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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, H-1518 Budapest 112, P.O. Box 7, 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.

Running title: Prolyl oligopeptidase catalysis

* This work was supported by the Wellcome Trust (grant no. 060923/Z/00/Z and

066099/01/Z) and the Human Frontier Science Program (RG0043/2000-M 102).

The atomic coordinates will be deposited in the Protein Data Bank.

¶ 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@enzim.hu.

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

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SUMMARY

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 pKa 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 kcat/Km 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:

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.

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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 of no longer than about 30 amino acid residues in total. Prolyl oligopeptidase (EC

3.4.21.26) is implicated in the metabolism of peptide hormones and neuropeptides (6-8).

Since 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 α/β hydrolase fold, and that its

catalytic triad (Ser 554, His 680, Asp 641) is covered by the central tunnel of an unusual β-

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

Arg643, Asp642, Arg252, Asp149 and Arg128. The complex electrostatic environment

created by these residues may considerably influence the binding and thus the specificity of

substrates. We have previously determined 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,

Abz-Gly-Phe-Ser-Pro-Phe(NO2)-Ala1, as the leader peptide, where Abz and Phe(NO2) were

the fluorescent donor and the quencher, respectively.

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EXPERIMENTAL PROCEDURES

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 (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 1.0 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

hydrolyses 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 Km and were calculated by non-linear regression data analysis, using the GraFit

software (21). The specificity rate constants (kcat/Km) were obtained by dividing the pseudo-

first-order rate constant by the total enzyme concentration in the reaction mixture.

The Michaelis-Menten parameters (kcat and Km) were determined with initial rate

measurements, using substrate concentrations in the range of 0.2-5 Km value. The kinetic

parameters were calculated with nonlinear regression analysis. With substrates exhibiting

very low Km values, initial rates below the Km could not be measured; therefore, the

parameters were calculated from a single progress curve, using the integrated Michaelis-

Menten equation (22, 23).

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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

kcat/Km(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), 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 composed of a bell-shaped and a sigmoid term.

kcat/Km = kcat/Km(limit)[1/(1 + 10pK1 - pH + 10pH - pK2)] (Eq. 1) kcat/Km = kcat/Km(limit)1[1/(1 + 10pK1 - pH + 10pH - pK2)] +

kcat/Km(limit)2[1/(1 + 10pK2 - pH + 10pH - pK3)] (Eq. 2)

kcat/Km = kcat/Km(limit)1[1/(1 + 10pK1 - pH + 10pH - pK2)] +

kcat/Km(limit)2[1/(1 + 10pK2 - pH)] (Eq. 3)

Extraction of individual rate constants from nonlinear temperature dependence –

Deviation from linearity of Arrhenius plot may be revealing of changes in rate limiting steps,

which allows for resolution of the individual rate constants that compose kcat/Km as defined by

Equations 4 and 5 (24, 25).

E + S ES EAk-1

k1 k2

(Eq. 4) kcat/Km = k1k 2/(k –1 + k 2) = k 1α/ (1 + α) (Eq. 5)

where k 1 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 α = k2/k–1 measures the

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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 kcat/Km approximates k1 whenever α >> 1. The

temperature dependence of the rate constants can be obtained from Equation 6 (24).

kcat/Km = k1,0 exp[(-E1/R)(1/T – 1/T0)]q/(1 + q) (Eq. 6)

where q = α0exp[Eα/R(1/T – 1/T0)], k1,0 is the value of k1 at the reference temperature T0 =

298.15 K, E1 is the activation energy associated with k1, and Eα = E–1 – E2. From the

temperature dependence of kcat/Km the value of k1 and the ratio of k2/k–1 can be obtained

together with the corresponding activation energies. Since T0 can be set to any value, the

parameters can be calculated for various temperatures.

Thermodynamic parameters – The temperature dependence of kcat/Km and kcat were

determined between 10 °C and 40 °C at concentrations of 2-20 nM prolyl oligopeptidase. The

thermodynamic parameters were calculated from Eyring plots (Equation 7), where k is the

rate constant, R is the gas constant (8.314 J/mol.K), T is the absolute temperature, NA is the

Avogadro number (6.022 × 1023/mol), h is the Planck constant (6.626 × 10-34 J.s), the enthalpy

of activation ∆H* = -(slope) × 8.3 14 J/mol, the entropy of activation ∆S* = (intercept -

23.76) × 8.3 14 J/mol.K. The free energy of activation, ∆G*, was calculated from Equation 8.

ln(k/T)= ln(R/NAh) + ∆S*/R - ∆H*/RT (Eq. 7)

∆G*’ = ∆H*-T∆S* (Eq. 8)

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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) (PDB code: 1qfm). Water molecules were added to the atomic model

automatically using ARP (30) at the positions of large positive peaks 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. Data collection and refinement statistics are given in

Table I.

