1
Chapter 1
Introduction
2
Introduction
Triosephosphate Isomerase (TIM or TPI; EC 5.3.1.1) is a nonregulatory glycolytic
enzyme that catalyzes the reversible isomerization of two triose phosphates:
dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (GAP).
With the tritium labeling of hydrogens of water in buffer or the substrate, the
disposition of tritium after the reaction suggested a cis-enediol(ate) phosphate
intermediate in the interconverstion of a ketose and a aldose (1).
HS
HR
OH
OH
C1
C2
H2C
O
C1
C2
O
3
H2C
OPO32-
C1
H
OH
C2
H
3
H2C
OPO32-
OH
3
OPO32-
GAP
DHAP
β -elimination
O
H
C1
C2
O
+
Pi
H2C3
Methyl Glyoxal
Figure 1.1 Reaction catalyzed by triosephosphate isomerase
TIM exists in all organisms that we know so far. Decreased activity of TIM leads to
triosephosphate isomerase (TPI) deficiency in humans, which includes chronic
hemolytic anemia and neuromuscular disorders (2).
3
CH2OPO32O H
H
H
OH H
OH
OH
H
OH
ATP ADP
CH2OH
O H
H
H
OH H
OH
OH hexokinase
H
OH
glucose
glucose 6-phosphate
glucose
6-phosphate
isomerase
22CH2OPO3
CH2OPO3
2CH
OPO
phosphofructoO
2
3
O CH2OH
kinase
H HO
H HO
H
OH
H
OH
OH H
ATP
OH
H
ADP
fructose 1,6-diphosphate
fructose 6-phosphate
aldolase
NAD ++Pi
2CH2OPO3
C
O
NADH+H+
CH2OH
C
O
ATP
2OPO3
HCOH
2CH2OPO3
C O
-
HCOH
phosphoglycerate
kinase
2-
CH2OPO3
glyceraldehyde
1,3-diphosphoglycerate
3-phosphoglycerate
3-phosphate
glyceraldehyde
phosphoglycerate
3-phosphate
mutase
dehydrogenase
O
C O
C
2CH2OPO3
dihydroxyacetone
phosphate
ADP
O
triosephosphate HC O
isomerase
HCOH
-
ATP
ADP
C O
O
CH3
pyruvate
O
O
-
H2O
2-
COPO3
pyruvate
kinase
CH2
enolase
phosphoenolpyruvate
Figure 1.2 Glycolysis pathway
C O
-
2HCOPO3
CH2OH
2-phosphoglycerate
4
In the glycolysis pathway (Figure 1.2), after fructose 1,6-biphosphate breaks down to
aldolase, TIM acts as a nonregulatory enzyme and converts DHAP to GAP in the
biologically significant direction. The direction of DHAP to GAP is biologically
relevant but thermodynamically uphill, and GAP to DHAP is biologically irrelevant
but thermodynamically downhill. The presence of TIM keeps a balance between
DHAP and GAP, with a DHAP ratio of about 96% at equilibrium for yeast (3). The
clinical data showed that deficiency of TIM does not slow down ATP synthesis,
however it causes a fatal accumulation of DHAP.
Figure 1.3 TIM α/β barrel structure with substrate (PDB 1NEY)
TIM is only active as a dimer of identical subunits, each composed of 247 residues
and of a molecular weight of about 26 kDa1 (4). Since the first three dimensional
crystal structure of chicken TIM was solved in 1975 (5), it has been the textbook
prototype for the (α/β)8 barrel (also known as TIM barrel). The outside eight parallel
1
Unless specifically noted otherwise all parameters are referring to the yeast TIM (S. cerevisiae TIM).
Yeast TIM was chosen in our studies instead TIM from other orgnisms because the biological profile
was best characterized.
5
α helices surround a cylinder core of eight parallel β strands (Figure 1.3). This
striking motif is surprisingly shared with more than 20 different classes of enzymes (6,
7). All of the enzymes in this family have their active sites located at the C-terminal
ends of the β strands; although they are not evolutionarily related, and few of them
exhibit significant sequence similarity.
