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Faraday Discuss., 1992, 93, 67-73
Structure and Mechanism of D-Xylose Isomerase David M. Blow, Charles A. Collyer,? Jonathan D. Goldberg and Oliver S. Smart$ Blackett Laboratory, Imperial College of Science Technology and Medicine, London S W7 2BZ, UK
The action of xylose isomerase depends on the presence of two divalent cations. Crystal structure analyses of the free enzyme, and of the enzyme bound to a variety of substrates and inhibitors, have provided models for a number of distinct intermediates along the reaction pathway. These models, in turn, have suggested detailed mechanisms for the various chemical steps of the reaction: a ring opening catalysed by an activated histidine, a hydrideshift isomerization,and a ring closure which may be facilitated by a polarised water molecule.
Xylose isomerase catalyses the isomerisation of the aldose, xylose, to the ketose xylulose. Many xylose isomerases, especially from Actinomycetes, are also efficient catalysts for isomerisation of the hexoses, glucose and fructose, and are very stable enzymes even at 50 "C. This gives them industrial importance in the processing of corn syrup for use in soft drinks. They also have potential application in production of ethanol from waste cellulose sources. Crystallographic analysis of this enzyme, complexed with a variety of substrates and substrate analogues, has provided a detailed hypothesis for the reaction mechanism of this cation-dependent enzyme. The molecular structure of xylose isomerase is based on the eight-stranded a / P barrel structure,1y2first observed in triose-phosphate i s ~ m e r a s eand , ~ subsequently found in many other enzymes. In each case the substrate binds in the centre of the cu/p barrel at its carboxy-terminal end, but there is little further similarity between the two enzymes. While triose phosphate isomerase is a simple monomer, xylose isomerase is a tetramer, in which pairs of barrels are roughly coaxial, with their two substrate-binding sites in proximity, and each peptide chain has a long carboxy-terminal extension which enfolds the other pair of barrels which make up the tetramer. Unlike triose phosphate isomerase, xylose isomerase is a metalloenzyme in which two cation-binding sites exist on each s ~ b u n i t .Site ~ 1 is a tight-binding site while site 2 binds its cation more loosely and, as will be shown, more flexibly. Each cation is coordinated octahedrally with ligands to four carboxylate groups and two substrate hydroxyls in site 1, and four carboxylates, a histidine and a water molecule in site 2. One carboxylate, Glu-216,$ coordinates both cation sites. The presence of two basic amino acids in the active site provides an equal number of positive and negative charges in the vicinity. These two cation sites lie to one side of a large cavity at the carboxy-terminal end of the eight 6-strands of the a l p barrel. The two cavities of the pair of barrels which face each other are adjacent, and share a common access route to bulk solvent. The side of the cavity facing the cation sites is hydrophobic, composed of several aromatic amino acid side chains, including
t Present address:
Farmitalia, Carlo Erba, via dei Gracchi, Milano, Italy.
$ Present address: Dept of Crystallography, Birkbeck College, Malet St., London WClE 7HX, UK.
