Daniel Hasenpusch

Uwe T Bornscheuer

Walter Langel
email: langel@uni-greifswald.de

Simulation on the structure of pig liver esterase

Keywords:

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Introduction

Pig liver esterase (PLE) is widely used as biocatalyst in organic synthesis [START_REF] Bornscheuer | Hydrolases in organic synthesis[END_REF]. The enzyme was studied in detail for the last 30 years and many applications were described [START_REF] De Maria | Pig liver esterase (ple) as biocatalyst in organic synthesis: From nature to cloning and to practical applications[END_REF]. The conversions of a broad range of racemic or prostereogenic esters proceed with high to excellent enantioselectivity, making PLE a very useful enzyme for sophisticated organic synthesis processes.

Typically the active sites of serine hydrolases is a catalytic triad with a SER, a HIS, and an ASP or GLU residue [START_REF] Bornscheuer | Hydrolases in organic synthesis[END_REF]. The side chain carboxyl group of ASP/GLU polarizes the adjacent HIS, which in turn accepts the hydroxyl proton from SER during the catalytic reaction. In early work the residues SER 200, HIS 440, and GLU 327 were assigned to the active triad of the acetylcholine esterase from Torpedo californica (TcAChE [START_REF] Sussman | Atomic-structure of acetylcholinesterase from torpedo-californica -a prototypic acetylcholine-binding protein[END_REF]). This was confirmed for further acetylcholine esterases by proving that the analogous GLU residue is essential for catalytic activity [START_REF] Shafferman | Mutagenesis of human acetylcholinesterase -identification of residues involved in catalytic activity and in polypeptide folding[END_REF]. Analogous assignments were made for lipases such as Candida rugosa Lipase (CRL) [START_REF] Monecke | Molecular modelling studies on the catalytic mechanism of candida rugosa lipase[END_REF]. In contrast to that the orientation of the side chain of the analogous GLU 353 residue in rabbit carboxyl esterase (rCE) (pdb codes 1THG and 2BCE [START_REF] Bencharit | Structural insights into cpt-11 activation by mammalian carboxylesterases[END_REF]) is opposite to that found for other esterases, and it is unlikely that this GLU 353 stabilizes the HIS 467 of the rCE active site.

No crystallization and crystal structure determination of pig liver esterase (PLE) was possible so far. The folding states of parts of the sequence to  and structures were assigned in analogy to known structures of typical , hydrolases [START_REF] Bornscheuer | Hydrolases in organic synthesis[END_REF][START_REF] Ollis | The alpha/beta-hydrolase fold[END_REF]]. Originally three isoenzymes (, , ) with different sequences and substrate specificities had been characterized in [START_REF] Heymann | Method for the estimation of esterase synthesis and degradation and its application to evaluate the influence of insulin and glucagon[END_REF][START_REF] Junge | Characterization of the isoenzymes of pig-liver esterase .2. Kinetic studies[END_REF].

The-isoenzyme of the PLE initially identified as proline--naphthyl amidase was cloned and overexpressed in P. pastoris X33 [START_REF] Musidlowska | By overexpression in the yeast pichia pastoris to enhanced enantioselectivity: New aspects in the application of pig liver esterase[END_REF] and E.coli Origami [START_REF] Bottcher | Functional expression of the gamma-isoenzyme of pig liver carboxyl esterase in escherichia coli[END_REF]. In 2008 additional isoenzymes were identified, and the amino acid sequence of six of them was determined.

They differ by a small number of mutations (Table 1) and were denoted PLE1-5 [START_REF] Hummel | Isoenzymes of pig-liver esterase reveal striking differences in enantioselectivities[END_REF]. PLE1 is identical to the former -PLE, whereas no unique assignment of the former and modifications was possible. In [START_REF] Hummel | Identifizierung von isoenzymen der schweineleberesterase[END_REF] the sequence of an additional modification, PLE6, is given. A further isoenzyme which complements the mutation pattern of PLE3 and PLE5 was described as alternative PLE (APLE) [START_REF] Hermann | Alternative pig liver esterase (aple) -cloning, identification and functional expression in pichia pastoris of a versatile new biocatalyst[END_REF]. Determination of the molecular weight has shown that under physiological conditions the enzyme mostly forms trimers which contain a random combination of different isoenzyme monomers [START_REF] Heymann | Method for the estimation of esterase synthesis and degradation and its application to evaluate the influence of insulin and glucagon[END_REF][START_REF] Junge | Characterization of the isoenzymes of pig-liver esterase .2. Kinetic studies[END_REF].