Please insert Table I here

RESULTS AND DISCUSSION

The effects of Glu at various P sites – We have previously shown that suc-Gly-Pro-Nan

is much worse as a substrate than Z-Gly-Pro-Nap (20). In the light of the basic environment

of the active site, a rationale of the large difference may be the effect of the negative charge of

the succinyl group. Therefore, we examined the effects on the kinetic and thermodynamic

parameters of the glutamic acid residue located at various sites of the leader peptide, Abz-

Gly-Phe-Ser-Pro-Phe(NO2)-Ala, which corresponds to the P1-P4 amino acids of bradykinin, a

possible natural substrate. Our previous X-ray crystallographic studies on the binding of an

octapeptide indicated that the P3-P2’ residues were clearly associated with the enzyme, while

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the P4 residue might weakly bind (18). Yet, we have extended the position of Glu also toward

subsites 4 and 5, as it is known from the three-dimensional structure of prolyl oligopeptidase

(20) that Arg252 and Arg128 are not far from the binding site, so that they may form

appropriate subsites for the binding of Glu in those positions. We have also analyzed the

effects of the opposite charge, using Arg at the P2 position.

Fig. 1 illustrates the pH dependences of kcat/Km 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 they display simple bell-shaped curves.

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 is the shift. The

parameters of the pH dependence curves are shown in Table II, which also shows the kinetic

parameters kcat and Km. The Km is highest for the peptide with P3 Glu and lowest with P2 Arg.

This suggests that the poor binding makes the peptide with P3 Glu the worst substrate of this

series, while the best substrate that possesses a P2 Arg exhibits the strongest binding. The

differences in the kcat values are less important. It may be noted that the C-terminal carboxyl

group of the substrate does not affect the pH-rate profiles as 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).

Please insert Fig. 1 and Table II here.

Kinetic properties of the R252S variant – Possible effects of Arg252 on the kinetic

parameters of the different substrates were examined by using the R252S variant, which

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eliminates a positive charge near the binding site. As seen from Table III it is the P3 Glu that

is most affected, in particular in the presence of 0.5 M NaCl. Interestingly, the rate constant

for the P4 Glu substrate is not affected, and the rate constant for the neutral Z-Gly-Pro- Nap is

only moderately decreased. Fig. 2 shows the pH dependence of kcat/Km for the P3 Glu

substrate. It is seen that not just the value of the specificity rate constant, but also its pH

dependence has changed considerably. The alterations were less impressive with P4 Glu and

P5 Glu. Nonetheless, it is clear from Table III and Fig. 2 that the electrostatic environment

created by Arg252 does influence the catalysis.

Please insert Fig. 2 and Table III here

Temperature dependence of rate constants – To gain a deeper insight into the kinetic

specificity of prolyl oligopeptidase, we have also investigated the temperature dependence of

the catalysis. For each substrate the Arrhenius plot deviated from a straight line, as shown in

Fig. 3 for the substrates with P2 Arg and P3 Glu, exhibiting the highest and lowest values for

kcat/Km, respectively. It should be emphasized that the decline in the curve with the increase in

temperature was not due to denaturation. Preincubation of the enzyme at high temperature, for

a longer time than that required for the activity measurement, did not affect the value of the

rate constant. The deviations from linearity were different for the various substrates, smaller

with P2 Glu, and greater with P2 Arg and P5 Glu, P3 Glu and P4 Glu being intermediates.

The nonlinear Arrhenius plot rendered it possible to determine the individual rate constants

that compose kcat and Km (24, 25). However, the moderate decline with P2 Glu, did not permit

to extract precise k2/k-1 values from the curve. Unfortunately, above 40 °C the enzyme tended

to denature, which prevented the accurate determination of the rate constants by using higher

temperature.

