcat/Km profiles. For
example, the pH dependence curves for the subtilisin and chymotrypsin reactions have revealed a pKa of ~7 for the
catalytic histidine. Unlike the sigmoid or bell-shaped pH rate profiles observed with the classic serine peptidases, the pH dependence for
prolyl oligopeptidase is more complicated. This has previously been
shown with Z-Gly-Pro-Nap (24, 25) and also demonstrated here with the
octapeptide substrate. As seen in Fig.
2A, the data conform to a
doubly bell-shaped curve, which arises from the modification of the
usual bell-shaped curve by an additional ionization event involving a
group with an apparent pKa of ~7
(pK2 in Table III).
The resulting pH dependence is composed of two active enzyme species,
which are illustrated by broken lines in Fig. 2A. Both
enzyme forms must bear the imidazole group as a base, because its
protonated form is catalytically inactive; therefore the
pK1 of ~5 (Table III) could be assigned to
His680. However, the pKa values of both
~5 and ~7 can be regarded as apparent dissociation constants, which
may be due to the presence of two enzyme forms of different activities.
This is supported by the observation that the relative activities of
the two forms may change with different substrates, leading to
the alteration in pKa
values.2 Similar effects on
the pKa values are also seen when the kinetic
parameters are compared in the absence and presence of 0.5 M NaCl (Table III). The possible structural differences between the two pH-dependent forms have also been detected
by intrinsic fluorescence measurements, which clearly indicate that the
low pH form is more unfolded (26).
Titration of His680--
In an attempt to obtain a
reliable pKa for the catalytic imidazole of prolyl
oligopeptidase, we made use of the interaction observed here between
prolyl oligopeptidase and its inhibitor, Z-Gly-Pro-OH (Fig.
1B). To this end, the association constant, 1/Ki of the enzyme-inhibitor complex has been
plotted against pH in Fig. 2B. The binding strength between
the enzyme and Z-Gly-Pro-OH greatly increases with decreasing pH, as
the imidazole N
It is known that high salt concentration depresses electrostatic
effects. Therefore it can be expected that addition of salt to the
reaction mixture decreases the value of the association constant,
provided that the contribution of the salt bridge is significant in the
formation of the enzyme-inhibitor complex. This has indeed been found,
as shown in Fig. 2B. Whereas the limiting value of the
association constant is reduced from 226 ± 7 to 179 ± 7 µM Conclusions--
Structure determination of the enzyme-substrate
complex allows us to describe the stereochemical features of prolyl
oligopeptidase in catalytic action. Because the binding of P3-P1
residues is very similar to that of the transition state inhibitor
Z-Pro-prolinal, we can conclude that substrate binding to the enzyme
favors the formation of the tetrahedral intermediate form. This
involves a nucleophilic attack and a general base catalysis as the
first elementary catalytic step. Decomposition of the tetrahedral
intermediate requires that the leaving group and the catalytic
histidine approach each other. The general acid catalysis is probably
promoted in a substrate-assisted manner, by the strong hydrogen bond
formation between the P1' amide and the P2 main chain oxygen atom. In
agreement with earlier kinetic experiments, the structure determination has also revealed that substrate binding is restricted to the P3-P2'
region only. Unlike in the enzymes chymotrypsin, subtilisin, and
papain, the main chain NH groups of P2-P4 residues of the substrate do
not form a 2 of the catalytic histidine 680 residue. It is argued that both hydrogen bonds promote proton transfer from the imidazolium ion to the leaving
group. Another complex formed with the product-like inhibitor benzyloxycarbonyl-glycyl-proline, indicating that the carboxyl group of the inhibitor forms a hydrogen bond with the N
2 of
His680. Because a protonated histidine makes a
stronger interaction with the carboxyl group, it offers a possibility
of the determination of the real pKa of the
catalytic histidine residue. This was found to be 6.25, lower than that
of the well studied serine proteases. The new titration method gave a
single pKa for prolyl oligopeptidase, whose
reaction exhibited a complex pH dependence for
kcat/Km, and indicated that
the observed pKa values are apparent. The procedure
presented may be applicable for other serine peptidases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
/
-hydrolase fold and that its catalytic triad
(Ser554, Asp641, His680) is covered
by the central tunnel of an unusual
-propeller, which operates as a
gating filter for the active site by excluding large structured
peptides (15).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and the GraFit software (19). In Equation 1 k stands
for kcat/Km, and (limit)
refers to the pH independent maximum rate constant.