TIM is a very efficient catalyst. It increases the rate of the isomerization by almost 10
orders of magnitude from the non-enzymatic value (7 × 10-5 M-1S-1) and βelimination by 5-8 orders of magnitude (8). In the thermodynamically unfavorable but
biologically significant direction, from DHAP to GAP, the catalytic rate is kcat =
7.5(±0.2) × 102 s-1, the Michaelis constant for DHAP Km = 1.4(±0.1) mM, and the
rate-limiting step is the loss of GAP, or a conformational change from closed loop to
open loop to release GAP; in the direction from GAP to DHAP, kcat = 8.7(±0.3) × 103
s-1, the Michaelis constant for GAP Km = 0.055(±0.004) mM, and the rate-limiting
step is the abstraction of a C2 proton by Glu165. When GAP serves as a substrate, the
second order rate constant kcat/Km is 1.5 × 108 M-1S-1, close to the diffusion limit for
the ligand to associate with the enzyme (9), which means that it almost has the
optimal catalytic efficiency for any enzyme.
There has been a lot of interest in experimental and computational studies of TIM. A
total number of 99 X-ray structures of TIM from various organisms have been
deposited into the RCSB Protein Data Bank so far. There are also a vast number of
mutagenic and biological kinetic studies, UV and EPR studies, NMR spectroscopy,
6
T-jump relaxation spectroscopy, as well as molecular dynamics simulations and
quantum mechanics simulations. Those studies have provided great opportunities to
pursue the ultimate and complete understanding to this model enzyme.
TIM Catalytic Reaction Mechanism and the Active Site Geometry
To perform the perfect glycolytic catalysis, a lot of residues in TIM are involved
together to drive the isomerization machine running. On the other hand, there are
three of them that really perform the key chemistry: the catalytic base Glu165 and the
electrophilic residues His95 and Lys12 (5, 10). In Figure 1.4, the three residues are
positioned perfectly towards the organic moiety of the substrate.
Figure 1.4 The active site geometry of TIM complexed with the substrate DHAP.
The structure is based on the PDB structure 1NEY (10). The three key residues
involved in catalysis are Glu165, His95, and Lys12. All the other residues provide
structural and electrostatic stabilization in the reaction coordinate.
7
When the enzyme is in the loop open form (when the enzyme is unligated or ligated
but in a scarcely populated open form), the carboxylate oxygens of Glu165 are
hydrogen bonded to the amide nitrogen and the side chain hydroxyl group of Ser96,
as well as to the Nε of His95. When the substrate is present, the Glu165 swings its
side chain by 2-3 Å towards the substrate organic moiety. Its hydrogen bond to Ser96
is broken, while the hydrogen bond with His95 remains. The side chain carboxylate
oxygen of Glu165 is well positioned to have nearly equal distances from C1 and C2
within the hydrogen bond regime: 3.06 Å to C1 and 2.99 Å to C22. This new position
is optimal for a reversible reaction – no matter whether the substrate is DHAP or
GAP, this carboxylate oxygen is able to interact with both C1 and C2.
As illustrated in Figure 1.5, in the conversion from DHAP to GAP, the reaction starts
with the abstraction of the pro-R proton from the C1 of DHAP by Glu165 as a
catalytic base (11). As such the substrate turns to an enediol(ate) intermediate, and the
active site loop 6 synchronically closes up to protect it from the bulk solvent. Side
chains of Asn10 and Lys12 serve to provide electrostatic stabilization to O1 and O2
of the substrate (12, 13). The side chain of the neutral His95 polarizes the carbonyl
group (O2) of the substrate (14, 15). Meanwhile the phosphate moiety of the substrate
is stabilized by the hydrogen bonds to Gly171, Ser211, Gly232 and Gly233.
2
The numbering scheme for DHAP starts from the hydroxyl group: O1-C1 (hydroxyl), O2-C2
(ketone), C3 (methylene).