In this paper we focus on 3D models for the enzyme structure without deformation by an embedded substrate as a first step towards understanding the reaction mechanism of the enzyme and its enantioselectivity. As the geometry of the active site might be modified by trimerization, we generated models of both monomer and trimer. A wide spread and valid method for generating 3D models of poorly crystallizing enzymes is homology modeling. The PLE1 has a high sequence homology (about 75%) to human liver carboxyl esterase (hCE) and rabbit liver esterase (rCE) whose crystal structures are known.

The structural basis of heroin and cocaine metabolism was also reported based on the crystal structure of hCE [START_REF] Bencharit | Structural basis of heroin and cocaine metabolism by a promiscuous human drug-processing enzyme[END_REF] and a possible entrance path to the hidden active site in hCE was described. Here, we verified the feasibility and stability of the homology model by extensive molecular dynamics simulations on the monomer. The originally modeled PLE1 was modified by several mutations transferring it to other isoenzymes. We traced the influence of single mutations and extended sequence variations on the secondary structure by classical molecular dynamics. This afforded extended simulations, since local rearranging and especially modification of the folding state around a newly introduced mutation may occur on a time scale of several ns, which was beyond the scope of earlier work. By runs of similar lengths the contacts between the trimeric subunits were characterized.

Methods

A homology model [START_REF] Gasteiger | Expasy: The proteomics server for in-depth protein knowledge and analysis[END_REF] of the PLE1 isoenzyme was generated on the basis of the human carboxyl esterase [START_REF] Bencharit | Crystal structure of human carboxylesterase 1 complexed with the alzheimer's drug tacrine: From binding promiscuity to selective inhibition[END_REF]. As a template we used the X-ray structures of hCE complexes with the heroin analogue naloxone methiodide (1MX9, chain A). The rabbit liver esterase rCE [START_REF] Bencharit | Structural insights into cpt-11 activation by mammalian carboxylesterases[END_REF] has an even greater similarity to PLE than hCE, but the only available crystal structure of its complex with 4-piperidino-piperidine (1K4Y) contains three hypervariable loops close to the active center, and 33 amino acids could not be localized. The full PLE sequence (molecular weight about 60 kDa) starts with a signal peptide with 18 residues, which is cleaved during processing of the enzyme. During homology modeling the sequence GQP at the N-terminus and ten residues in addition to the ER-retention signal HAEL at the C-terminus were omitted.

According to standard numbering our models contains the PLE residues 4 to 534.

For the isoenzymes PLE2-6 and APLE the respective side chain atoms of the mutated residues in the optimized PLE1 structure were stripped, and their names in the pdb file replaced. This data set was reloaded into the xLeap program of the AMBER package (versions 7 to 10) [START_REF] Case | AMBER 10[END_REF], which adds the missing side chain atoms of the mutated residues according to predefined standard structures of the respective amino acid.

The crystal structure of human carboxyl esterase trimers in complex with taurocholate (2DR0) was used as a template for the PLE trimer, and three identical PLE1 monomers from a trajectory with 6 ns simulation time were aligned to each of the three chains of this model. No hydrogen bonds between the PLE1 monomers were predefined in this initial configuration.

The 531 residues in the identical monomers are denominated 4-534/1, 4-534/2 and 4-534/3.

Molecular dynamics simulations

Models for molecular dynamics simulations were prepared in xLeap using the parm94 force field. Orthorhombic box dimensions for periodic boundary conditions were 72x84x92 Å 3 and 85x137x134 Å 3 for single monomers and trimers, respectively. This allowed for a spacing of at least 10 Å between enzyme and cell boundaries. The box was filled with about 50000 and 40000 discrete TIP3P water molecules for monomers and trimers, respectively. Bond lengths of the water molecules were fixed using a SHAKE algorithm. Under physiological conditions all proteins considered here had negative total charges, which were compensated by appropriate addition of Na + counter ions (CIO) to the system. The geometry of each system was optimized during at least three runs, relaxing (1) the positions of the water molecules and the counter ions, and (2) the enzyme structure only in fixed water environment, and (3) minimizing the energy of the whole system. Starting structures for all mutations were derived from the homology model of PLE1 after geometry optimization. By molecular dynamics at constant pressure and temperature (NpT) for 20ps cell size and water density were adjusted. Production runs scanned 14 ns at constant energy with an initial temperature of 300 K and a time step of 1 fs. All images of 3D structures were generated with VMD [START_REF] Humphrey | Vmd: Visual molecular dynamics[END_REF] and its plug-ins [START_REF] Stone | An efficient library for parallel ray tracing and animation[END_REF][START_REF] Frishman | Knowledge-based protein secondary structure assignment[END_REF][START_REF] Eargle | Multiple alignment of protein structures and sequences for vmd[END_REF][START_REF] Gille | Strap: Editor for structural alignments of proteins[END_REF].