K1 and K2 are the dissociation constants of a catalytically competent base and acid, respectively. The
pH rate profiles composed of two bell-shaped curves were fitted to the
following equation
(Eq. 1)
(Eq. 2)
where k(limit)1 and
k(limit)2 gave the limiting values of the rate
constant for the low pH and high pH forms of the enzyme.
where ki and k0 are pseudo
first-order rate constants determined at substrate concentrations at
least 10-fold less than Km in the presence and
absence of inhibitor (I), respectively.
(Eq. 3)
) in the difference electron
density, only at places where the resulting water molecule fell
into an appropriate hydrogen bonding environment. Restrained isotropic
temperature factor refinements were carried out for each individual
atom. The final model contains all the 710 amino acid residues in both
complexes, the bound peptides, and a large number of solvent
(glycerol and water) molecules. Statistics for the data processing and
refinement are given in Table I.
Data collection and refinement statistics
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1 atom of Arg643.
Interestingly, there is no interaction between main chain NH groups of
the P2-P4 residues and the enzyme molecule. The amino-terminal Abz does not bind to the protein; consequently the resulting electron density is not defined at this residue (Fig. 1A). The
P1'-P2' (Phe-Gly) portion is bound close to the imidazolium ion of the active site histidine residue. In the ground state of the reaction, the
N
2 atom of His680 is poised to accept the proton from
the OH of Ser554, with the simultaneous generation of the
tetrahedral transition state. In the second step, which occurs during
decomposition of the transition state, N
2 of His680 is
required to be at a hydrogen bond distance from the main chain NH of
the P1' phenylalanine to facilitate its protonation. The P1'-NH to
His680-N
2 distance in the present S554A structure is
longer (3.84 Å) than a normal hydrogen bond. Of course, this distance
should be shortened in the tetrahedral intermediate, in which the
proton transfer takes place from the N
2 of His680 to the
P1' NH group. Most interestingly, a further catalytic contribution
seems to arise from the main chain NH of the P2' glycine, which also
forms a hydrogen bond with the N
2 of His680, thereby
promoting the proton transfer from the imidazolium ion to the substrate
leaving group. This contribution may be regarded as a
substrate-assisted catalysis. If Ala554 of the present
mutant was converted back to Ser, the additional oxygen atom would be
in a proper position to provide nucleophilic attack on the substrate
carbonyl carbon while donating its proton to N
2 of
His680. The aromatic side chain of the Phe residue at the
P1' position adopted the most favorable conformation; it is projected
back to the cavity to stack against the peptide bond of the P1 and P2
residues. The carboxyl-terminal P3' and P4' residues presumably did not
bind to the enzyme and must be rather mobile, because they are not seen
in the electron density map. Therefore it could be concluded that the
enzyme binds no more than six residues (P4-P2') even from a longer
substrate. This is fairly consistent with kinetic investigations,
indicating that extension of the substrate over the P3-P2' region fails
to enhance the kinetic specificity constant (3). Detailed interactions
between the enzyme and the substrate are listed in Table
II.
View larger version (58K):
[in a new window]
Fig. 1.
Stereo view of the peptide/inhibitor binding
site of prolyl oligopeptidase. A, octapeptide binding.
B, Z-Gly-Pro-OH binding to the S554A variant. The bound
ligands are shown darker than the protein residues. The SIGMAA (28)
weighted 2mFo
Fc electron
density using phases from the final model is contoured at 1
level,
where
represents the root-mean-square electron density for
the unit cell. Contours more than 1.4 Å from any of the displayed
atoms have been removed for clarity. C, covalently bound
inhibitor Z-Pro-prolinal to Ser554 of the wild type enzyme
(drawn from Protein Data Bank code 1qfs (14)). Dashed lines
indicate hydrogen bonds (drawn with MolScript (29, 30)).