Ei Amino acid numering is for xylose isomerase of Arthrobacter B3278, throughout. 67
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Structure and Mechanism of Xylose Isomerase
68
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Table 1 Pyranose-substrates and substrate analogues used as ligands
2
I
xylose glucose 5-thioglucose 1-deoxynojirimycin
X
Y
Z
OH OH OH H
0 0 S NH
H CH20H CH20H CHZOH
1
H
OH
Fig. 1 Electron density for 5-thioglucose bound to the active site. All electron-density maps are displayed as Fderiv - Fcalcmaps, where FcaIc is the refined structure in the absence of the ligand
one (Phe-25) from an adjacent subunit, but near to this hydrophobic surface is an activated histidine (His-53) which is polarised by a buried aspartate (Asp-56). X-Ray analysis of crystalline complexes of the xylose isomerase of Arthrobacter B3278 with a variety of ligands (Table l), and using various cations, has allowed us to create realistic models which represent a number of steps in the isomerisation of an aldose to a ketose.’ Binding 5-thioglucose to enzyme crystals provided the first model of a complex of the enzyme with a closed-ring aldose (Fig. 1). The electron density in the presence of 1-deoxynojirimycin indicated a similar ring orientation. In these structures cation 1 coordinates to O(3) and O(4) of the pyranose ring while cation 2 makes no direct interaction with the sugar. In the 5-thioglucose and 1-deoxynojirimycin complexes, His-53, which is polarized by the side chain of Asp-56, provides a correctly oriented nucleophile to catalyse ring opening by transfer of a proton from O( 1) to O(5) (Fig. 2). When xylose bound to enzyme crystals, the electron density plainly showed the sugar in an open-chain conformation’ (Fig. 3). Similar electron density distributions were observed on binding the five- and six-carbon polyols, xylitol and sorbitol, to the crystal^.^ These electron-density distributions were interpreted as showing cation 1 bound to O(2)
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D.M. Blow et al.
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Fig. 2 Scheme for ring-opening catalysed by His-53'
Fig. 3 Electron density for xylose bound at the active site
and O(4) of the open-chain sugar. The quality of crystals of the Arthrobacter enzyme limited the attainable resolution to 2.3 A at least (often in practice, to 2.5 A), and at this resolution details of the conformation could not be assigned. However, Whitlow et aL,6 in a similar experiment using crystals of the enzyme from Streptomyces rubiginosus, complexed with an equilibrium mixture of xylose-xylulose, were able to obtain diffraction data to 1.6 A resolution, and noted a planar conformation at the site we assumed to be occupied by C(2), indicating the open-chain ligand to be predominantly in the ketose form. In the above experiments the cation at site 2 still had no direct ligands to the substrate. However, when xylose was incubated with enzyme crystals in the presence of A13+,a somewhat different structure was observed' (Fig. 4). Under the conditions of the experiment, there was evidence that the site 1 cation is A13', while the loosely bound cation at site 2 was Mg2+. Small structural changes of the enzyme around site 2 were evident, including a significant rearrangement of Asp-254 and smaller movements of Glu-216 and His-219, which accompanied a 1 A movement of the second cation to site 2', where it coordinates O( 1) and O(2) of the substrate. This A13' complex is considered to be a transition-state analogue in which the cation at 2' is equidistant from 0(1) and O(2) and 2', 0(1), C(1), C(2) and O(2) are all approximately coplanar. Cation 1 also coordinates 0(2), while N H l ( Lys-182) coordinates O( l), strongly polarising both C-0 bonds towards C+-O-. One hydroxyl proton [say OH(2)] is assumed to be lost (possibly to a water molecule), creating conditions where the hydrogen H(2) can readily transfer
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70