Conclusions from our simulations on a limited number of structures were cross checked by comparison with super families of proteins with related sequences and functions. The database program 3DM allows data to be organized around a single class of molecules, and a common numbering scheme for structurally equivalent amino acids is used enabling the identification of conserved residues and distributions of amino acids for specific residues.

First a subfamily of mammalian carboxyl esterases similar to PLE and hCE was derived from the full  folded hydrolases superfamily with about 12000 structures. This subfamily contained about 170 structures, but showed only a small number of 3DM core residues, for which conservation statistics may be evaluated. The superfamily comprises a wide spectrum of different structures with little overlap. We cured this problem by starting from a family of about 1300 structures around the prototype acetylcholine esterase. Subfamilies such as lipases are now omitted, and the members of the acetylcholine esterase family are much more similar to each other than those of the superfamily. Consequently the number of 3DM core amino acids is much higher in this new family. Still its subfamily with PLE and hCE has nearly as many members (150) as the one derived from all  folded hydrolases with known 3D structure.

Results and discussion

Monomer structure, mutations and their position relative to the entrance helix

The secondary structure of the nearly globular protein (Fig. 1) consists of a series of helices and -folds. The residues in the range 72-87 are referred to as entrance helix. In contrast to the lipases the active site is not freely accessible in all esterases, and substrates have to pass between this α helix and its counterpart, formed by residues 338-346. Folding in this range thus is crucial for the formation of the Michaelis-Menten complex and for the activity of the enzyme.

The 25 mutations between the isoenzymes are correlated in part forming six independent groups (Table 1 and Fig. 2). The β-folds are highly conserved and not affected by any of the mutations considered here:

(1) For residues 73, 75-77, 80 on the entrance helix, three combinations are possible, (i) ASP, VAL-VAL-GLU, THR (ii) ASP, VAL-ALA-GLY, THR or (iii) GLU, ILE-GLY-GLY.

LEU. ASP and VAL or GLU and ILE on positions 73 and 75 are pair wise correlated. Very important for the stability of the helix should be the transition from VAL and GLU to GLY or ALA on position 76 and GLY on 77, respectively.

(2) Eleven mutations in three different strands are strictly correlated: 87, 92 and 93 are close to the entrance helix. 129, 133, 134, 138 and 139 are on a strand which is parallel to the entrance helix and has partial helix folds. Residue 138 is close (within 5 Å) to 139 and 462.

The residue 139 in turn is near to 461 and 462 and can interact with 461 directly. This may affect the positions of 459 and 463 and have an indirect influence on the structure of the entrance helix. 234, 236 and 237 have distances of more than 7 Å to the entrance helix, and no obvious correlation between their configuration and the structure of the entrance helix was found.

Three further groups of mutations, (3-5) are found on a single strand between residues 285 and 302:

(3) The only uncorrelated single point mutation transfers PHE on position 285 to PRO and might affect the residues 286 and 287 next to the entrance helix.

(4) For 286 and 287 the sequences LEU-THR or PHE-ALA were found, which mainly differ by an exchange of the aliphatic and aromatic side chains.

(5) A triple mutation on 290, 294 and 302 in the same strand as groups (3) and (4) transfers PHE, GLN, PRO into LEU, PRO, THR. Amino acid 290 is close to the loop of 285 to 287 and its mutation may have an impact on the substrate entrance. Two further mutations shift a proline from position 302 to 294. This switches the -angles at both positions by 180° and will essentially rotate the very flexible backbone structure between them by 180°. Outside of this window 294 to 302, the effects of both mutations on the backbone conformation probably compensate to a wide extent. [START_REF] Bencharit | Structural insights into cpt-11 activation by mammalian carboxylesterases[END_REF] The group of three mutations on the positions 459, 461 and 463 transfers PHE, LEU, LYS into ALA, PHE, ARG shifting the aromatic side chain from 459 to 461. This might deform an -helix in the proximity of the entrance helix.

RMSD of the monomer

Root mean square displacements (RMSD) of the coordinates of single residues were evaluated by comparing the atomic positions in the last frames of the total simulation time of 14 ns with those every 2 ns before, for trajectories (Fig. 3) from the seven PLE isoenzyme monomers (PLE1-6 and APLE) as well as the three PLE1 monomers in the trimer. MD calculations induced significant relaxation in most parts of the secondary structures derived from geometry optimized PLE1. Typically initial RMSD values of 5-10 Å decreased to 1-3 Å during the interval from 8 to 12 ns (Fig. 3a). This indicates relaxation of the mutated parts and a stabilization of the whole isoenzyme. Only during the last 2-4 ns the RMSD value further decreased due to convergence of the structure to the final configuration. In some cases fluctuations with periods of 2-5 ns were observed which may be due to slow rearranging of larger structures of the enzyme (Fig. 3b).