Figure 1.5 Catalytic pathway of DHAP to GAP catalyzed by TIM. In the yellow box, Glu165 abstracts the pro-R proton to
initiate the reaction. In the green boxes, three different proposals for the proton transfer from hydroxyl to carbonyl were
illustrated. The last step, in the black box, is the donation of the Glu165 side chain hydrogen to C2.
8
9
The mechanism of the later step, the transfer of the proton from O1 to O2 in the
intermediate, is still unknown. As illustrated in Figure 1.5, several experimental and
theoretical groups provide three proposals (16-18):
(A) As first proposed by Knowles et al. (9) and was widely accepted (19-22), the
neutral His95 donates a proton from NƐ to the substrate O2 atom to form an
enediol(ate) intermediate, and subsequently it abstracts a second proton from the
substrate O1 atom;
(B) A direct internal proton transfer from O1 to O2 forms the intermediate (11, 20,
21). However the QM/MM studies pointed out that this transfer generates a
significantly larger energy barrier than the other two mechanisms. Therefore this path
was least considered;
(C) To generate the same enediol(ate) intermediate as the one in path (A), the proton
transferred to the substrate O2 atom could also come from Glu165 with its
carboxylate group of the side chain rotates onto the O2 atom (18, 19, 22). The second
proton transfer from the substrate O1 atom to Glu165 side chain is followed.
The last step in catalysis is generally believed to have the proton on the Glu165
carboxylate group transferred to the substrate C2 atom to produce GAP.
Energetically, the first step is highly unfavorable in the absence of the enzyme due to
the high pKa of the substrate C1 atom (~17-19) (23). In the presence of the enzyme,
Glu165 is exposed to the bulk solvent in the open state with a pKa value of 3.9, and is
isolated by residues Ala163, Ile170, Gly209, and Leu230 from the solvent in the
10
closed form with a pKa value of 6.3 (24, 25). But still, ‘the α-protons of carbon acids
are not very acidic and the general base catalysts are not very basic’ (26), which
generate a high activation energy that is not compatible with the observed kcat values.
This mismatch is removed by the protonation onto the substrate’s carbonyl group by
the imidazole of His95. Consequencly the α-proton of the aldehyde or ketone has a
reduced pKa value of ~2 or ~5 respectively (23). In addition, Nϛ of Lys12 if the
substrate is DHAP or NƐ2 of Asn10 if the substrate is GAP serves as electrophile to
stabilize the negative charge on O2.
The site-directed mutation of Glu165Asp in Chicken TIM only moves the carboxylate
group only by ~ 1 Å further to the intermediate analogue with the rest of the structure
unchanged (27), but reduces the catalytic activity of the enzyme by 1000 fold (28, 29).
Other mutations of Glu165 to shorter residues without the carboxylate group, such as
alanine and glycine, abolish the enzyme catalysis completely.
The infrared spectrum of DHAP bound to TIM provided the first direct evidence that
an enzyme electrophile is responsible to polarize the carbonyl group of DHAP and
promotes catalysis (14). Using infrared spectroscopy and X-ray crystallography, with
the mutation of His95Gln and His95Asn as well as a series of secondary mutations,
Komives, et al. further concluded that His95 was the responsible electrophile, and
additionally, it plays a second role as a general acid-base in the proton transfer from
O1 to O2 of the substrate based on the observation that Glu95 in the mutant His95Glu
acts as both base and acid (15). Meanwhile, in the mutants His95Gln and His95Asn,
11
the activity of the enzyme is reduced by 100 for His95Gln and 104 for His95Asn,
compared to the wild type enzyme (15). The 15N NMR titration studies demonstrated
that the imidazole ring of His95 is neutral over the entire pH range where TIM is
active, which rules out the widely accepted proposal that it donates and abstracts a
proton to O2 and from O1 during catalysis (30). The crystal structure (31) as well as
computer simulation studies (13, 18) also stand for this point of view.
Lys12 for DHAP, and Asn10 for GAP, are also in contact with the organic moiety of
the substrate. With their side chains hydrogen bonded to the oxygens of the substrate,
they provide a positive electrostatic environment in the proximity of the substrate
oxygens, and thereby stabilize the charged transition state. The mutation of Lys12Met
reduces kcat to 0.018 s-1. Other mutations, Lys12Met·Gly15Ala, Lys12Arg, and
Lys12His, all reduce the enzymatic activity significantly (32).