Structure and Mechanism of Xylose Isomerase
Fig. 4 Electron density for xylose incubated with the enzyme in the presence of A13+ and Mg2’
1)2+
It ,.o +dH
LyS-182-N, A H H
H ‘
M(2I2+
Fig. 5 Scheme for isomerisation by hydride shift5
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Fig. 6 Electron density for 2,5-dideoxy-2,5-imino-~-glucitol (DDIG) bound to the active site
HO
H
HO
H a-0-xylulduranose
HOCH2H
CH2OH
HOCHp
CH20H
‘~
DDIG
~~H
aFF
H4
HO
H
HO
H
HOCH2
OH
‘ HO H W H zCH-Enz O H
3-deoxy-3-fluoromethylene -&glucose
Fig. 7 Schematic formulae for sugar analogues. The dashed line indicates the pseudosymmetry is the form considered of DDIM. The structure labelled ‘3-deoxy-3-fluoromethylene-~-glucose’ to be bound to the enzyme12
as a hydride ion between the carbonium-like C(2) and C(1). This makes a plausible model for the isomerization by a hydride-shift mechanism (Fig. 5 ) . Lee et aL7 demonstrated that when deuteriated glucose ([2-2H]glucose) is used as a substrate, the rate of isomerisation is reduced by a factor of four. This shows that the rate-limiting step of the enzyme-catalysed reaction is the isomerisation, and that it requires a hydrogen transfer from C(2). This experiment was done using xylose isomerase from Clostridium thermosulfurogenes,but we have observed the same effect using enzyme from Arthrobacter.’ It has proved difficult to visualise directly the complex of the enzyme with a furanose such as xylulose or fructose in the closed-ring form. Crystallographic experiments using 5-thio-~-fructose(kindly synthesized by F. J. Montgomery and P. Grice) and 1,4-dideoxy1,4-imino-~-arabinitol(a gift from Dr R. Nash’) did not show electron density for the
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72
I
Structure and Mechanism of Xylose Isomerase
Y; I
I
Fig. 8 Proposed scheme for the ring-opening, isomerization and ring-closure reactions
ligand in a unique bound state.' 2,5-Dideoxy-2,5-imino-~-mannitol (DDIM) and 2,5dideoxy-2,5-imino-~-glucitol (DDIG) were synthesized by N. G. Ramsden and G. W. J. Fleet, and DDIG (Ki = 50 mmol drnh3) was found to inhibit the enzyme much more strongly than DDIM." DDIG and DDIM are analogues of a - ~and - p-D-fructofuranose (aFF and PFF), in which the ring oxygen is replaced by an imino group, and in which O(2) is absent. The electron density of DDIG when bound to the enzyme is shown in Fig. 6. This structure cannot simply be interpreted as a model for the binding of aFF to the enzyme, since insertion of the extra oxygen at O(2) leads to a severe steric clash with the six-membered ring of Trp-15. It may be noted that the DDIM molecule has almost precise two-fold symmetry about an axis through the ring heteroatom and the mid-point of C(3)-C(4) (Fig. 7: the symmetry could be precise if the pucker obeyed it). In the case of DDIG, the corresponding symmetry is destroyed by the opposite chirality at C(2). Looked at in this way, PFF is rather like DDIM with an oxygen added at O(2) (and of course, an oxygen in the ring). aFF bears the same relation to DDIG. Makkee et a l l 1 showed that aFF is the form of fructose produced by the enzyme. In nature, the product of the enzyme is D-xylulose, and following Makkee we might assume it is a-D-xylulofuranose. This molecule may be considered somewhat closer to two-fold symmetry than DDIG or aFF (Fig. 7). Carrel1 et aLI2 analysed a structure formed when the inhibitor 3-deoxy-3fluoromethylene-D-glucose (Fig. 6) was bound to xylose isomerase from Streptomyces rubiginosus. The ligand was observed covalently bound to the enzyme, in a p-furanose form. The conformation observed was similar to that created if the observed conformation of bound DDIG is rotated 180" about the pseudo-two fold axis. We have hypothesised" that aFF is bound in an analogous manner, namely similar to the observed conformation of bound DDIG, rotated 180" about this axis. In such a
I
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D. M.Blow et al.