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Please insert Fig. 3 and Table IV here

It should be noted that conformational changes might also result in nonlinear Arrhenius

plots (31, 32). For example, sharp changes in slope were found at about 14 °C for D-amino

acid oxidase; at the same temperature sedimentation velocity and UV spectroscopy indicated

a change in conformational state of the enzyme (31). In the present case, however, the data

points well fit to the theoretical curve, which is not expected when conformational changes

predominate. In addition, the activation energies seem to be independent of temperature as

required in a short temperature range employed here, whereas reactions with different enzyme

conformations could proceed with different activation energies. It is likely, however, that

conformational changes are associated with the change in the rate constants. A further

problem may arise from the cis-trans isomerization of proline-containing substrates (3, 33),

which may also change with the increase of temperature. However, our experimental

conditions were not rate determining for cis-trans isomerization (3).

The kinetic (k1, k2/k-1) and activation (E1, E-1 – E2) parameters calculated from the

temperature dependence of kcat/Km are listed in Table IV, which shows two interesting

phenomena. Firstly, the k1 is close to the specificity rate constant, independently 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 E1 for the Glu-containing substrates having lower k1 values. Since the faster reaction

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displays too high an activation energy, the activation entropy should compensate for the

unexpected effect.

The activation parameters (∆H* and ∆S*) were calculated from Eyring plots (ln(k/T) vs.

1/T), using the k1 values of Table IV. The plots gave perfect straight lines (not shown), and

the calculated parameters are shown in Table II. Indeed, the ∆S* is higher for the substrate

having the P2 Arg compared with the ∆S* for the slower reactions of the Glu-containing

substrates. Most importantly, the ∆S* values are positive for all substrates, which indicates

that the transition states of the reactions are less ordered than the ground states, an atypical

phenomenon in enzyme catalysis. The release on binding of the ordered water molecules that

are associated with the substrates and the enzyme can account for the most part of the highly

positive values.

Table II also shows ∆H* and ∆S* for kcat, 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, which must be frozen out on 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 kcat/Km, the kcat yields linear Arrhenius plots (Fig. 4), which indicates

that the substrate acylation is rate limiting over the temperature range investigated, and that k2

determines kcat (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

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value of k2 agrees reasonably well with kcat 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 (Ks = k-1/k1). In the reaction for the substrate with the P2 Arg, the

Ks is practically the same as the Km value obtained from the Michaelis-Menten kinetics (Table

II). The k1 and k-1 are considerably smaller with the P3 Glu, the k1 decreasing to a greater

extent. Hence, the difference between the Ks and Km 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.

Please insert Fig. 4 and Table V here

Substrate binding modes – Determination of the three-dimensional structures of the

complexes formed between prolyl oligopeptidase and its substrates were essential for

understanding the differences in the specificity rate constant. We have determined the crystal

structures with substrates having P2 Arg, and P2, P3, P4, and P5 Glu (Fig. 5). Instead of the

wild type enzyme, we used the inactive prolyl oligopeptidase, in which an alanine residue

replaced the catalytic serine (18). Nevertheless, during the long crystallization period the

substrates were cleaved, so that only the acyl parts of the substrates were seen in the electron

density maps. These enzyme-product complexes share identical binding modes to the

enzyme-substrate complexes previously determined (18). In the case of subtilisin modified

also at the active site serine residue, about a 106-fold decrease in activity was demonstrated

with kinetic analysis (34), indicating a small remaining activity but with a different

mechanism. Using the S554A variant, we succeeded in measuring a similarly very low

kcat/Km, namely 0.98±0.03 M-1s-1, for the reaction with the substrate containing Arg at the P2

position. This represents a decrease of seven orders of magnitude. It may be noted that the

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remaining activity is not due to prolyl oligopeptidase impurity in the preparation, since the

active enzyme can be inhibited by diisopropyl-phosphofluoridate, whereas the inhibitor does

not affect the remaining activity. Also, the E. coli cells in which the enzyme is expressed do

not contain prolyl oligopeptidase, which eliminates the possibility of active enzyme

contaminating the inactive form.

Please insert Fig. 5 here.

The crystal structures assumed similar binding modes for all substrates involving

important interactions with the peptide bonds of the P2 and P3 residues, as described earlier

(16, 18). Of course, the P1 Pro ring interacting with the side chain of Trp595 determines the

primary specificity. The P2 side chains of both Arg and Glu are disordered, pointing towards

the bulk water. Residues P4 and P5 are also disordered, indicating that they do not interact

with the potential Arg binding sites. The substrate containing P3 Glu was not seen at all,

suggesting that the binding constant is higher for this substrate than for the others This is

consistent with the highest Km value for this substrate (Table II). The weak binding of the

substrate with P3 Glu is reasonable in terms of the hydrophobic S3 subsite, which has a

preference for Phe, and large hydrophobic groups such as the benzyloxycarbonyl group, but

disfavors the succinyl group, although this charged group binds at the same place as the

aromatic ring (20).