Conformational Change
For the big picture, how structure decides function, TIM has attracted keen interest to
study its conformational change, due to the conceptually simplicity of the TIM
reaction: it’s all about the shuttling of protons. The conformational change of TIM
was first observed in the chicken TIM by soaking the enzyme into the substrate
solution (33). The first atomic level conformational change was detected on the yeast
TIM complexed with the substrate analogue 2-phosphoglycolate at 2.5 Å (12). The
12
structural change was seen in both experimental binding methods: soaking (34-36),
and cocrystallization (12, 31).
The conformational change of the enzyme has been probed by many crystal structures,
indicating that it involves a conformational change of the active site loop 6. In the
high resolution unligated TIM structure, a dominant conformation of loop 6 was
observed as in the so-called open form (37). In the high resolution TIM structures
where the protein is ligated by the substrate or substrate analogues ligated TIM
structures, with some exceptions (34, 38-40), nearly all structures indicate that the
loop is dominantly populated in the loop closed form (10). The comparison of open
and closed conformations in chicken, yeast and trypanosomal TIM structures shows
that the open form of chicken, yeast, and trypanosomal TIM are essentially identical
to each other; and the closed form of yeast and trypanosomal TIM are essentially
indistinguishable (41). Solid state deuterium NMR studies have suggested that the
loop conformational change is not ligand-gated, however, it affects the relative
populations of the open and closed conformations (42). After deletion of this loop, the
‘loopless’ mutant can no longer enolize the substrate to form the intermediate, or
reprotonate the intermediate to form product. This enzyme without loop has a specific
catalytic activity about 105-fold lower than the wild type, which was speculated to be
due to much higher activation energy barriers for the enolization of the substrate by
the enzyme (43).
13
loop 5
loop 7
Unligated state: 1YPI
Ligated state: 7TIM
Figure 1.6 Loop 6 conformational change upon binding of the ligand PGH
(phosphoglycolohydroxamate). The structures shown are yeast unligated (PDB entry
1YPI (37)) contrasted with the enzyme complexed with PGH (PDB entry 7TIM (13)).
Loop 5, 6, 7 are labeled in the figure.
Loop 6 is composed of 11 residues (Pro166-Val167-Trp168-Ala169-Ile170-Gly171Thr172-Gly173-Leu174-Ala175-Ala176 in yeast) (44). Upon ligand binding, the loop
clamps down over the active site as a rigid entity, with the tip of it (Thr172) moving
by more than 7 Å (Figure 1.6). At the same time, adjacent loop 5 and loop 7 also have
been observed to have conformational flexibility (40). The small loop 5 adjusts
slightly in response to the hydrogen bond partner change from loop 6. Loop 7 also has
a synchronous dramatic conformational rearrangement, to make room for Glu165 to
swing to its functionally competent position. The combination of conformational
change in loop 5, loop 6, and loop 7 causes a rearrangement of hydrogen bond
interactions among these loops.
14
There are at least two major functions for the loop 6:
(1) The conformation of the closed loop effectively sequesters the formed
intermediate from the bulk solvent (45, 46). This shielding could immediately
decrease the dielectric constant in the vicinity of the intermediate, thereby
increasing the electrostatic interactions in the active site (12), and lowering
down the energy required for enolization.
(2) The closed loop largely prevents the β-elimination from the intermediate that
could happen easily if the catalysis was a more simple acid-base reaction by
organic catalysts (8).
The tip of the loop 6 (Thr172) shifts up to 8 Å upon the loop closure, however, the
ends of the loop change little. For example, the distance from Cα of Pro166 to Cα of
Ala176 is 7.3 Å in the open loop, and 7.5 Å in the closed loop (44). Also, there is a
significant similarity of the internal loop conformations in the open and closed forms
measured by the superposition of the two structures by least-squres optimization of
the loop atoms (44). Therefore the entire loop movement could be described as a lid
closure with two fixed hinges which only change in angles.
