Structures of Prolyl Oligopeptidase Substrate/Inhibitor Complexes

USE OF INHIBITOR BINDING FOR TITRATION OF THE CATALYTIC HISTIDINE RESIDUE*

Vilmos FülöpDagger §, Zoltán Szeltner, Veronika Renner, and László Polgár||

From the Dagger  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



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 Nepsilon 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 Nepsilon 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

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 alpha /beta -hydrolase fold and that its catalytic triad (Ser554, Asp641, His680) is covered by the central tunnel of an unusual beta -propeller, which operates as a gating filter for the active site by excluding large structured peptides (15).

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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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
k=k(<UP>limit</UP>)[1/(1+10<SUP><UP>p</UP>K<SUB>1</SUB>−<UP>pH</UP></SUP>+10<SUP><UP>pH−p</UP>K<SUB>2</SUB></SUP>)] (Eq. 1)
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
k=k(<UP>limit</UP>)<SUB>1</SUB>[1/(1+10<SUP><UP>p</UP>K<SUB>1</SUB>−<UP>pH</UP></SUP>+10<SUP><UP>pH−p</UP>K<SUB>2</SUB></SUP>)]+ (Eq. 2)

k(<UP>limit</UP>)<SUB>2</SUB>[1/(1+10<SUP><UP>p</UP>K<SUB>2</SUB>−<UP>pH</UP></SUP>+10<SUP><UP>pH−p</UP>K<SUB>3</SUB></SUP>)]
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.

The Ki values, the dissociation constant of the enzyme-inhibitor complex, were calculated from the following equation
k<SUB>i</SUB>/k<SUB>0</SUB>=1/(1+I/K<SUB>i</SUB>) (Eq. 3)
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.

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 (>3.0sigma ) 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.


                              
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Table I
Data collection and refinement statistics



    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 Neta 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 Nepsilon 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, Nepsilon 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-Nepsilon 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 Nepsilon 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 Nepsilon 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 Nepsilon 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.



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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 - Delta Fc electron density using phases from the final model is contoured at 1sigma level, where sigma  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)).


                              
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Table II
Hydrogen bonds between prolyl oligopeptidase and the bound substrate/inhibitor

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-Nepsilon 2, P1'-NH···P2-CO···Arg643-Neta 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.

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 Nepsilon 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.

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).



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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 (open circle ) 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 (open circle ) 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.


                              
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Table III
Kinetic parameters for the reactions of prolyl oligopeptidase with the octapeptide substrate

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 Nepsilon 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).

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-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.

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 beta -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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

* 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.


    ABBREVIATIONS

The abbreviations used are: Z, benzyloxycarbonyl; Abz, o-aminobenzoyl; Mes, 4-morpholineethanesulfonic acid; Nap, 2-naphtylamide.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES


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