The impact of the mutations on the final isoenzyme structures was analyzed by using in turn one of them as template and evaluating the RMSD for the others. In major parts of the isoenzymes, mutations incite little rearranging and fluctuation, and the RMSD values between different isoenzymes were below 2 Å, in part even below 1 Å. All β-folds and a few helices form a rigid and highly conserved backplane of the enzyme, which is its basic structure (Fig. 4). In contrast to that the flexible front consists of a major part of the helices and of several loops, containing all mutations. Many variable structural elements strongly affect the enzyme structure, especially near the entrance and around the active site (Fig. 5a).

Influence of the mutations on the structure of the enzyme

In the isoenzymes considered here, the entrance helix is affected by up to five mutations with respect to PLE 1 (Fig. 2), and some of them are known to affect the experimental substrate specificity (e.g. VAL to ALA and GLU to GLY on positions 76 and 77, respectively [START_REF] Musidlowska-Persson | Site directed mutagenesis of recombinant pig liver esterase yields mutants with altered enantioselectivity[END_REF]).

With the exception of PLE3, (Fig. 5b, Fig. 6), the residues 74-83 form a well-defined α-helix (Fig. 5a, yellow). In this isoenzyme the dihedrals for four residues (74, 75, 77 and 80) are not in the range of α-folds, and the helix is broken into a coil structure. We speculate that this is a collective effect of the mutations in groups (1) and ( 3). The replacement of residues 76 and 77 by glycines certainly weakens the entrance helix, but this has no effect on its folding in PLE6 and APLE. If the additional mutation of residue 285 into a proline shifts the helix (272 to 284), some stress on the entrance helix might induce unfolding.

The residues 89-96 formed a random coil in the homology model and in most isoenzymes but arranged to a short helix in the PLE1 monomer (Fig. 7). This folding is reflected by high RMSD values above 6 Å very close to the entrance helix (blue in Fig. 4). The secondary structure of this flexible zone seems to be the result of a complicated interplay of several mutations.

Trimer

The trimer built from three PLE1 monomers has a propeller like structure (Fig. 8) and is stable during the simulation time of 14 ns. The rigid and soft parts of the monomers are found on the bottom and on the top of the propeller blades, respectively. All three interfaces between the trimers have similar structures involving nearly the same corresponding residues (Table 2). The contact site between subunits 1 and 2 is shown in detail in Fig 8 (insert). The residues are contained on three and four strands for the clockwise (Table 2a) and counterclockwise oriented monomer faces (Table 2b), respectively.

The PLE monomers contain five cysteine residues which form two disulfide-bridges (70-99, and 256-267) close to the contacts between trimer subunits (cf. Table 2). According to [START_REF] Lange | Cloning, functional expression, and characterization of recombinant pig liver esterase[END_REF], residue 70 rather than the adjacent CYS 71 is involved in the first bridge. It is conserved to 100% in the subfamily of PLE, and this high degree of conservation indicates substantial importance for the enzyme structure. We assume that it maintains the connection between the strands 5' and 6' (red and orange in Fig. 8), whereas 256-267 close a loop inside strand 2 (blue). The cystine bridges seem to stabilize the contact areas between the subunits and thus may be essential for trimerization.

The RMSD pattern of the final structure of the trimer was obtained by comparing the amino acid in each of the three monomers with their correspondents in the relaxed PLE1 configuration, from which the trimer had been assembled. Deviations in the RMSD pattern between the trimer components and monomer PLE1 are similar as between the mutated and original monomers.

Assignment of the catalytic triad and structure of the active site

SER 204 has been identified earlier as the nucleophilic residue in the active site, and the acceptor for its proton is HIS 449. The two tautomeric forms  and  of histidine with the N  and N  atoms protonated, respectively, are equilibrated at physiological pH-values by proton transfers [START_REF] Hass | Characterization of conformational exchange of a histidine side chain: Protonation, rotamerization, and tautomerization of his61 in plastocyanin from anabaena variabilis[END_REF]. This cannot be reproduced in a force field calculation, and AMBER provides three predefined residues, HID, HIE, and HIP, denoting the  and -tautomers and the doubly protonated form of the imidazol ring, respectively. According to the homology model, only a proton at N  points towards negatively charged oxygen atoms, whereas an H atom at the -species would be directed versus the SER hydroxyl group hindering proton transfer from SER to HIS. Rotation of the ring around the C  -C  bond by 180° does not cure this problem, since the two N-atoms are not in symmetrical positions with respect to this axis. We used the -tautomer (HID) as residue 449 enabling the formation of a very stable hydrogen bond between the O  of SER 204 and the deprotonated histidine N  . The O  from SER 204 can also be stabilized by GLU 229 and GLU 203, the H  proton forming transient Hbonds to the backbone O of these residues (Fig. 9). The hydrogen bonds to HIS 449 and to GLU 229 are alternating during simulation.