The internal hydrogen bonding network is responsible for this rigidity of loop 6 (47):
The carbonyl oxygen of Pro166 is hydrogen bonded to the amide nitrogen of Ala169;
the carbonyl oxygen of Val167 is hydrogen bonded to the amide nitrogen of Ile170;
the carbonyl oxygen of Trp168 and the amide nitrogen of Leu174 are hydrogen
bonded to the hydroxyl of Thr172. In the loop open structure, Gly171, Gly173,
15
Ala175, and Ala176 don’t have any hydrogen bond partners in the rest of the enzyme,
whereas in the loop closed structure, except Ala175, all the other residues have
hydrogen bond partners (44). When the substrate is present, it is hydrogen bonded to
only one resiude in the active site loop 6 – the amide nitrogen of Gly171 is hydrogen
bonded to the phosphate oxygen of the substrate. Upon loop closure, the hydrogen
bond between NƐ1 of Trp168 and phenolic OηH of Tyr164 is broken and instead the
NƐ1 of Trp168 is hydrogen bonded to carboxylate of Glu129. Meanwhile Trp168
forms an extra hydrogen bond the ring of Pro166. Newly formed hydrogen bonds in
loop 6 upon loop closure also include Gly173 to Ser211, and Ala176 to Tyr208. To
reach the substrate organic moiety, the side chain of Glu165 moves significantly from
the open form to the closed form, with the Cδ shifting by 3 Å (44).
With the movement of loop 6, loop 5 also adjusts slightly largely due to the fact that
the carboxylate oxygens of Glu129 mediate the interaction between Tyr164 OηH and
Trp168 NƐ1, by forming hydrogen bonds with each of them. Whereas in the open
loop, Tyr164 OηH and Trp168 NƐ1 are directly hydrogen bonded (48).
The interaction between the C-terminal of loop 6 and loop 7 plays an important role
in the loop opening and closing: computational studies have showed that the
loosening of the hydrogen bonds between the C-terminal of loop 6 and the YGGS
motif in the loop 7 actually initiates the loop opening (44, 49). The hydrogen bonds
between Gly173 to Ser211, and Ala176 to Tyr208 therefore should be moderately
strong, so that the loop 6 is easy to open, thus insures the catalysis efficiency. In the
16
unligated enzyme, the amide nitrogen of Gly210 points towards the protein core, the
aromatic ring of Tyr208 (40). In the ligated enzyme, it points outwards and forms a
hydrogen bond with the phosphate oxygen on the substrate via a water molecule. The
peptide plane of Gly209-Gly210 rotates to make room for the Glu165 side chain to
reach to its optimal catalysis ‘swing-in’ position.
The Dimer Interface
The native TIM is only competent as a homodimer even though the two identical
subunits don’t cooperate in the catalysis (50). The dimerization induces the
rearrangement in the monomers (51, 52), as well as the key residues involved in the
dimer interface interactions have been studied (40, 52-54). However, it is still
unknown whether one monomer participates in the catalysis event of the other
monomer. The interface residues have been defined as having at least one atom in
contact with an atom of the other subunit within 4 Å (40). There amino acids that are
involved in the interface interactions have considerable sequence variability (41). The
32 interface residues in the yeast TIM are illustrated in Figure 1.7 and tabulated in
Table 1.1. Most of those residues are located in loop1, loop2, loop3, and loop4,
especially in loop 3.
17
Figure 1.7 The dimer interface residues illustrated in a homodimer TIM structure
(PDB entry 1NEY (10)). The 32 interface residues (yeast) are marked in blue in the
picture. Loop 1, loop 2, loop 3, loop 4, and the active site loop 6 have been pointed
out.
Although there is much sequence variability of the interface residues, the direct
hydrogen bonds between the two monomers are highly conserved (41). In yeast TIM
24 direct hydrogen bonds have been observed, most of which are with the loop 3
residues (Table 1.2). Hidden in the interface, there are seven cavities, filled with 18
water molecules (40).