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 enzyme and substrate main chain groups (35).

The uniform binding of the different substrates is apparently at variance with the

significant differences in rate constants and with their dissimilar pH dependencies. This

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indicates that the substrate specificity is not entirely controlled by the binding mode, but it is

also dependent on the route to the active site, which is markedly influenced by the charged

residues of the substrate and the enzyme.

Acknowledgements – Thanks are due to Ms. I. Szamosi for excellent technical

assistance. D.R. thanks the BBSRC for the award of a studentship. 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 (Hamburg), and SRS (Daresbury).

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38. Esnouf, R. M. (1997) J. Mol. Graphics 15, 133-138

39. Brünger, A. T. (1992) Nature 355, 472-474

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1 Abbreviations: Abz, 2.aminobenzoyl; Phe(NO2), p-nitrophenylalanine; Nap, 2-

naphthylamide; Nan, 4-nitroanilide

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LEGENDS TO FIGURES

Fig. 1. The pH-dependences of kcat/Km. (A) P2 Arg ( , left ordinate); the points were fitted

to Equation 3, the broken line is a simple sigmoid curve. P2 Glu ( , right ordinate); the

points were fitted to Equation 2, the broken line represents a bell-shaped curve. (B) P3 Glu

( , right ordinate), P4 Glu ( , left ordinate), and P5 Glu ( , left ordinate).

Fig. 2. The pH-rate profiles for the reactions of prolyl oligopeptidase ( ) and its R252S

variant ( ) with the substrate containing P3 Glu.

Fig. 3. Arrhenius plots for kcat/Km. (A) With Arg at the P2, (B) with Glu at the P3 position of

the leader peptide Abz-Gly-Phe-Ser-Pro-Phe(NO2)-Ala. The broken lines were calculated

with k1 and E1 values shown in Table IV.

Fig 4. Arrhenius plots for kcat. Arg at P2 ( ), Glu at P3 ( ), and Glu at the P5 ( )

positions.

Fig. 5. Stereo view of the peptide binding site of prolyl oligopeptidase. Peptides with

amino acids of A, P2 Arg, B, P2 Glu, C, P4 Glu 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 Figs. A, B and C,

respectively, are only partially in the density, and so should be considered fairly disordered. The

bound ligands are shown darker than the protein residues. The SIGMAA (36) weighted 2mFo-

∆Fc electron density using phases from the final model is contoured at 1σ level, where σ

represents the rms electron density for the unit cell. Contours more than 1.4 Å from any of the

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displayed atoms have been removed for clarity. Dashed lines indicate hydrogen bonds (Drawn

with MolScript, (37, 38). The close proximity of the charged groups, Arg 128, Arg 252, Arg

643, Asp 149, Asp 641, and Asp 642 are also illustrated.

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Table I Data collection and refinement statistics

P P2R P2E P4E Data collection

Radiation, detector DESY BW7B DESY BW7B SRS 14.1 and wavelength (Å) MAR345 IP, 0.845 MAR345 IP, 0.845 ADSC Q4 CCD 1.488

Unit cell dimensions (Å) 70.2, 98.7, 109.4 70.7, 99.3, 110.7 70.3, 99.1, 109.8 Resolution (Å) 51-2.35 (2.35-2.41) 35-1.85 (1.85-1.90) 36-2.1 (2.10-2.15) Observations 117,435 247,702 153,386 Unique reflections 31,460 66,292 43,221 (I/σ(I) 12.6 (2.2) 14.6 (2.5) 13.8 (3.7) Rsym

a 0.099 (0.467) 0.076 (0.406) 0.089 (0.129) Completeness (%) 97.6 (98.7) 98.7 (97.0) 95.1 (76.8)

Refinement

Non-hydrogen atoms 6,053 (including 1 glycerol and 300 water molecules)

6,906 (including 6 glycerol and 1125 water molecules)

6,291 (including 2 glycerol and 532 water molecules)

Rcrystb 0.193 (0.247) 0.156 (0.194) (0.147 (0.145)

Refletions used 30,095 (2,182) 63,593(4,515) 41,394 (2,376) Rfree

c 0.237 (0.307) 0.194 (0.222) 0.205 (0.234) Reflections used 1,365 (94) 2,699 (187) 1,827 (101) Rcryst (all data)b 0.195 0.158 0.149 Mean temperature factor (Å2) 32.2 16.0 26.4

