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J. Biol. Chem., Vol. 278, Issue 49, 48786-48793, December 5, 2003

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Electrostatic Environment at the Active Site of Prolyl Oligopeptidase Is Highly Influential during Substrate Binding^*

Zoltán Szeltner, Dean Rea¶, Veronika Renner, Luiz Juliano||, Vilmos Fülöp**, and László Polgár

From the Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, P. O. Box 7, H-1518 Budapest 112, Hungary, the Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom, and the ^||Universidade Federal de Sao Paulo, Escola Paulista de Medicina, Departamento de Biofisica, Rua Tres de Maio, 100, 04044-020 Sao Paulo S.P., Brasil

Received for publication, August 28, 2003 , and in revised form, September 23, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
The positive electrostatic environment of the active site of prolyl oligopeptidase was investigated by using substrates with glutamic acid at positions P2, P3, P4, and P5, respectively. The different substrates gave various pH rate profiles. The pK_a values extracted from the curves are apparent parameters, presumably affected by the nearby charged residues, and do not reflect the ionization of a simple catalytic histidine as found in the classic serine peptidases like chymotrypsin and subtilisin. The temperature dependence of k_cat/K_m did not produce linear Arrhenius plots, indicating different changes in the individual rate constants with the increase in temperature. This rendered it possible to calculate these constants, i.e. the formation (k₁) and decomposition (k_–1) of the enzyme-substrate complex and the acylation constant (k₂), as well as the corresponding activation energies. The results have revealed the relationship between the complex Michaelis parameters and the individual rate constants. Structure determination of the enzyme-substrate complexes has shown that the different substrates display a uniform binding mode. None of the glutamic acids interacts with a charged group. We conclude that the specific rate constant is controlled by k₁ rather than k₂ and that the charged residues from the substrate and the enzyme can markedly affect the formation but not the structure of the enzyme-substrate complexes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Prolyl oligopeptidase is a member of a relatively new group of serine peptidases unrelated to the well known trypsin and subtilisin families (1–4). The new family includes enzymes of different specificities like the prolyl oligopeptidase itself, dipeptidyl-peptidase IV, acylaminoacyl-peptidase, and oligopeptidase B (5). These enzymes selectively cleave substrates that are no longer than 30 amino acid residues in total. Prolyl oligopeptidase (EC 3.4.21.26 [EC] ) is implicated in the metabolism of peptide hormones and neuropeptides (6–8). Because specific inhibitors relieve scopolamine-induced amnesia (9–12), the enzyme is of pharmaceutical interest. The activity of prolyl oligopeptidase has also been associated with depression (13, 14) and blood pressure regulation (15).

The crystal structure determination of prolyl oligopeptidase has revealed that the carboxyl-terminal peptidase domain of the enzyme displays an / hydrolase fold and that its catalytic triad (Ser-554, His-680, and Asp-641) is covered by the central tunnel of an unusual -propeller (16). Recent engineering of the enzyme provided evidence for a novel strategy of regulation in which oscillating propeller blades act as a gating filter during catalysis, letting small peptide substrates into the active site while excluding large proteins, thereby preventing accidental proteolysis in the cytosol (17).

The active site region of prolyl oligopeptidase exhibits several charged residues such as Arg-643, Asp-642, Arg-252, Asp-149, and Arg-128. The complex electrostatic environment created by these residues may considerably influence the binding and thus the specificity of substrates. We have determined previously that, principally, five subsites of the enzyme (S3–S2') interact with a polypeptide substrate (18). In this work we have examined the effects of charged residues at different subsites. Because of the mainly positive environment around the active site, we substituted glutamic acid for the residues of the internally quenched substrate Abz¹-Gly-Phe-Ser-Pro-Phe(NO₂)-Ala, as the leader peptide, where Abz and Phe(NO₂) were the fluorescent donor and the quencher, respectively.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Enzyme Preparations—Prolyl oligopeptidase from porcine brain and its variants S554A and R252S were expressed in Escherichia coli JM105 cells and purified as described previously (19, 20). The enzyme concentrations were determined at 280 nm (3).