The protonated N  of HIS 449 forms hydrogen bonds to negatively charged atoms. The previous assignment [START_REF] Shafferman | Mutagenesis of human acetylcholinesterase -identification of residues involved in catalytic activity and in polypeptide folding[END_REF][START_REF] Bencharit | Structural insights into cpt-11 activation by mammalian carboxylesterases[END_REF] provides stabilization by the GLU 336 side chain. In none of the present simulations this group pointed to the active site, and no stable hydrogen bonds to HIS 449 were formed. In most PLE structures the carboxyl O atoms of the GLU 336 side chain had distances of about 8 Å to the HIS N  after molecular dynamics. The group is oriented into the opposite direction forming stable hydrogen bonds to LYS 521 NZ or backbone N atoms of GLU 336, LYS 334 and ASN 333. For the bonding in the active site we distinguish three different patterns:

(1) In PLE1, PLE2 and APLE the carboxyl group of GLU 452 is close to the protonated N  atom of HIS 449 with O-N-distances of less than 3 Å, forming very stable hydrogen bonds.

The side chain carboxyl group reorients in some cases and alternating bonds to O  and O  are observed. When assuming that GLU 336 is replaced by GLU 452 in the catalytic triads, the two characteristic distances between SER and HIS, as well as HIS to GLU have the typical values, and the exchange should have small impact on the catalytic mechanism. Molecular mechanics calculations depend on the parameterization of the respective force field. We verified if the reorientation of the GLU 336-GLU 452 is an artifact of the force field used here. In the experimental structures of rCE [START_REF] Bencharit | Structural insights into cpt-11 activation by mammalian carboxylesterases[END_REF] the side chains of the two residues equivalent to 336 and 452 are oriented towards the HIS equivalent to 449 in PLE, but neither carboxyl group is close enough to the proton at N  for a hydrogen bond. Due to the missing positions for 33 residues, rCE could not be used as starting structure. We instead simulated four hydrolases, for which the active centers are fully known: hCE (1MX9), TcAchE (2VJA), aged form of human butyrylcholinesterase (hBuChE, 2WSL), and CRL (1LPM). The same protocol as for PLE was used and in the unconstrained simulations all enzymes had stable hydrogen bonds between their HIS N  and SER H  in the active sites (Table 3a). In each case except of TcAchE alternative bonding of the SER H  proton to second acceptors was also observed. They mostly consisted of a backbone O of GLU 229 (PLE), SER 222 (hBuChE), and GLY 123 (CRL), or a side chain carboxyl group of GLU 200 (hCE), Only in the case of hBuchE this alternative hydrogen bond was more stable than the SER-HIS contact.

In the experimental structure of hCE [START_REF] Bencharit | Crystal structure of human carboxylesterase 1 complexed with the alzheimer's drug tacrine: From binding promiscuity to selective inhibition[END_REF], GLU 334 (equivalent to position 336 in PLE) forms a weak hydrogen bond (3 Å) to HIS 447 (equivalent 449). After a very short simulation time of only 100 ps this GLU relaxes and a hydrogen bond to GLU 450 (equivalent 452) is formed, which is close to HIS 447 already in the starting configuration (Table 3c). In unrestrained simulations of TcAChE, hBuChE and CRL, histidine in the active site is stabilized by the GLU residue equivalent to 336, and the calculation confirms the assignment given in the literature [START_REF] Sussman | Atomic-structure of acetylcholinesterase from torpedo-californica -a prototypic acetylcholine-binding protein[END_REF][START_REF] Shafferman | Mutagenesis of human acetylcholinesterase -identification of residues involved in catalytic activity and in polypeptide folding[END_REF][START_REF] Bencharit | Structural insights into cpt-11 activation by mammalian carboxylesterases[END_REF][START_REF] Schrag | Ser-his-glu triad forms the catalytic site of the lipase from geotrichum-candidum[END_REF][START_REF] Manco | Residues at the active site of the esterase 2 from alicyclobacillus acidocaldarius involved in substrate specificity and catalytic activity at high temperature[END_REF][START_REF] Hosokawa | Structure and catalytic properties of carboxylesterase isozymes involved in metabolic activation of prodrugs[END_REF] for the majority of hydrolases (Table 3b). We thus obtained a stable active site with the equivalents of GLU 336 and HIS 449 bonding for at least three hydrolases from our simulation protocol, whereas in an early force field calculation over just 400 ps this structure could only be stabilized by restraints [START_REF] Monecke | Molecular modelling studies on the catalytic mechanism of candida rugosa lipase[END_REF]. This makes it unlikely that the AMBER force field parametrization artificially induces the switching from GLU 336 to GLU 452 in PLE.