Rmsds from ideal values

Bonds (Å) 0.006 0.008 0.026 Angles (°) 0.9 1.1 1.9 DPI coordinate error (Å) 0.18 0.08 0.10 PDB code 1uoo 1uop 1uoq Numbers in parentheses refer to values in the highest resolution shell.

aRsym = ΣjΣh|Ih,j - <Ih>|/ΣjΣh<Ih> where Ih,j is the jth observation of reflection h, and <Ih> is

the mean intensity of that reflection.

bRcryst = Σ||Fobs|-|Fcalc||/Σ|Fobs| where Fobs and Fcalc are the observed and calculated structure

factor amplitudes, respectively.

cRfree is equivalent to Rcryst for a 4 % subset of reflections not used in the refinement (39)

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Table II

Kinetic and activation parameters for the reactions of prolyl oligopeptidasea Parameter P2 Arg P2 Glu P3 Glu P4 Glu P5 Glu kcat (s-1) 3.20±0.09 2.40±0.03 1.82±0.03 1.70±0.07 7.22±0.21 Km (µM) 0.40±0.07 1.54±0.04 6.3±0.2 1.32±0.12 3.7±0.3 kcat/Km (µM-1s-1) 8.0±1.4 1.55±0.08 0.26±0.01 1.56±0.15 1.95±0.17 kcat/Km(lim)1 (µM-1s-1) 3.3±1.1 1.2±0.2 – 1.3±0.3 – kcat/Km(lim)2 (µM-1s-1) 10.4±0.4 3.4±0.2 0.35±0.01 2.9±0.2 2.87±0.03 pK1 5.8±0.5 5.1±0.5 5.37±0.07 4.7±1.0 4.5±0.1 pK2 7.6±0.2 7.1±0.2 – 7.1±0.3 – pK3 > 9 8.91±0.08 8.73±0.05 8.94±0.09 8.68±0.02 pH optimum > 8.5 7.9 7.0 7.9 6.6 ∆H* (kJ/mol) for k1 115.6 72.4 102.2 80.7 106 ∆S* (J/mol⋅K) for k1 274 120 202 148 238 ∆G* (kJ/mol) for k1 33.8 36.4 42.0 36.4 35.1 ∆H* (kJ/mol) for kcat 48.7 85.7 71.3 ∆S* (J/mol⋅K) for kcat -74.0 45.1 9.7 ∆G* (kJ/mol) for kcat 70.8 72.3 68.5 a Abz-Gly-Phe-Ser-Pro-Phe(NO2)-Ala was used as leader peptide, except with the P5 Glu

substrate, where a Glu was inserted between Abz and Gly.

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Table III

Effects of Arg252 on specificity rate constants for prolyl oligopeptidasea Enzyme P3 Glu

(µM-1s-1) P4 Glu (µM-1s-1)

P5 Glu (µM-1s-1)

Z-Gly-Pro-Nap (µM-1s-1)

Wild type 0.332 (7.0) 1.81 (7.0) 3.09(6.5) 4.84 (8.0) R252S variant 0.129 (6.5) 1.80 (7.5) 1.62 (7.0) 3.77 (7.5) Wild type (NaCl) 0.575 (7.6) 5.50 (7.6) R252S variant (NaCl) 0.125 (7.0) 1.14 (7.8) a The reactions were measured in the absence or in the presence of 0.5 M NaCl at substrate

concentrations of 0.2–0.3 µM. The numbers in brackets stand for the pH values close to the

pH optimum where the kcat/Km values were determined. The amino acid residues of the leader

peptide, Abz-Gly-Phe-Ser-Pro-Phe(NO2)-Arg-Ala, are substituted at the P3, P4, and P5

positions, respectively, by a glutamic acid residue. P5 Glu = Abz-Glu-Gly-Phe-Gly-Pro-Phe-

Gly-Phe(NO2)-Ala.