Kinetics—The reaction of prolyl oligopeptidase with Z-Gly-Pro-Nap (Bachem Ltd., Bubendorf, Switzerland) was measured fluorometrically using a Cary Eclipse fluorescence spectrophotometer equipped with a Peltier four-position multicell holder accessory and a temperature controller. The excitation and emission wavelengths were 340 and 410 nm, respectively. Cells with excitation and emission path lengths of 1 and 0.4 cm, respectively, were used. The substrates with internally quenched fluorescence, Abz-Gly-Phe-Ser-Pro-Phe(NO₂)-Ala and its derivatives, were prepared with solid phase synthesis, and their hydrolyzes were followed as in the case of Z-Gly-Pro-Nap, except that the excitation and emission wavelengths were 337 and 420 nm, respectively.

The pseudo-first order rate constants were measured at substrate concentrations lower than 0.1 K_m and calculated by non-linear regression data analysis using the GraFit software (21). The specificity rate constants (k_cat/K_m) were obtained by dividing the pseudo-first order rate constant by the total enzyme concentration in the reaction mixture.

The Michaelis-Menten parameters (k_cat and K_m) were determined with initial rate measurements using substrate concentrations in the K_m value range of 0.2–5. The kinetic parameters were calculated with nonlinear regression analysis. With substrates exhibiting very low K_m values, initial rates below the K_m could not be measured; therefore, the parameters were calculated from a single progress curve using the integrated Michaelis-Menten equation (22, 23).

Theoretical curves for the bell-shaped pH rate profiles were calculated by nonlinear regression analysis using Equation 1 and the GraFit software (21). In Equation 1, shown here,

(Eq. 1)

k_cat/K_m(limit) stands for the pH-independent maximum rate constant, and K₁ and K₂ are the dissociation constants of the catalytically competent base and acid, respectively. When an additional ionizing group modifies the bell-shaped character of the pH dependence curve, the data were fitted to Equation 2 (doubly bell-shaped curve), shown here,

(Eq. 2)

where the limiting values stand for the pH-independent maximum rate constants for two active forms of the enzyme, and K₁, K₂, and K₃ are the apparent dissociation constants of the enzymatic groups whose state of ionization controls the rate constants. The points for the rate constants for the substrate with Arg at position P2 were fitted to Equation 3, depicted here,

(Eq. 3)

which is composed of a bell-shaped and a sigmoid term.

Extraction of Individual Rate Constants from Nonlinear Temperature Dependence—Deviation from the linearity of an Arrhenius plot may be indicative of changes in rate-limiting steps, which allows for resolution of the individual rate constants that compose k_cat/K_m as defined by Equations 4 and 5 (24, 25), shown here,

(Eq. 4)

(Eq. 5)

where k₁ and k_–1 are the rate constants for binding and dissociation of the substrate, k₂ is the first-order acylation rate constant, EA is the acyl enzyme, and = k₂/k_–1 measures the stickiness of the substrate (26), which indicates that the substrate dissociates more slowly from its complex formed with the enzyme than it reacts to yield product (i.e. stickiness is high if k_–1 < k₂). It follows from Equation 5 that k_cat/K_m approximates k₁ whenever » 1. The temperature dependence of the rate constants can be obtained from Equation 6 (24), presented here,

(Eq. 6)

where q = ₀exp((E/R(1/T – 1/T₀)), k_1,0 is the value of k₁ at the reference temperature T₀ = 298.15 K, E₁ is the activation energy associated with k₁, and E = E_–1 – E₂. From the temperature dependence of k_cat/K_m, the value of k₁ and the ratio of k₂/k_–1 can be obtained together with the corresponding activation energies. Because T₀ can be set to any value, the parameters can be calculated for various temperatures.

Thermodynamic Parameters—The temperature dependence of k_cat/K_m and k_cat were determined between 10 and 40 °C at concentrations of 2–20 nM prolyl oligopeptidase. The thermodynamic parameters were calculated from Eyring plots as shown in Equation 7,

(Eq. 7)

where k is the rate constant, R is the gas constant (8.314 J/mol·K), T is the absolute temperature, N_A is the Avogadro number (6.022 x 10²³/mol), h is the Planck constant (6.626 x 10^–34 J·s), the enthalpy of activation H* = –(slope) x 8.3 14 J/mol, and the entropy of activation S* = (intercept – 23.76) x 8.3 14 J/mol·K. Equation 8, shown here,

(Eq. 8)

demonstrates how the free energy of activation, G*, was calculated.