Interpretation of the results on the basis of the 3DM database

According to [START_REF] Cygler | Relationship between sequence conservation and 3-dimensional structure in a large family of esterases, lipases, and related proteins[END_REF], GLU 452 is conserved and has a crucial importance. The survey with 3DM supported quantitatively the high conservation of amino acids at the core position equivalent to 452 in PLE. In the AChE family 63.2% GLU / 33.8 % ASP and in its subfamily 62.4% GLU / 35.6% ASP were found. In the hydrolases superfamily this position is no core residue indicating that it has specific importance in the AChE family, but not in all other members of the superfamily. It would be consistent with our simulations if this residue was part of the active site in most members of the AChE family, but not of the superfamily.

In contrast to that the position 336 in PLE is highly conserved in the superfamily having scores of 74.4% ASP / 19.9% GLU. As ASP is prevailing at this position, the mechanism of hydrolysis is in general described with this amino acid in the catalytic triad. Whereas GLU in this position is an exception in the superfamily, it is nearly quantitatively found in the respective positions in the acetylcholine family (91.2 % GLU / 6.7% ASP) and in its subfamily (96.0% GLU and 2.6% ASP).

Mutations affecting the orientations of GLU 336

Two differences between the sequences in the acetylcholinesterase family and in its subfamily of mammalian esterases with hCE and PLE might contribute to the flip of the GLU 336 side chain:

(1) The PLE subfamily preferably contains a LYS at position 521 instead of ARG in acetylcholine and butyrylcholine esterases. The ARG side chain forms a very stable salt bridge to ASP 404, which is a highly conserved residue in both families (98.7 % and 80% in the subfamily and the acetylcholine family, respectively). The link between residues 404 and 521 is weakened by replacing ARG with LYS, and the LYS side chain can form a hydrogen bond to the reoriented GLU 336 side chain. This was not possible in enzymes with ARG at position 521.

(2) At the positions 337 and 338 next to GLU 336, significant differences are found between family and subfamily. In the acetylcholineesterase family GLY is dominant on position 337 (65.9%), where only 4.0% of the members of the subfamily have a GLY residue, PHE being favored (63.6%). At the neighboring position 338 the situation is reversed with 11.3 % and 87.3 % of glycine in the AChE family and its subfamily, respectively. This position is not conserved in the AChE family, and several other amino acids than GLY could be located here, mainly LEU, THR and SER with frequencies of 20.1, 18.4 and 13.6%, respectively.

Shifting of GLY by one position from the neighbor of the glutamic acid in the AChE-family to the second residue after GLU 336 in PLE, might modify the flexibility of the strand close to GLU 336 and favor the reorientation of its side chain away from the active site.

Exchanging only these two amino acids, as suggested by results from the 3DM-evaluation (PHE337GLY and GLY338THR) did not have significant influence on the orientation of the GLU 336 side chain in a further simulation, and additional differences in the sequences between family and subfamily should be relevant: The residues 337-346 were replaced with two corresponding sequences from other hydrolases including the 10 amino acids following the glutamate/aspartate residue corresponding to GLU 336 in PLE. In these enzymes it is known that this residue indeed is part of the active site. The first sequence contained typical residues for choline esterases (Fig. 10, Mutation 1) and the second one was taken from CRL (Fig. 10, Mutation 2). In the homology model used as starting configuration the side chain of GLU 336 is oriented towards HIS 449. First a reorientation like in PLE1 was observed, but then the mutation of ten amino acids in this helix resulted in a breakdown of the whole helix.

Surprisingly a new orientation of the GLU 336 side chain back to the HIS 449 was observed.

Also the deformation of this counter helix induces unfolding of the entrance helix.

We assume that the sequence of these ten amino acids is crucial for the access of the substrates to the active site of PLE, determining the shape of the entrance channel together with the entrance helix (72-87). It thus seems to be possible that the evolution of the entrance channel in PLE induced a reorientation of the GLU 336 side chain away from the active site, being replaced by the GLU 452 carboxyl group.

Conclusions

Simulations for more than 10 ns are sufficient to change local conformations and thus to reveal significant differences between the enzyme and its template for homology modeling. In one case the simulation time was even sufficient for modifying the fold of a heptapeptide.

