Inversion of the roles of the nucleophile and acid/base catalysts in the covalent binding of epoxyalkyl xyloside inhibitor to the catalytic glutamates of endo-1,4-ß-xylanase (XYNII): a molecular dynamics study

Tuomo Laitinen2, Juha Rouvinen and Mikael Peräkylä1

Department of Chemistry, University of Joensuu, PO Box 111, Joensuu, FIN-80101 and 1 Department of Chemistry, University of Kuopio, PO Box 1627, Kuopio, FIN-70211, Finland


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
X-Ray crystal structures have revealed that 2,3-epoxypropyl-ß-D-xyloside reacts with endo-1,4-ß-xylanase (XYNII) by forming a covalent bond with Glu86. In contrast, 3,4-epoxybutyl-ß-D-xyloside forms a covalent bond with Glu177. In the normal enzyme reaction Glu86 acts as the catalytic nucleophile and Glu177 as the acid/base catalyst. To rationalize the observed reactivity of the two mechanism-based inhibitors, we carried out eight 300 ps molecular dynamics simulations for different enzyme–inhibitor complexes. Simulations were done for both stereo isomers (R and S) of the inhibitors and for enzyme in which the protonation state of the nucleophile and acid/base catalyst was normal (Glu86 charged, Glu177 neutral) and in which the roles of the catalytic residues were reversed (Glu86 neutral, Glu177 charged). The number of reactive conformations found in each simulation was used to predict the reactivity of epoxy inhibitors. The conformation was considered to be a reactive one when at the same time (i) the proton of the catalytic acid was close (<2.9/3.4/3.9 Å) to the oxirane oxygen of the inhibitor, (ii) the nucleophile was close to the terminal carbon of the oxirane group (<3.4/3.9/4.4 Å) and (iii) the nucleophile approached the terminal carbon from a reactive angle (<30/45/60° from an ideal attack angle). On the basis of the number of reactive conformations, 2,3-epoxypropyl-ß-D-xyloside was predicted to form a covalent bond with Glu86 and 3,4-epoxybutyl-ß-D-xyloside with Glu177, both in agreement with the experiment. Thus, the MD simulations and the X-ray structures indicate that in the covalent binding of 3,4-epoxybutyl-ß-D-xyloside the roles of the catalytic glutamates of XYNII are reversed from that of the normal enzyme reaction.

Keywords: enzyme catalysis/epoxyl-inhibitors/molecular dynamics/xylanase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Endo-1,4-ß-xylanase II (XYNII) from Trichoderma reesei is an enzyme that hydrolyzes the ß-1,4-glycosidic linkage between two xylose residues by a mechanism in which the configuration at the anomeric carbon of the substrate is retained. According to the classification of Henrissat and Bairoch (1993), XYNII belongs to the family of G/11 glycosidases, which contains only endo-1,4-xylanases. G/11 xylanases are small protein molecules (MW ~20 000). The basic fold of these xylanases contains one {alpha}-helix and two ß-sheets packed against each other, forming a so-called ß-sandwich which is capable of binding to at least three xylose residues. The structure resembles a right hand, the ß-sheets forming the palm and the fingers, and one long loop forming the thumb, which partially closes the cleft. The three-dimensional structures of the family of G/11 xylanases have revealed two conserved glutamic acid residues on different sides of the cleft (Figure 1Go). In the enzymatic reaction the active site Glu177 is assumed to act as the acid/base catalyst and Glu86 as the nucleophile, which stabilizes the carbonium ion intermediate formed (McIntosh et al., 1996Go).



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Fig. 1. A snapshot structure of the active site of XYNII when the protonation state of the XYNII was inverted and X-O-C4 (3R) was used.

 
Epoxyalkyl oligosaccharides have been widely used to label the nucleophilic residue of the retaining glycosidases (Withers and Aebersold, 1995Go). These compounds are active site-directed inhibitors, which consist of a reactive epoxide group linked by an alkyl chain to the saccharide unit (Figure 2Go) (Legler and Bause, 1973Go; Rodrigues and Stick, 1990; Rodrigues et al., 1990Go; Liotta et al., 1991Go; Yu et al., 1996Go). The rate of inactivation is dependent on the structural properties of inhibitor molecules, such as the number of saccharide units, the length of the aglycon chain and the stereochemistry of the epoxyl group (Rodrigues et al., 1990Go; Høj et al., 1991Go). The X-ray crystal structures of three covalently bound epoxyalkyl xyloside inhibitor–XYNII complexes have recently been determined by our research group (Havukainen et al., 1996Go). The X-ray structures showed that two of the inhibitors, 2,3-epoxypropyl-ß-D-xyloside (X-O-C3) and 4,5-epoxypentyl-ß-D-xyloside, form a covalent bond with the nucleophile Glu86. This mode of binding is in agreement with the expectation that the epoxyalkyl compounds are mechanism-based inhibitors and become covalently linked to the catalytic nucleophile. In contrast, the third inhibitor, 3,4-epoxybutyl-ß-D-xyloside (X-O-C4), was found to form a covalent bond with the acid/base catalyst Glu177.