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conformation, there are no steric clashes. This conformation brings a firmly bound water molecule (Wat-519), observed in other states of the enzyme, within hydrogenbonding distance of 0(2), where it may act as a base to facilitate ring closure/opening. Cation 2 coordinates Wat-519 while cation 1 coordinates both O(5)and O(2). 0(1) and O(2) occupy positions close to those observed in the various open-chain conformations. Fig. 8 presents a complete scheme for the ring-opening, isomerisation and ring-closure reaction. Ligands have been observed in all the proposed conformations except the a F F form which is still hypothetical. Another possibility is that a product might leave the active site in the (open-chain) ketose form, so that no state corresponding to Fig. 8(f) exists. Molecular mechanics calculations have shown that the active site cavity is large enough to accommodate the required changes from the pyranose to the open-chain conformation, and that there is no major energy barrier for the conformational change.' Preliminary energy calculations suggest a low energy for a-D-xylulofuranose in the proposed conformation of Fig. 8 (f). References 1 F. R. Salemme, Prog. Biophys. Mol. Biol., 1983,42, 95. 2 A. M. Lesk, C. I. Branden and C. Chothia, Proteins, 1989, 5, 139. 3 D. W. Banner, A. C. Bloomer, G. A. Petsko, D. C. Phillips, C. I. Pogson and I. A. Wilson, Nature (London), 1975,255,609. 4 K. Henrick, C. A. Collyer and D. M. Blow, J. Mol. Biol, 1989, 208, 129. 5 C. A. Collyer, K. Henrick, and D. M. Blow, J. Mol. Biol., 1990, 212, 211. 6 M. Whitlow, A. J. Howard, B. C. Finzel, T. L. Poulos, E. Winborne and G. L. Gilliland, Proteins, 1991, 9, 153 7 C. Lee, M. Bagdasarian, M.Meng, and J. G. Zeikus, J. Biol. Chem., 1990,265, 19082. 8 0. S. Smart, J. Akins and D. M. Blow, Proteins, in the press. 9 R. J. Nash, E. A. Bell and J. M. Williams Phytochem., 1985, 24, 1620. 10 C. A. Collyer, J. D. Goldberg, H. Viehmann, D. M. Blow, N. G. Ramsden, G. W. J. Fleet, F. J. Montgomery and P. Grice Biochemistry, submitted. 11 M. Makkee, A. P. G. Kieboom and H. van Bekkum, Recl. Trav. Chim. Pays Bus, 1984, 103, 361. 12 H. L. Carrell, J. P. Glusker, V. Burger, F. Manfre, D. Tritsch and J-F. Biellmann, Proc. Natl. Acad. Sci USA, 1989,86,4440. Paper 1/06423A; Received 18th December, 1991
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Faraday Discuss., 1992, 93, 67-73
Structure and Mechanism of D-Xylose Isomerase David M. Blow, Charles A. Collyer,? Jonathan D. Goldberg and Oliver S. Smart$ Blackett Laboratory, Imperial College of Science Technology and Medicine, London S W7 2BZ, UK
The action of xylose isomerase depends on the presence of two divalent cations. Crystal structure analyses of the free enzyme, and of the enzyme bound to a variety of substrates and inhibitors, have provided models for a number of distinct intermediates along the reaction pathway. These models, in turn, have suggested detailed mechanisms for the various chemical steps of the reaction: a ring opening catalysed by an activated histidine, a hydrideshift isomerization,and a ring closure which may be facilitated by a polarised water molecule.
Xylose isomerase catalyses the isomerisation of the aldose, xylose, to the ketose xylulose. Many xylose isomerases, especially from Actinomycetes, are also efficient catalysts for isomerisation of the hexoses, glucose and fructose, and are very stable enzymes even at 50 "C. This gives them industrial importance in the processing of corn syrup for use in soft drinks. They also have potential application in production of ethanol from waste cellulose sources. Crystallographic analysis of this enzyme, complexed with a variety of substrates and substrate analogues, has provided a detailed hypothesis for the reaction mechanism of this cation-dependent enzyme. The molecular structure of xylose isomerase is based on the eight-stranded a / P barrel structure,1y2first observed in triose-phosphate i s ~ m e r a s eand , ~ subsequently found in many other enzymes. In each case the substrate binds in the centre of the cu/p barrel at its carboxy-terminal end, but there is little further similarity between the two enzymes. While triose phosphate isomerase is a simple monomer, xylose isomerase is a tetramer, in which pairs of barrels are roughly coaxial, with their two substrate-binding sites in proximity, and each peptide chain has a long carboxy-terminal extension which enfolds the other pair of barrels which make up the tetramer. Unlike triose phosphate isomerase, xylose isomerase is a metalloenzyme in which two cation-binding sites exist on each s ~ b u n i t .Site ~ 1 is a tight-binding site while site 2 binds its cation more loosely and, as will be shown, more flexibly. Each cation is coordinated octahedrally with ligands to four carboxylate groups and two substrate hydroxyls in site 1, and four carboxylates, a histidine and a water molecule in site 2. One carboxylate, Glu-216,$ coordinates both cation sites. The presence of two basic amino acids in the active site provides an equal number of positive and negative charges in the vicinity. These two cation sites lie to one side of a large cavity at the carboxy-terminal end of the eight 6-strands of the a l p barrel. The two cavities of the pair of barrels which face each other are adjacent, and share a common access route to bulk solvent. The side of the cavity facing the cation sites is hydrophobic, composed of several aromatic amino acid side chains, including
t Present address:
Farmitalia, Carlo Erba, via dei Gracchi, Milano, Italy.