Unrestrained runs result in stable structures with well defined active sites. Reliable models are obtained for enzymes, for which no experimental structural data are available. The stability of the trimer in the simulation and the identification of contacts between its monomers are a further validation of the simulation.

Our enzyme model provides a rigid carapace made up from β-strands and a soft body of αhelices containing the mutations and determining the experimental substrate specificity.

Similar to hCE the access of the substrate to the active site passes through two -helices.

Their structure and even the folding state of one of them are specifically affected by mutations which modify the substrate specificity. This makes it likely that the entrance channel rather than the structure of the active site determines the substrate specificity of the enzyme.

In addition to that it was shown that the exchange of the GLU residue within the families of hydrolases is not essential for the reaction mechanism as long as the composition of the triad is conserved. This model of enzyme specificity sheds a new light on the mechanisms of esterase reactions and on the possibilities to tailor substrate specific enzymes. Table 1 Differences in the sequences of the PLE-isoenzymes. For PLE1 all residues are shown, which undergo mutations in any of the other isoenzymes. In the other columns, only exchanged residues are indicated,"-" means no change with respect to PLE1. Three letter code is used for the amino acids. Groups of correlated mutations are indicated (see text) The insert shows the contact between subunits 1 and 2 (view from the bottom).

Tables

Strands forming the contact are colored according to Table 3 Click here to download high resolution image

Figure 3a

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Figure 3b

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Figure 5a

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Figure 5b

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( 2 )

 2 In PLE4 and PLE6 the HIS 449 ring rotates by 180° around the C  -C  axis. Still the Hbond between SER 204 O and HIS 449 N  is stable, but the proton at N  forms an H-bond to backbone GLU 336 O. (3) The active sites of PLE3 and PLE5 differ from those of the other isoenzymes. Even though PLE3 formed stable hydrogen bonds between HIS 449 N  and GLU 452 OE1 or OE2, the N  of HIS 449 and the O  of SER 204 were too far apart for a hydrogen bond. The SER 204 is rather stabilized by the GLU 229 backbone O. PLE 5 has only one stable H-bond in the catalytic triad between the two protonated atoms HIS 449 N  to SER 204 O  , since the histidine ring is stuck in a position which makes the formation of the expected H-bond to HIS 449 N  impossible. In all isoenzymes but PLE5 and PLE6 the carboxyl group of GLU452 is fixed by additional hydrogen bonds and has a stable position. One of the carboxyl oxygen atoms, O 1 or O 2 , is hydrogen bonded to backbone N atoms of GLY 450, ASP 451 and GLU 452. The second oxygen, O 2 or O 1 , forms hydrogen bonds to ASN 333 ND2 and SER 230 O  in all runs. GLU 336 and GLU 452 are cross linked via ASN 333, and their side chain orientations are correlated. In PLE5 the respective distances are elongated to 3-4 Å, whereas in PLE6 the side chain of 452 has reoriented.

Figure captions Fig. 1 Fig. 2 Fig. 3 aFig. 4 Fig. 5 aFig. 6 Fig. 7 Fig. 8

 captions12345678 Figure captions

Fig. 9 Fig. 10

 910 Fig. 9 Time history of characteristic atom-atom distances in the active site of the PLE1 monomer during the molecular dynamics run. The black line indicates high stability of the SER204-HIS449 hydrogen bond. In competition to this interaction, the serine residue forms hydrogen bonds to the backbone oxygen atoms of the glutamic acids 203 and 229 (cf.Table 3a)