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Fig. 2. Chemical structure of XYNII ligands X-O-C3 and X-O-C4.

 
In this work, we applied MD simulations to study the interactions between XYNII and active site-directed epoxyalkyl inhibitors and how the interactions are affected by changes in the length of the alkyl chain linkages and the chirality of the inhibitors. The specific aim of this work was to examine whether MD calculations can explain the binding of 3,4-epoxybutyl-ß-D-xyloside to the acid/base catalyst of XYNII which was observed in the X-ray study. MD simulations were done for both chiral forms (2R and 2S) of 2,3-epoxypropyl-ß-D-xyloside and (3R and 3S) 3,4-epoxybutyl-ß-D-xyloside. In addition, simulations were done for XYNII in which the roles of the catalytic residues were normal (Glu86 charged, Glu177 neutral) and inverse (Glu86 neutral, Glu177 charged). Our simulations showed how stereoisomers of the two epoxyalkyl inhibitors differ in their active site interactions and we were able to provide an explanation for the unexpected binding of 3,4-epoxybutyl-ß-D-xyloside.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Simulations

All the MD simulations were done with the AMBER (version 4.1) program (Pearlman et al., 1995Go) and using the force field described in Cornell et al. (1995) and the saccharide parameters of the GLYCAM_93 set (Woods et al., 1995Go). The atomic charges for the inhibitor molecules were calculated with the RESP method (Bayly et al., 1993Go; Cornell et al., 1993Go) at the HF/6-31G* level (Frish et al., 1995). Quantum mechanical (QM) calculations (Frish et al., 1995) at the MP2/6-31G*//HF/6-31G* level were used to derive the missing torsion parameters for the oxirane parts of the inhibitor molecules (Cornell et al., 1995Go; Fox and Kollman, 1998Go). The atomic point charges and parameters of the oxirane parts of the inhibitors are available from the authors. The initial coordinates of XYNII were obtained from the X-ray crystal structure of endo-1,4-ß-xylanase (XYNII) from Trichoderma reesei complexed with 2,3-epoxypropyl-ß-D-xyloside (X-O-C3) determined at 1.8 Å resolution. The inhibitors studied in this work were placed at the active site using a 2,3-epoxypropyl-ß-D-xyloside of the crystal structure as a template. The orientation of the epoxy group was manually built in a reactive conformation (see below for a definition of the reactive conformation) for each of the inhibitors using the LEAP program (Schafmeister et al., 1995Go). The XYNII–inhibitor complexes were solvated with a TIP3P (Jorgensen et al., 1983Go) water cap of 20.0 Å centered on the carboxylic group of Glu86. Residues and waters inside the sphere of 20 Å from the carboxylic group of Glu86 were allowed to move in the MD simulations. We used a time step of 1.5 fs and the SHAKE algorithm (Ryckaert et al., 1977Go) to constrain bonds to hydrogen atoms at their equilibrium values. The system was heated to a simulation temperature of 300 K during 15 ps of simulation, the water–enzyme–substrate complex was equilibrated an additional 45 ps and the final data were collected between 60 and 360 ps of the simulation. The trajectory files were collected in steps of 15 fs and from each simulation a number of 20 000 snapshot structures were included in the analysis of geometric parameters. MD simulations were carried out for (2R)- and (2S)-2,3-epoxypropyl-ß-D-xyloside and (3R)- and (3S)-3,4-epoxybutyl-ß-D-xyloside. For all four molecules we carried out MD simulations in which the catalytic glutamates were in normal (Glu86 charged, Glu177 neutral) and in inverse-protonated-states (Glu86 neutral, Glu177 charged). This resulted in a total of eight MD simulations.