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 (16). Crystals belong to the orthorhombic space group P2₁2₁2₁. X-ray diffraction data were collected using synchrotron radiation. Data were processed using the HKL suite of programs (27). Refinement of the structures was carried out by alternate cycles of REFMAC (28) and manual refitting using O (29) based on the 1.4-Å resolution model of wild type enzyme (16) (Protein Data Bank code 1QFM [PDB] ). Water molecules were added to the atomic model automatically using ARP (30) at the positions of large positive peaks in the difference electron density, but 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. Data collection and refinement statistics are given in Table I.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Fig. 1 illustrates the pH dependences of k_cat/K_m for the substrates containing P2 Arg and P2, P3, P4, and P5 Glu residues. The P2 Arg has a high pH optimum. The pH dependence does not follow a simple ionization curve, as indicated by the points deviated at low pH. The reaction of the peptide with a Glu residue at positions P2 or P4, in particular at P4, conforms to a double bell curve, whereas at positions P3 and P5 simple bell-shaped curves are displayed. The acidic limb of the pH dependence curve for substrates with Glu is shifted toward the lower pH region. The further the Glu is from the scissile bond, the greater the shift. The parameters of the pH dependence curves are shown in Table II, which also shows the kinetic parameters k_cat and K_m. The K_m is highest for the peptide with P3 Glu and lowest with P2 Arg. This suggests that poor binding makes the peptide with P3 Glu the worst substrate of this series, whereas the best substrate that possesses a P2 Arg exhibits the strongest binding. The differences in the k_cat values are less important. It may be noted that the carboxylterminal carboxyl group of the substrate does not affect the pH rate profiles because its ionization is outside of the pH range studied. Moreover, we did not observe any difference between the leader peptide and its amide derivative (not shown).

View larger version (23K): [in this window] [in a new window]   FIG. 1. The pH dependences of kcat/Km. A, P2 Arg (, left ordinate); the points were fitted to Equation 3, and the broken line is a simple sigmoid curve. P2 Glu (, right ordinate); the points were fitted to Equation 2, and the broken line represents a bell-shaped curve. B, P3 Glu (+, right ordinate), P4 Glu (, left ordinate), and P5 Glu (, left ordinate).

View larger version (13K): [in this window] [in a new window]   FIG. 2. The pH rate profiles for the reactions of prolyl oligopeptidase () and its R252S variant () with the substrate containing P3 Glu.

View larger version (13K): [in this window] [in a new window]   FIG. 3. Arrhenius plots for kcat/Km. Shown are Arg at the P2 position (A) and Glu at the P3 position (B) of the leader peptide Abz-Gly-Phe-Ser-Pro-Phe(NO2)-Ala. The broken lines were calculated with the k1 and E1 values shown in Table IV.

The kinetic (k₁, k₂/k_-1) and activation (E₁, E_-1 – E₂) parameters calculated from the temperature dependence of k_cat/K_m are listed in Table IV, which shows two interesting phenomena. First, the k₁ is close to the specificity rate constant, independent of its value, and this suggests that the rate-limiting step is the formation of the enzyme-substrate complex. This may be surprising, because usually the substrate binding is a very fast, diffusion-controlled process. In the present case, it appears that the nature of substrate controls the access to the active site. Secondly, the k₁ values are not consistent with their activation energies (E₁). For example, the higher k₁ for P2 Arg is associated with a higher E₁ compared with the E₁ for the Glu-containing substrates having lower k₁ values. Because the faster reaction displays too high an activation energy, the activation entropy should compensate for the unexpected effect.

Table II also shows H* and S* for k_cat, which is characteristic of the breakdown of the enzyme-substrate complex and does not principally involve the removal of water molecules. As expected, a negative S* was obtained with the substrate containing P2 Arg. However, the rate constants for the negatively charged compounds display moderately or slightly positive values. Possibly, the enzyme-substrate complexes that include glutamic acids are more disordered, and their motions must be frozen out upon going to the transition state. Indeed, the ratios between side chain and main chain B-factors calculated from the crystal structures of the enzyme-substrate complexes are considerably higher in the P2 Glu and P4 Glu complexes than in P2 Arg (1.7, 1.6, and 1.2, respectively), indicating the higher mobility of the glutamate side chains.