18
Residues
Asn10
Lys12
Leu13
Asn14
Gly15
Ser16
Lys17
Pro43
Als44
Thr45
Tyr46
Asp48
Gln64
Asn65
Tyr67
Ser71
Conservation of
residue type
**
**
**
**
**
**
Residues
Gly72
Ala73
Phe74
Thr75
Gly76
Glu77
Asn78
Ser79
Gln82
Asp85
Val86
His95
Glu97
Arg98
Tyr101
Phe102
Conservation of
residue type
**
*
*
**
**
**
**
*
**
**
**
Table 1.1 The residues on the dimer interface for the yeast wild type TIM (41, 52).
Interface residues have at least 1 atom within 4 Å of an atom of the other subunit (40).
** means the residue is conserved in all 13 TIM sequences.
* means the residue is conserved in yeast TIM, chicken TIM, and trypanosomal TIM.
19
2° structure
Subunit 1
Subunit 2
Loop 1
Asn10 Nδ2
Thr75 Oγ1
Asn10 Nδ2
Gly72 N
Leu13 O
Gly72 N
Leu13 O
Gly72 O
Leu13 N
Gly72 N
Lys17 Nϛ
Asp48 Oδ1
Lys17 Nϛ
Asp48 Oδ2
Lys17 Nϛ
Asp85 Oδ1
Loop 2
Tyr46 OH
Asp85 Oδ1
Loop 3
Gln64 OƐ1
Gly76 N
Ser71 Oγ
Asn14 O δ1
Gly72 N
Leu13 O
Gly72 N
Leu13 N
Thr75 Oγ1
Asn10 Nδ2
Gly76 N
Gln64 OƐ1
Glu77 OƐ1
Arg98 NH1
Gln82 NƐ2
Gly15 O
Asp85 Oδ2
Lys17 N
Asp85 Oδ2
Tyr46 OH
Glu97 OƐ1
Thr75 N
Glu97 OƐ1
Thr75 Oγ1
Arg98 NH1
Thr75 O
Arg98 NH1
Glu77 OƐ1
Loop 4
Table 1.2 Interface direct hydrogen bonds in yeast TIM (41).
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Methylglyoxal Production
In the titration experiment of the substrate DHAP to chicken TIM, it was first
observed the formation of methylglyoxal and orthophosphate from the substrate
elimination by 1H NMR (55). Since then this side reaction has been studied
extensively under different conditions: without catalyst, with buffer catalysts, with
TIM as a catalyst, and with methylglyoxal synthase as a catalyst (8, 56-60). Without
TIM, this elimination is much faster (8). TIM does a ‘less than perfect’ job by
surrounding its flexible loop to the phosphate moiety of the intermediate, therefore
suppressing this elimination. The kinetic parameters of TIM catalyzed phosphate
elimination for rabbit TIM are kcat = 0.011 s-1, Km = 0.76 mM, kcat/Km = 14 M-1s-1
(59). If the enediolate phosphate binds to TIM, this phosphate elimination decreases
its rate constant by 105 – 108 fold. Despite the slow rate of the reaction, it has
significant mechanistic and physiological consequences. Methylglyoxal is a highly
reactive compound. It can potentially modify the active site of the enzyme by reacting
with the arginine residues in the protein to form imidazolone adducts, and with the
lysine residues to form N-epsilon-(carboxyethyl)lysine (CEL) and the imidazolium
crosslink, methylglyoxal-lysine dimer (MOLD) (61).
There was an interesting study that employed methylglyoxal synthase to investigate
the relationship between TIM and methylglyoxal (62). Methylglyoxal synthase is an
enzyme that uses the same initial chemical steps as the TIM reaction to form the
enediol(ate) of DHAP to catalyze the elimination of phosphate and to produce
21
methylglyoxal. 2-phosphoglycolate is a competitive inhibitor of both methylglyoxal
synthase and TIM, therefore the active site structure of methylglyoxal synthase bound
to 2-phosphoglycolate and TIM bound to 2-phosphoglycolate were compared. The
result showed surprising similarities in the active site catalysis residues.