Reactive conformations

In enzyme–substrate complexes the reactive groups are typically oriented in such a way that the formation of a transition state from a ground state conformation requires only a few structural changes. Such a disposition of reactive functional groups explains a large part of the rate of enhancement in enzyme-catalyzed reactions compared with the corresponding solution reactions. Recently, such arguments have been used to explain reactivities in intramolecular reactions and to analyze MD simulations of enzyme–substrate complexes (Lightstone and Bruice, 1994Go, 1996Go, 1997Go; Torres and Buice, 1998; Lau and Bruice, 1998Go). In this work, the number of reactive ground state conformations in each MD simulation was used to estimate the reactivity of different epoxyalkyl inhibitors. Here we have used the principle that if other things are equal, the system which has the largest number of reactive ground state conformations has the largest reaction rate. More accurate modeling of the covalent binding would require QM treatment of the transition states of the reactions and the inclusion of protein and water environment in the QM calculations. Such calculations were not thought to be necessary for the purpose of the present study.

The inhibitory mechanism of epoxyalkyl inhibitors is assumed to follow the enzymatic mechanism of retaining glycosidases (Figure 3Go). Our recent ab initio QM calculations indicated that a suitable geometric arrangement of both the nucleophilic and the acid/base catalyst is needed for efficient binding of the epoxy inhibitors (Laitinen et al., 1998Go). The criteria for the definition of the reactive conformations used here is based on the most favorable reactant distances and attack angles of the nucleophiles in the epoxide ring opening reactions (Na et al., 1993Go) and in the acid- and nucleophile-catalyzed opening reaction of the oxirane ring (Lightstone and Bruice, 1994Go, 1996Go, 1997Go). The reactive conformation had to meet three criteria (Figure 4Go). (i) The distance of the approach of the acidic proton to the oxirane oxygen had to be <2.9/3.4/3.9 Å, (ii) the distance of the approach of nucleophilic oxygen to the reactive terminal carbon of oxirane had to be <3.4/3.9/4.4 Å and (iii) the approach of the nucleophilic glutamate to the oxirane carbon had to be within 30/45/60° from the normal to the plane of the terminal carbon and its hydrogens. The tightest set of distance criteria (2.9 Å for the acid and 3.4 Å for the nucleophile distance) for the reactive conformation is based on distances which are the sum of the van der Waal's radii of interacting atoms plus 0.2 Å. The enlarged sets of the reactive conformation limits are used because the tightest set of limits was found to result in only a few reactive conformations and no difference between the isomers was observed.



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Fig. 3. Proposed reaction scheme for inhibitory mechanism.

 


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Fig. 4. Definition of reaction parameters.

 
Both experimental (Havukainen et al., 1996Go) and molecular dynamic studies (Muilu et al., 1998Go) suggest that the closed-conformation is dominant when substrate or inhibitor is introduced to the active site of the XYNII. It is also most likely that the closed conformation is the reactive conformation for the enzyme (Muilu et al., 1998Go). Therefore, the X-ray structure in which the protein is in the closed conformation was chosen as a starting structure for the MD simulations of the active site region.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
2,3-Epoxypropyl-ß-D-XYLOSIDE

The crystal structure shows that X-O-C3 is covalently bound to the nucleophilic glutamate (Glu86), the hydroxyl group is coordinated with an acid/base catalyst (Glu177) which has donated a proton to the former epoxide oxygen, and the saccharide unit is docked above the tryptophane ring (Trp18) (Havukainen et al., 1996Go). The xylose ring makes several hydrogen bonds with the active site residues and, therefore, is firmly bound at the active site (Wakarchuk et al., 1994Go; Havukainen et al., 1996Go). During all the simulations of 2,3-epoxypropyl-ß-D-xyloside and also 3,4-epoxybutyl-ß-D-xyloside the xylose unit stays above Trp18 while a flexible aglycon unit is able to adopt different rotational conformations. The most stable non-covalent complex structures were formed when the oxirane oxygen pointed to the hydrogen of the neutral glutamate. In the reactive conformations the epoxide end of the inhibitor molecule is positioned between the catalytic residues so that the oxirane oxygen is coordinated with the proton of the neutral glutamate and the reactive terminal carbon of the epoxyl group with the charged glutamate (Figure 4Go).