$ Present address: Dept of Crystallography, Birkbeck College, Malet St., London WClE 7HX, UK.
Ei Amino acid numering is for xylose isomerase of Arthrobacter B3278, throughout. 67
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Structure and Mechanism of Xylose Isomerase
68
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Table 1 Pyranose-substrates and substrate analogues used as ligands
2
I
xylose glucose 5-thioglucose 1-deoxynojirimycin
X
Y
Z
OH OH OH H
0 0 S NH
H CH20H CH20H CHZOH
1
H
OH
Fig. 1 Electron density for 5-thioglucose bound to the active site. All electron-density maps are displayed as Fderiv - Fcalcmaps, where FcaIc is the refined structure in the absence of the ligand
one (Phe-25) from an adjacent subunit, but near to this hydrophobic surface is an activated histidine (His-53) which is polarised by a buried aspartate (Asp-56). X-Ray analysis of crystalline complexes of the xylose isomerase of Arthrobacter B3278 with a variety of ligands (Table l), and using various cations, has allowed us to create realistic models which represent a number of steps in the isomerisation of an aldose to a ketose.’ Binding 5-thioglucose to enzyme crystals provided the first model of a complex of the enzyme with a closed-ring aldose (Fig. 1). The electron density in the presence of 1-deoxynojirimycin indicated a similar ring orientation. In these structures cation 1 coordinates to O(3) and O(4) of the pyranose ring while cation 2 makes no direct interaction with the sugar. In the 5-thioglucose and 1-deoxynojirimycin complexes, His-53, which is polarized by the side chain of Asp-56, provides a correctly oriented nucleophile to catalyse ring opening by transfer of a proton from O( 1) to O(5) (Fig. 2). When xylose bound to enzyme crystals, the electron density plainly showed the sugar in an open-chain conformation’ (Fig. 3). Similar electron density distributions were observed on binding the five- and six-carbon polyols, xylitol and sorbitol, to the crystal^.^ These electron-density distributions were interpreted as showing cation 1 bound to O(2)
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Fig. 2 Scheme for ring-opening catalysed by His-53'
Fig. 3 Electron density for xylose bound at the active site
and O(4) of the open-chain sugar. The quality of crystals of the Arthrobacter enzyme limited the attainable resolution to 2.3 A at least (often in practice, to 2.5 A), and at this resolution details of the conformation could not be assigned. However, Whitlow et aL,6 in a similar experiment using crystals of the enzyme from Streptomyces rubiginosus, complexed with an equilibrium mixture of xylose-xylulose, were able to obtain diffraction data to 1.6 A resolution, and noted a planar conformation at the site we assumed to be occupied by C(2), indicating the open-chain ligand to be predominantly in the ketose form. In the above experiments the cation at site 2 still had no direct ligands to the substrate. However, when xylose was incubated with enzyme crystals in the presence of A13+,a somewhat different structure was observed' (Fig. 4). Under the conditions of the experiment, there was evidence that the site 1 cation is A13', while the loosely bound cation at site 2 was Mg2+. Small structural changes of the enzyme around site 2 were evident, including a significant rearrangement of Asp-254 and smaller movements of Glu-216 and His-219, which accompanied a 1 A movement of