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Table IV

Kinetic and activation parameters for the reactions of prolyl oligopeptidasea Parameter 15°C 20°C 25°C 30°C 35°C P2 Argb k1 (µM-1s-1) 1.5±0.1 3.5±0.5 7.9±1.5 17±4 36±9 k2/k-1 5±2 2±1 1.1±0.4 0.6±0.2 0.3±0.1 E1 (kJ/mol) 118±9 120±9 120±9 118±8 118±8 E-1 – E2 (kJ/mol) 104±5 104±5 104±5 104±5 104±6 P2 Glu k1 (µM-1s-1) 0.89±0.03 1.5±0.1 2.5±0.3 4.2±0.8 6.8±1.6 k2/k-1 63±149 28±56 13±21 6±8 3±3 E1 (kJ/mol) 75±8 75±8 75±8 75±8 75±8 E-1 – E2 (kJ/mol) 113±51 113±51 113±51 113±51 113±51 P3 Gluc k1 (µM-1s-1) 0.064±0.014 0.13±0.04 0.27±0.10 0.55±0.32 1.1±0.8 k2/k-1 7.9±9.9 4.9±8.3 2.6±4.1 1.4±2.0 0.8±1.0 E1 (kJ/mol) 106±24 105±21 105±18 105±21 105±21 E-1 – E2 (kJ/mol) 92±14 94±22 95±23 94±22 94±22 P4 Glu k1 (µM-1s-1) 0.81±0.94 1.5±0.3 2.6±0.9 4.5±1.9 7.7±3.5 k2/k-1 16±31 8±13 3.7±5.3 1.8±2.3 0.8±1.1 E1 (kJ/mol) 83±17 83±17 83±17 83±17 83±16 E-1 – E2 (kJ/mol) 108±31 108±31 108±31 108±31 107±35 P5 Glu k1 (µM-1s-1) 0.95±0.04 2.05±0.15 4.3±0.5 8.9±1.3 18±3 k2/k-1 7.7±2.2 3.3±0.9 1.5±0.4 0.7±0.2 0.31±0.08 E1 (kJ/mol) 109±6 109±6 109±6 109±6 109±6 E-1 – E2 (kJ/mol) 119±3 119±3 119±3 119±3 119±3 a The parameters were determined at pH 8.0 and calculated by using Equation 6. The leader

peptide: Abz-Gly-Phe-Ser-Pro-Phe(NO2)-Arg-Ala, except for the longer substrate with P5

Glu, Abz-Glu-Gly-Phe-Ser-Pro-Phe(NO2)-Arg-Ala

b pH 8.6

c pH 7.0

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Table V

Kinetic and activation parameters for the reactions of prolyl oligopeptidase Parameter P2 Arg

(pH 8.6) P3 Glu (pH 7.0)

P5 Glu (pH 7.0)

k1 (µM-1s-1) 7.9±1.6 0.27±0.10 2.9±0.7 k-1 (s-1) 2.3±0.1 0.49±0.04 2.9±0.2 k2 (s-1) 2.46±0.09 1.27±.0.14 6.2±0.3 E1 (kJ/mol) 120±9 105±18 120±12 E-1 (kJ/mol) 163±3 195±7 190±4 E2 (kJ/mol) 59±3 100±7 74±4 Ks = k-1/k1 (µM) 0.29 1.81 0.99

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Fig. 1

pH6 8 10

kcat

/Km

(µM

-1s-1

)

0

4

8

12

kcat

/Km

(µM

-1s-1

)

1

1.5

2

2.5

A

pH6 8 10

kcat

/Km

(µM

-1s-1

)

0

0.5

1

1.5

2

2.5

3

kcat

/Km

(µM

-1s-1

)

0.15

0.2

0.25

0.3

0.35B by guest on February 5, 2018http://w

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Fig. 2

pH6 8 10

kcat

/Km

(µM

-1s-1

)

0

0.2

0.4

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Fig. 3

1/T0.0032 0.0034 0.0036

ln(k

cat/K

m)

14

15

16A

1/T0.0032 0.0034 0.0036

ln(k

cat/K

m)

11

12

13

B

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Fig. 4

1/T0.0032 0.0033 0.0034 0.0035

ln (k

cat)

(s-1

)

-2

0

2

4

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W595

D642

D641

R643

R252

D149

S554AN555

H680Y473

R128

W595

D642

D641

R252

R643

N555S554A

D149

H680Y473

R128

A

W595

D642

D641

R643

R252

D149

S554AN555

H680Y473

R128

W595

D642

D641

R252

R643

N555S554A

D149

H680Y473

R128

B

W595

D642

D641

R643

R252

D149

S554AN555

H680Y473

R128

W595

D642

D641

R252

R643

N555S554A

D149

H680Y473

R128

C

Fig. 5

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PolgárZoltán Szeltner, Dean Rea, Veronika Renner, Luiz Juliano, Vilmos Fülöp and László

influential during substrate bindingElectrostatic environment at the active site of prolyl oligopeptidase is highly

published online September 25, 2003J. Biol. Chem. 

  10.1074/jbc.M309555200Access the most updated version of this article at doi:

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