On the basis of the number of reactive conformations, 2,3-epoxypropyl-ß-D-xyloside was predicted to form a covalent bond with Glu86, which is in agreement with the experimental results. The (2R)-isomer of the X-O-C3 has a small average value of 55° (Table IGo) for the attack angle, and it has suitable distance parameters to the nucleophile Glu86 (average 3.82 Å) and to the acid/base catalyst (average 4.31 Å). In addition, a number of 0/209/4056 reactive conformations (Table IIGo) were found during 300 ps of simulation with three sets of limit values. The reactive conformations mainly occurred during the period before 200 ps. In the case of the (2S)-isomer, a large variation (Figure 5Go) in the attack angle was observed, and the average value was calculated to be 92°. The average distances between the catalytic residues and the oxirane group are 4.00 Å for the nucleophile and 4.84 Å for the acid/base catalyst (Table IGo). In the case of the (2S)-isomer a number of 0/409/2078 reactive conformations were observed (Table IIGo). The reactive conformations were mainly detected between 160 and 230 ps of the simulation when the reactive angle had small values (Figure 5Go) and the distance parameters were in a reactive range (Figure 6Go). After 230 ps the protonated Glu177 turned into a conformation in which the distance to the proton donor stayed between 7 and 8 Å (Figure 6Go). The main difference between these two isomers was that while the (2R)-isomer stayed for a longer period close to the reactive conformations, the (2S)-isomer meets those conformations during a short period of the simulation, after which the acid/base catalyst flips to a less reactive conformation.


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Table I. Average values for the nucleophilic and acid/base distances (Å) and for the attack angle (°)
 

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Table II. The number of reactive structures
 


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Fig. 5. The changes in the reactive angle (definition in the Figure 3Go) between the terminal carbon of X-O-C3 (2S)-inhibitor and nucleophilic residue (Glu86), Glu177 is protonated.

 


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Fig. 6. The reactive distances (Figure 3Go) between X-O-C3 (2S)-inhibitor and the catalytic residues of XYNII.

 
In the simulations of the inverted protonation state (Glu86 neutral, Glu177 charged), the (2R)-isomer of X-O-C3 was observed to have a large variation in the values of the reactive angle (from 20° to almost 180°, with an average of 99°). The acid/base distance had short average values of 2.68 Å, but this inhibitor stayed further away from the nucleophile (an average value of 5.80 Å). The number of reactive conformations was 0/27/492. With the (2S)-isomer reactive conformations were found 0/0/13. In this case the average distance values were fairly large, 5.3 Å for acid/base and 4.18 for nucleophilic distance. The reactive angle stayed close to its average value (81°) throughout the simulation. Results from the MD simulations led us to conclude that the normal binding was more favorable for 2,3-epoxypropyl-ß-D-xyloside. In addition, the simulations predicted that (2S)- and (2R)-isomers are almost equally reactive. It must be noted that, although the relatively short MD simulations are thought to be able to differentiate between the reactivity of the different protonation states, they are not expected to reproduce the subtle differences (both have a large number of reactive conformations) that exist between the (2S)- and (2R)-isomers of 2,3-epoxypropyl-ß-D-xyloside.

3,4-Epoxybutyl-ß-D-XYLOSIDE

The crystal structure revealed that the inhibitor X-O-C4 binds to the Glu177 of XYNII. This was an unexpected observation, because Glu177 acts as an acid/base catalyst in an enzyme reaction. Four different MD simulations were carried out for this inhibitor: both the (3S)- and the (3R)-isomer of the inhibitor were simulated with the enzyme in which either Glu86 or Glu177 was set as an acid/base catalyst (neutral). In the simulation of XYNII in the normal protonation state (Glu87 charged, Glu177 neutral), the (3R)-isomer of the X-O-C4 had an average reaction angle of 88°, an average acid/base distance of 3.03 Å, and an average nucleophilic distance of 5.01 Å. With both tighter limits there were zero reactive conformations, but one such structure was observed with the largest set of the limit values. A closer look at the snapshot structures showed that the oxirane's reactive terminal carbon pointed away from the nucleophilic Glu86 most of the time. In the case of the (3S)-isomer of X-O-C4, the average acid/base distance between the Glu177 and oxirane oxygen was 3.96 Å and the average nucleophilic distance between Glu86 and the reactive carbon of the oxirane ring was 5.17 Å. The reactive angle was measured to give an average value of 62°. Although the angle values were close to the reactive ones, the number of reactive conformation was zero with both tighter limits due to the long oxirane-to-acid and oxirane-to-nucleophile distances. A total of 175 reactive conformations were found with the largest set of the limit values for the X-O-C4 when the catalytic residues of XYNII were in the normal protonation state. In the simulations of the normal protonation state the X-O-C4 clearly had fewer reactive conformations than in the corresponding simulations of X-O-C3.