In contrast to the k_cat/K_m, the k_cat yields linear Arrhenius plots (Fig. 4), which indicates that the substrate acylation is rate-limiting over the temperature range investigated and that k₂ determines k_cat (25). This plot yields k₂ and E₂, thereby allowing for resolution of six independent parameters as listed in Table V. In accordance with the above argument, the value of k₂ agrees reasonably well with k_cat calculated in a different way (Table II). The knowledge of k₁ and k_–1 renders it possible to calculate the dissociation constant of the enzyme-substrate complex (K_s = k_–1/k₁). In the reaction for the substrate with the P2 Arg, the K_s is practically the same as the K_m value obtained from the Michaelis-Menten kinetics (Table II). The k₁ and k_–1 are considerably smaller with the P3 Glu, with the k₁ decreasing to a greater extent. Hence, the difference between the K_s and K_m is greater in this case. However, the k₂ alters only slightly, so that the major difference between the two substrates resides in the formation and decomposition of the enzyme-substrate complex.

View larger version (16K): [in this window] [in a new window]   FIG. 4. Arrhenius plots for kcat. Shown are Arg at the P2 (x), Glu at the P3 (), and Glu at the P5 () positions.

View larger version (53K): [in this window] [in a new window]   FIG. 5. Stereo view of the peptide binding site of prolyl oligopeptidase. Peptides with amino acids of P2 Arg (A), P2 Glu (B), and P4 Glu (C) are bound to the S554A variant. The substitutions are made in the leader peptide Abz-Gly-Phe-Ser-Pro-Phe(NO2)-Ala, of which only the P1–P4 residues are seen. Note that P2 Arg, P2 Glu, and P4 Glu in panels A, B, and C, respectively, are only partially in the density and, thus, should be considered fairly disordered. The bound ligands are shown darker than the protein residues. The SIGMAA-weighted (36) 2 mFo – Fc electron density using phases from the final model is contoured at the 1 level, where represents the root mean square electron density for the unit cell. Contours >1.4 Å from any of the displayed atoms have been removed for clarity. Dashed lines indicate hydrogen bonds (drawn with MolScript; Refs. 37 and 38). The close proximity of the charged groups Arg-128, Arg-252, Arg-643, Asp-149, Asp-641, and Asp-642 are also illustrated.

Like prolyl oligopeptidase, the classical serine proteases also lack interactions between enzyme groups and substrate side chains. Apart from the primary specificity determinant (P1 residue), interactions tend to be between the enzyme and the substrate main chain groups (35).

The uniform binding of the different substrates is apparently at variance with the significant differences in rate constants and their dissimilar pH dependences. This indicates that substrate specificity is not entirely controlled by the binding mode but is also dependent on the route to the active site, which is markedly influenced by the charged residues of the substrate and the enzyme.

	FOOTNOTES

 
The atomic coordinates and structure factors (code 1UOO [PDB] , 1UOP [PDB] , and 1UOQ [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by Wellcome Trust Grants 060923/Z/00/Z and 066099/01/Z and the Human Frontier Science Program Grant RG0043/2000-M 102. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of a studentship from the Biotechnology and Biological Science Research Council.

^** A Royal Society University Research Fellow.

To whom correspondence should be addressed. Tel.: 36-1-279-3110; Fax: 36-1-466-5465; E-mail: polgar{at}enzim.hu.

¹ The abbreviations used are: Abz, 2.aminobenzoyl; Phe(NO₂), p-nitrophenylalanine; Nap, 2-naphthylamide; Z, benzyloxycarbonyl.

	ACKNOWLEDGMENTS

 
Thanks are due to Ms. I. Szamosi for excellent technical assistance. We are grateful for access to the facilities of beam line European Molecular Biology Laboratory BW7B at the DORIS storage ring of Deutsches Elektronen Synchrotron (DESY; Hamburg, Germany) and the Synchrotron Radiation Source (SRS; Daresbury, United Kingdom).

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

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This Article Abstract Full Text (PDF) All Versions of this Article: 278/49/48786    most recent M309555200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Szeltner, Z. Articles by Polgár, L. Search for Related Content PubMed PubMed Citation Articles by Szeltner, Z. Articles by Polgár, L. Social Bookmarking                      What's this?