the second cation to site 2', where it coordinates O( 1) and O(2) of the substrate. This A13' complex is considered to be a transition-state analogue in which the cation at 2' is equidistant from 0(1) and O(2) and 2', 0(1), C(1), C(2) and O(2) are all approximately coplanar. Cation 1 also coordinates 0(2), while N H l ( Lys-182) coordinates O( l), strongly polarising both C-0 bonds towards C+-O-. One hydroxyl proton [say OH(2)] is assumed to be lost (possibly to a water molecule), creating conditions where the hydrogen H(2) can readily transfer
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70
Structure and Mechanism of Xylose Isomerase
Fig. 4 Electron density for xylose incubated with the enzyme in the presence of A13+ and Mg2’
1)2+
It ,.o +dH
LyS-182-N, A H H
H ‘
M(2I2+
Fig. 5 Scheme for isomerisation by hydride shift5
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Fig. 6 Electron density for 2,5-dideoxy-2,5-imino-~-glucitol (DDIG) bound to the active site
HO
H
HO
H a-0-xylulduranose
HOCH2H
CH2OH
HOCHp
CH20H
‘~
DDIG
~~H
aFF
H4
HO
H
HO
H
HOCH2
OH
‘ HO H W H zCH-Enz O H
3-deoxy-3-fluoromethylene -&glucose
Fig. 7 Schematic formulae for sugar analogues. The dashed line indicates the pseudosymmetry is the form considered of DDIM. The structure labelled ‘3-deoxy-3-fluoromethylene-~-glucose’ to be bound to the enzyme12
as a hydride ion between the carbonium-like C(2) and C(1). This makes a plausible model for the isomerization by a hydride-shift mechanism (Fig. 5 ) . Lee et aL7 demonstrated that when deuteriated glucose ([2-2H]glucose) is used as a substrate, the rate of isomerisation is reduced by a factor of four. This shows that the rate-limiting step of the enzyme-catalysed reaction is the isomerisation, and that it requires a hydrogen transfer from C(2). This experiment was done using xylose isomerase from Clostridium thermosulfurogenes,but we have observed the same effect using enzyme from Arthrobacter.’ It has proved difficult to visualise directly the complex of the enzyme with a furanose such as xylulose or fructose in the closed-ring form. Crystallographic experiments using 5-thio-~-fructose(kindly synthesized by F. J. Montgomery and P. Grice) and 1,4-dideoxy1,4-imino-~-arabinitol(a gift from Dr R. Nash’) did not show electron density for the
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72
I
Structure and Mechanism of Xylose Isomerase
Y; I
I
Fig. 8 Proposed scheme for the ring-opening, isomerization and ring-closure reactions
ligand in a unique bound state.' 2,5-Dideoxy-2,5-imino-~-mannitol (DDIM) and 2,5dideoxy-2,5-imino-~-glucitol (DDIG) were synthesized by N. G. Ramsden and G. W. J. Fleet, and DDIG (Ki = 50 mmol drnh3) was found to inhibit the enzyme much more strongly than DDIM." DDIG and DDIM are analogues of a - ~and - p-D-fructofuranose (aFF and PFF), in which the ring oxygen is replaced by an imino group, and in which O(2) is absent. The electron density of DDIG when bound to the enzyme is shown in Fig. 6. This structure cannot simply be interpreted as a model for the binding of aFF to the enzyme, since insertion of the extra oxygen at O(2) leads to a severe steric clash with the six-membered ring of Trp-15. It may be noted that the DDIM molecule has almost precise two-fold symmetry about an axis through the ring heteroatom and the mid-point of C(3)-C(4) (Fig. 7: the symmetry could be precise if the pucker obeyed it). In the case of DDIG, the