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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: enzyme catalysis/epoxyl-inhibitors/molecular dynamics/xylanase
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
All the MD simulations were done with the AMBER (version 4.1) program (Pearlman et al., 1995) and using the force field described in Cornell et al. (1995) and the saccharide parameters of the GLYCAM_93 set (Woods et al., 1995
). The atomic charges for the inhibitor molecules were calculated with the RESP method (Bayly et al., 1993
; Cornell et al., 1993
) 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., 1995
; Fox and Kollman, 1998
). 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., 1995
). The XYNIIinhibitor complexes were solvated with a TIP3P (Jorgensen et al., 1983
) 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., 1977
) 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 waterenzymesubstrate 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 enzymesubstrate 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 enzymesubstrate complexes (Lightstone and Bruice, 1994, 1996
, 1997
; Torres and Buice, 1998; Lau and Bruice, 1998
). 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 3). 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., 1998
). 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., 1993
) and in the acid- and nucleophile-catalyzed opening reaction of the oxirane ring (Lightstone and Bruice, 1994
, 1996
, 1997
). The reactive conformation had to meet three criteria (Figure 4
). (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.
|
|
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1996). The xylose ring makes several hydrogen bonds with the active site residues and, therefore, is firmly bound at the active site (Wakarchuk et al., 1994
; Havukainen et al., 1996
). 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 4
).
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 I) 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 II
) 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 5
) 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 I
). In the case of the (2S)-isomer a number of 0/409/2078 reactive conformations were observed (Table II
). The reactive conformations were mainly detected between 160 and 230 ps of the simulation when the reactive angle had small values (Figure 5
) and the distance parameters were in a reactive range (Figure 6
). 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 6
). 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.
|
|
|
|
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 7 and the variation in the reactive distances in Figure 8
. 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 1
. 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 II
), 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.
|
|
It has been well demonstrated (Høj et al., 1991) 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, 1995
). These specific enzymesubstrate 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 |
---|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cornell,W.D., Cieplak,P., Bayly,C.I., Gould,I.R., Merz,K.M., Ferguson,D.M., Spellmeyer,D.C., Fox,T., Caldwell,J.W. and Kollman,P.A. (1995) J. Am. Chem. Soc., 117, 51795197.[ISI]
Cornell,W.D., Cieplak,P., Bayly,C.I., Kollman,P.A. (1993) J. Am. Chem. Soc., 115, 96209631.[ISI]
Fox,T., Kollman,P.A. (1998) J. Phys. Chem., 102, 80708079.[ISI]
Frisch,M.J. et al. (1995) Gaussian 94. Gaussian, Pittsburgh.
Havukainen,R., Törrönen,A., Laitinen,T. and Rouvinen,J. (1996) Biochemistry, 35, 96179624.[ISI][Medline]
Henrissat,B. and Bairoch,A. (1993) Biochem. J., 293, 853859.
Høj,P.B., Rodrigues,E.B., Iser,J.R., Stick,R.V. and Stone,B.A. (1991) J. Biol. Chem., 266, 1162811631.
Jorgensen,W.L., Chandrasekhar,J., Madura,J., Impley,R.W. and Klein,M.L. (1983) J. Chem. Phys., 79, 926933.[ISI]
Laitinen,T., Rouvinen,J. and Peräkylä,M. (1998) J. Org. Chem., 63, 81578162.[ISI]
Lau,E.Y. and Bruice,T.C. (1998) J. Am. Chem. Soc., 120, 1238712394.[ISI]
Legler,G. and Bause,E. (1973) Carbohydr. Res., 28, 4552.[ISI][Medline]
Lightstone,F.C. and Bruice,T.C. (1994) J. Am. Chem. Soc., 116, 1078910790.[ISI]
Lightstone,F.C. and Bruice,T.C. (1996) J. Am. Chem. Soc., 118, 25952605.[ISI]
Lightstone,F.C. and Bruice,T.C. (1997) J. Am. Chem. Soc., 119, 91039113.[ISI]
Liotta,L.J., Lee,J. and Ganem,B. (1991) Tetrahedron, 47, 24332447.[ISI]
McIntosh,L.P., Hand,G., Johnson,P.E., Joshi,M.D., Kàrner,M., Plesniak,L.A., Ziser,L., Wakarchuk,W.W., Withers,S.G. (1996) Biochemistry, 35, 99589966.[ISI][Medline]
Muilu,J., Törrönen,A., Peräkylä,M., Rouvinen,J., (1998) Proteins, 31, 434444.[Medline]
Na,J., Houk,K.N., Shevlin,C.G., Janda,K.N., Lerner,R.A. (1993) J. Am. Chem. Soc., 115, 84538454.[ISI]
Pearlman,D.A., Case,D.A., Caldwell,J.W., Ross,W.S., Cheatham III,T.E., Ferguson,D.M., Seibel,G.L., Singh,U.C., Weiner,P.K. and Kollman,P.A. (1995) AMBER 4.1. University of California, San Francisco.
Rodriques,E.B. and Stick,R.V. (1990) Aust. J. Chem., 43, 665679.[ISI]
Rodrigues,E.B., Scally,G.D. and Stick,R.V. (1990) Aust. J. Chem., 43, 13911405.[ISI]
Ryckaert,J.P., Ciccotti,G. and Berendsen,H.J.C. (1977) J. Comput. Phys., 23, 327341.[ISI]
Schafmeister,C.E.A.F., Ross,W.S. and Romanovski,V. (1995) LeaP. University of California, San Francisco.
Torres,R.A. and Bruice,T.C. (1998) Proc. Natl Acad. Sci. USA, 95, 1107711082.
Törrönen,A. and Rouvinen,J. (1995) Biochemistry, 34, 847856.[ISI][Medline]
Wakarchuk,W.W., Campbell,R.L., Sung,W.L., Davoodi,J. and Yaguchi,M. (1994) Protein Sci., 3, 467475.
Withers,S.G. and Aebersold,R. (1995) Protein Sci., 4, 361372.
Woods,R.J., Dvek,R.A., Edge,C.J. and Fraser-Reid,B. (1995) J. Phys. Chem., 99, 38223846.
Yu,Z., Caldera,P., McPhee,F., De Voss,J.J., Jones,P.R., Burlingame,A.L., Kuntz,I.D., Craik,C.S. and de Montellano,P.R.O. (1996) J. Am. Chem. Soc., 118, 58465956.[ISI]
Received November 4, 1999; revised January 18, 2000; accepted February 8, 2000.