Figure 1

 1 Figure 1Click here to download high resolution image

Figure 2

 2 Figure 2 Click here to download high resolution image

  Bonding of the residue GLU 333 and its ASP/GLU analogs in selected hydrolases

	Tab 3a hydrolases Tab 3b Enzyme	Residues in the active site: Hydrogen bonding of SER 204 analogs in selected ASP/GLU Atom H-bond donor Remarks
	Pig liver esterase	336		OEX	GLU 336 N	oriented away from the active site (stable)
	Enzyme		Serin 336	Atom OEX	H-bond acceptor LYS 521 NZ (stable) Remarks
	Pig liver esterase Human Carboxyl-	204 334	OG	OEX	HIS 449 NE2 GLU 334 N	active site (mostly stable) oriented away from the active site
	Group Residue (1) 73 esterase I	PLE1 ASP 204	OG	PLE2 -	PLE3 GLU GLU 229 O	PLE4 -alternative PLE5 -	PLE6 GLU	APLE GLU
	75 76 Human Carboxyl-	VAL VAL 201 334	OG	--OEX	ILE GLY HIS 447 NE2 LYS 519 NZ	-ALA active site -ALA	ILE GLY	ILE GLY
	77 80 Torpedo californica esterase I	GLU THR 324		--OEX	GLY LEU HIS 437 ND1 part of the active site (stable) GLY GLY GLY --LEU	GLY LEU
	(2) Acetylcholinesterase 87	GLY	OG	-	ARG GLU 200 OEX	ARG alternative ARG	ARG	ARG
		92	THR		-	ILE	ILE		ILE	ILE	ILE
	93 Torpedo californica	LEU 197 324	OG	-OEX	PRO HIS 437 NE2 ASN 321 N	PRO active site (stable) PRO (stable)	PRO	PRO
		129	LEU		-	VAL	VAL	VAL	VAL	VAL
	133 Acetylcholinesterase	PRO 324		-OEX	SER SER 223 OG	SER	SER	SER	SER
		134	MET		-	THR	THR	THR	THR	THR
	Human Human	138	VAL 196 323	OG	-OEX	LEU HIS 436 NE2 HIS 436 ND1 part of the active site (stable) LEU LEU LEU active site (not stable)	LLEU
	139 234 Butyrylcholinesterase Butyrylcholinesterase	VAL LEU		--	ALA PHE	ALA PHE	ALA PHE	ALA PHE	ALA PHE
		236 237	VAL ALA 323		--OEX	ALA GLY SER 222 O SER 222 OG	ALA GLY alternative (more stable) ALA ALA GLY GLY	ALA GLY
	(3) Candida rugosa lipase 209 285 PHE 323	OG	-OEX	PRO HIS 449 NE2 ASN 320 ND	PRO active site (mostly stable) --	-
	(4)	286	LEU		-	-	-		PHE	PHE	PHE
		287	THR 209 323	OG	-OEX	-GLY 123 O ASN 320 N	-	ALA alternative	ALA	ALA
	(5)	290	PHE		-	LEU	LEU	LEU	LEU	LEU
	294 Candida rugosa lipase 341 GLN		-OEX	PRO HIS 449 ND1 part of the active site (stable) PRO PRO PRO	PRO
		302	PRO		-	THR	THR	THR	THR	THR
	(6)	459	PHE 341		ALA OEX	-SER 241 OG	ALA	ALA	ALA	-
		461	LEU		PHE	-	PHE	PHE	PHE	-
		463	LYS 341		ARG OEX	-GLN 338 N	ARG	ARG	ARG	-

Table 3c

 3c Bonding of the residue GLU 449 and its ASP/GLU analogs in selected hydrolases

	Enzyme	ASP/GLU Atom	H-bond donor
	Pig liver esterase	452	OEX	HIS 449 ND1
		452	OEX	SER 230 OG
		452	OEX	GLY 450 N
		452	OEX	ASP 451 N
		452	OEX	GLU 452 N
	Human Carboxyl-	450	OEX	HIS 447 ND1
	esterase I
		450	OEX	ASN 331 ND2
		450	OEX	GLY 448 N
		450	OEX	ASP 449 N
	Torpedo californica	440	OEX	GLN 222 NE2
	Acetylcholinesterase
		440	OEX	ASN 321 ND2
		440	OEX	TYR 418 OH
	Human	439	OEX	ASN 320 ND2
	Butyrylcholinesterase
		439	OEX	GLN 221 NE2
	Candida rugosa lipase 452	ODX	GLN 338 OE1
		452	ODX	SER 436 OG
		452	ODX	GLN 438 NE2
		452	ODX	TYR 467 OH

Acknowledgments

We gratefully acknowledge support of Markus Gall (Institute of Biochemistry, Greifswald) for an introduction into the 3DM database, and fruitful discussions with Dr. Elke Brüsehaber, Anke Hummel and Dr. Robert Kourist (Institute of Biochemistry, Greifswald).

Table 2a

Residues linking the monomer subunits 1-3 to its neighbor in clockwise sense.

Connections are found between 1 and 5' (cf. Table 2b) (cyanred in Fig. 8), 2 and 6' (blueorange), the first 2 amino acids of 2 and 7' (blueorange), the remaining amino acids of 2 and 4' (blueyellow) and between 3 and 5' (icebluered). Only the SER 45 (magenta) and GLU 270 residues (cyan) of each of the three monomers in the center of the structure form contacts to the two other subunits both in clockwise and counterclockwise sense. Colors refer to Fig. 8.

Amino acid

Number contacts su1 to su2 contacts su2 to su3

Tab 2b

Residues linking the monomer subunits 1-3 to its neighbor in counterclockwise sense Amino acid Number contacts su1 to su3 contacts su3 to su2 contacts su2 to su1 strand 4' (yellow