In the simulation of the (3R)-isomer in the inverted protonation state (Glu87 neutral, Glu177 charged), the average attack angle was 68° and the average distance values were 3.63 Å for acid/base catalyst and 5.00 Å for nucleophile. In the simulation there were 3/242/1837 reactive conformations. These numbers are close to those found for the X-O-C3 inhibitors in the normal protonation state. The variation in the attack angle in the simulation of X-O-C4 in the inverted protonation state is presented in Figure 7Go and the variation in the reactive distances in Figure 8Go. It can be seen from the figures that a large number of reactive conformations existed during the first 100 ps of simulation, after which the system stayed in slightly less reactive conformations. A snapshot structure of such a conformation is presented in Figure 1Go. The (3S)-isomer of the X-O-C4 inhibitor had a less suitable geometry with respect to the acid/base catalyst Glu86 (an average distance of 4.72 Å) and the nucleophile Glu177 (an average attack distance 6.20 Å). In addition, the attack angle had a large average value of 133°. Consequently, the number of reactive conformations is zero with all sets of limit values. Based on the number of reactive conformations (Table IIGo), the (3R)-3,4-epoxybutyl-ß-D-xyloside inhibitor was predicted to react with Glu177, the catalytic residue acting as an acid/base catalyst in the normal catalytic reaction. This prediction is in agreement with the experiment. Thus, in the covalent binding of 3,4-epoxybutyl-ß-D-xyloside the roles of the catalytic residues of XYNII have reversed from those of the normal enzyme reaction.



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Fig. 7. The changes in the reactive angle (Figure 3Go) between the terminal carbon of the X-O-C4 (3R)-inhibitor and nucleophilic residue (Glu177), Glu86 is protonated (the inverted protonation state).

 


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Fig. 8. The reactive distances (Figure 3Go) between X-O-C4 (3R)-inhibitor and catalytic residues of XYNII (the inverted protonation state).

 
Epoxyalkyl inhibitors and the catalytic mechanism

It has been well demonstrated (Høj et al., 1991Go) that both the stereochemistry and the chain length of aglycon in the mechanism-based epoxide-bearing inhibitors have a significant effect on their activity. The MD simulations of the inhibitors showed that the saccharide unit is firmly bound above the active-site tryptophan (-2 binding site of XYNII) and, therefore, the reactivity differences between the molecules studied are caused by the differences in the structure of the aglycon chain of the molecules. In the case of the epoxyalkyl inhibitors the flexible aglycon chain is able to adopt different conformations. The populations of the conformations and location of the reactive epoxy group relative to the catalytic glutamates in these conformations determine the reactivity and specificity of the inhibitors. The MD simulations indicated that (3R)-3,4-epoxybutyl-ß-D-xyloside adopts conformations, in which the reactive epoxy group is suitably positioned to react only with Glu177 and which seem to cause the catalytic amino acids to change their functional roles compared with the normal enzyme reaction. In contrast to epoxyalkyl inhibitors, there are probably five binding sites for the saccharide units of the natural substrates of XYNII responsible for substrate recognition (Törrönen and Rouvinen, 1995Go). These specific enzyme–substrate interactions position the catalytic groups and the reactive glycosidic bond suitable for an efficient enzyme reaction.

By changing the immediate surroundings of the glutamates the enzyme is able to fine tune the pKa values of the carboxylic group and to achieve a situation where one glutamate is neutral and one is charged. The differences in the pKa's are still so small that in suitable circumstances the glutamates may exist in a reversed protonation state and be able to catalyze a different reaction. The finely tuned active-site interactions and hydrogen bond network may be disturbed when a polar and flexible epoxide-bearing inhibitor molecule is introduced into the active site. It is possible that binding of 3,4-epoxybutyl-ß-D-xyloside to XYNII increases the population of the reverse-protonated state of the catalytic glutamates and, consequently, the roles of the catalytic residues are reversed compared with the normal enzyme reactions.


    Notes
 
2 To whom correspondence should be addressed Email: tuomo.laitinen{at}joensuu.fi Back


    Acknowledgments
 
This work was supported by the Academy of Finland and the Finnish Graduate School `Protein Structure and Function' (to T.L.).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received November 4, 1999; revised January 18, 2000; accepted February 8, 2000.





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