Hydrogen bonds between prolyl oligopeptidase and the bound
substrate/inhibitor
2,
P1'-NH···P2-CO···Arg643-N
1···P4-CO)
has not been observed with other serine peptidases. In summary, the
unique intramolecular hydrogen bond (P1'-NH···P2-CO) can possibly
help the general acid catalysis, i.e. the proton transfer
from the imidazolium ion to the leaving group.
2 of His680. The
strength of this interaction obviously depends on the protonation state
of the imidazole group, which can form a strong salt bridge only if it
is protonated. This phenomenon offers a possibility to determine the
pKa of the imidazole group by titrating with the
inhibitor, as discussed below.
View larger version (17K):
[in a new window]
Fig. 2.
A, the pH rate profiles for the reaction
of prolyl oligopeptidase with the octapeptide. The reactions were
performed in the presence ( ) and absence (
) of 0.5 M
NaCl. The broken lines calculated from Equation 1 stand for
the two pH-dependent forms in the presence of 0.5 M NaCl. B, formation of enzyme-inhibitor complex
as a function of pH. The association constants
(1/Ki) were calculated from Equation 3 for prolyl
oligopeptidase and Z-Gly-Pro-OH in the presence (
) and absence (
)
of 0.5 M NaCl. First-order rate constants were measured
with 2-20 nM enzyme and 0.29 µM
Z-Gly-Pro-Nap as substrate.
Kinetic parameters for the reactions of prolyl oligopeptidase with the
octapeptide substrate
2 becomes protonated and creates a stronger salt
bridge with the inhibitor. The proline carboxyl group is fairly acidic (pKa ~2), which ensures that its ionized form
remains unchanged over the experimentally available pH range. The data fit to the ionization of a group with pKa of
6.25 ± 0.04, which can be assigned to His680. This
approach of titrating the catalytic histidine may be of use for other
serine peptidases. For example, streptogrisin A, which is structurally
related to chymotrypsin, forms complexes with Ac-Pro-Ala-Pro-Phe-OH and
Ac-Pro-Ala-Pro-Tyr-OH, so that their carboxylate ions are bonded to the
catalytic histidine, if it is protonated (27).
1
s
1, the pKa of
His680 is not changed (6.22 ± 0.05). These results
confirm that the kinetic pKa values extracted from
Fig. 2A do not represent the His680 ionization.
-sheet with the enzyme. The pH dependence of the rate
constant (kcat/Km) gave a
complex curve and did not permit the determination of the
pKa of the catalytic histidine. The observed
interaction between the product-like inhibitor Z-Gly-Pro-OH and prolyl
oligopeptidase, however, allowed us to titrate the enzyme with the
inhibitor by measuring the association constant as a function of pH.
This resulted in a pKa of 6.2, lower than that found
for chymotrypsin and subtilisin. This titration method for
pKa determination could be employed for other peptidases.
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ACKNOWLEDGEMENTS |
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We thank J. Fejes and I. Szamosi for technical assistance. We are grateful for access to the facilities of beam line European Molecular Biology Laboratory X11 at the DORIS storage ring of Deutsches Elektronen-Synchrotron, Hamburg, Germany.
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FOOTNOTES |
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* This work was supported by the Wellcome Trust (Grant 055178/Z/98/Z), NATO (HTECH.CRG 970581), the Royal Society, the British-Hungarian Science and Technology Program (BP/885/5/18), and the Training and Mobility of Researches/Large Scale Facilities program (reference number ERBFMGECT980134).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.
The atomic coordinates and the structure factors (code 1e8m and 1e8n) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ A Royal Society University Research Fellow.
To whom correspondence should be addressed. Tel.:
36-1-466-5633; Fax: 36-1-466-5465; E-mail: polgar@enzim.hu.
Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M007003200
2 L. Polgár, unpublished result.
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ABBREVIATIONS |
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The abbreviations used are: Z, benzyloxycarbonyl; Abz, o-aminobenzoyl; Mes, 4-morpholineethanesulfonic acid; Nap, 2-naphtylamide.
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