corresponding symmetry is destroyed by the opposite chirality at C(2). Looked at in this way, PFF is rather like DDIM with an oxygen added at O(2) (and of course, an oxygen in the ring). aFF bears the same relation to DDIG. Makkee et a l l 1 showed that aFF is the form of fructose produced by the enzyme. In nature, the product of the enzyme is D-xylulose, and following Makkee we might assume it is a-D-xylulofuranose. This molecule may be considered somewhat closer to two-fold symmetry than DDIG or aFF (Fig. 7). Carrel1 et aLI2 analysed a structure formed when the inhibitor 3-deoxy-3fluoromethylene-D-glucose (Fig. 6) was bound to xylose isomerase from Streptomyces rubiginosus. The ligand was observed covalently bound to the enzyme, in a p-furanose form. The conformation observed was similar to that created if the observed conformation of bound DDIG is rotated 180" about the pseudo-two fold axis. We have hypothesised" that aFF is bound in an analogous manner, namely similar to the observed conformation of bound DDIG, rotated 180" about this axis. In such a
I
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conformation, there are no steric clashes. This conformation brings a firmly bound water molecule (Wat-519), observed in other states of the enzyme, within hydrogenbonding distance of 0(2), where it may act as a base to facilitate ring closure/opening. Cation 2 coordinates Wat-519 while cation 1 coordinates both O(5)and O(2). 0(1) and O(2) occupy positions close to those observed in the various open-chain conformations. Fig. 8 presents a complete scheme for the ring-opening, isomerisation and ring-closure reaction. Ligands have been observed in all the proposed conformations except the a F F form which is still hypothetical. Another possibility is that a product might leave the active site in the (open-chain) ketose form, so that no state corresponding to Fig. 8(f) exists. Molecular mechanics calculations have shown that the active site cavity is large enough to accommodate the required changes from the pyranose to the open-chain conformation, and that there is no major energy barrier for the conformational change.' Preliminary energy calculations suggest a low energy for a-D-xylulofuranose in the proposed conformation of Fig. 8 (f). References 1 F. R. Salemme, Prog. Biophys. Mol. Biol., 1983,42, 95. 2 A. M. Lesk, C. I. Branden and C. Chothia, Proteins, 1989, 5, 139. 3 D. W. Banner, A. C. Bloomer, G. A. Petsko, D. C. Phillips, C. I. Pogson and I. A. Wilson, Nature (London), 1975,255,609. 4 K. Henrick, C. A. Collyer and D. M. Blow, J. Mol. Biol, 1989, 208, 129. 5 C. A. Collyer, K. Henrick, and D. M. Blow, J. Mol. Biol., 1990, 212, 211. 6 M. Whitlow, A. J. Howard, B. C. Finzel, T. L. Poulos, E. Winborne and G. L. Gilliland, Proteins, 1991, 9, 153 7 C. Lee, M. Bagdasarian, M.Meng, and J. G. Zeikus, J. Biol. Chem., 1990,265, 19082. 8 0. S. Smart, J. Akins and D. M. Blow, Proteins, in the press. 9 R. J. Nash, E. A. Bell and J. M. Williams Phytochem., 1985, 24, 1620. 10 C. A. Collyer, J. D. Goldberg, H. Viehmann, D. M. Blow, N. G. Ramsden, G. W. J. Fleet, F. J. Montgomery and P. Grice Biochemistry, submitted. 11 M. Makkee, A. P. G. Kieboom and H. van Bekkum, Recl. Trav. Chim. Pays Bus, 1984, 103, 361. 12 H. L. Carrell, J. P. Glusker, V. Burger, F. Manfre, D. Tritsch and J-F. Biellmann, Proc. Natl. Acad. Sci USA, 1989,86,4440. Paper 1/06423A; Received 18th December, 1991