The distances between the functional groups of Asp71, His98, His19, and the
carboxylate oxygens of 2-phosphoglycolate in methylglyoxal synthase are similar to
the distances between the functional groups of Glu165, His95, Lys13, and the
carboxylate oxygens of 2-phosphoglycolate in TIM, with enantiomorphic spatial
relationships.
The methylglyoxal formation from the reaction catalyzed by TIM has a cellular rate
of 0.4 mM/day. If methylglyoxal were left to accumulate, its concentration produced
by TIM would go as high as the concentration that of cellular triosephosphates in
about 2 hours. In vivo, methylglyoxal is metabolized to D-lactate by glyoxalases I and
II (63). However, in our solid state NMR experiments, this pair of enzymes was not
present in the buffer. In another word, in the conditions where there are no such
enzymes to consume the byproduct in the TIM reaction as it does in the natural
metabolism, the side product from the phosphate elimination would be inevitable at
high temperatures.
22
Studies on the TIM Loop Motion
Albery and Knowles first determined the rate constants along the reaction coordinate
by the free energy profile deduced from deuterium and tritium isotope exchange
experiments (9). In the direction from DHAP to GAP, kcat = 7.5(±0.2) × 102 s-1, while
the ligand release step was determined to have a rate of ~ 4000 s-1, which means the
rate of loop movement is on the same time scale as catalysis. Therefore it’s likely the
rate limiting step from DHAP to GAP, is the loss of GAP, or the conformational
change from closed loop to open loop to release GAP.
The upper limit of the reaction rates for many enzyme-substrate and protein-ligand
associations should be diffusion controlled, with a second-order rate constant kcat/Km
close to 108 – 1010 s-1 (64, 65). Blacklow et al. used buffers with different viscosities,
as well as both wild type and mutant enzyme, to confirm that TIM catalysis is
diffusion controlled, reaching the perfection of catalysis (66).
Another approach to study dynamics of macromolecules is computational methods.
Molecular dynamics calculations showed that loop 6 of the unligated TIM opens and
closes more like a lid than a flexible loop (44, 67). With the binding of the substrate
DHAP (68), or the substrate analogue PGH (phosphoglycolohydroxamate) (44), the
loop remained closed throughout the simulation. They suggested that the loop motion
occurs in a time scale of microseconds, and is rate-limiting in the catalysiss from
23
DHAP to GAP (49). Theoretical calculations also provided assumptions for the
catalysis mechanism (13, 17-20, 69).
Experimentally, since the application of NMR and fluorescence spectroscopy,
especially NMR, in the macromolecular dynamics field, a big progress has been made
addressing the dynamics of the loop motion. Solid state deuterium NMR was used to
determine the time scale of the loop motion. The motion was observed in both of the
ligated and unligated enzyme, which indicated that the loop motion is not ligandgated (42). Solution state NMR later confirmed this ligand unrelated motion, with the
populations skewed toward the open conformation in the unligated enzyme and
toward the closed conformation in the ligated enzyme (70). With the absolutely
conserved Trp168 as the marker for loop 6, using substrate analogues and a simplified
two-step model, studies including 1D Solid state deuterium NMR (42, 71), 1D
solution state 19F and 31P NMR (72), and T-jump fluorescence relaxation
spectroscopy (73), revealed that the loop opening has a rate constant of ~ 104 s-1 and
is rate-limiting or partially rate-limiting.
Since the employment of newly developed solution state NMR techniques for
quantitatively characterizing motions with microsecond-to-millisecond time scales
(74), the motions in TIM have been probed extensively and site-specifically. The apo
yeast TIM was site-specifically assigned by solution state NMR (BMRB entry 7216);
the G3P ligated yeast TIM was assigned for the backbone nitrogens (70). TROSY
Hahn spin-echo pulse sequence was used to measure the kex and the population ratio
24
for the open and closed conformations. The conformational exchange rates for the
residues on the active site loop 6 were observed to be similar, and a value of kex equal
to 3500 ± 200 s-1 was calculated for loop motion at 25 °C. Both of the backbone
chemical shifts of the unligated and G3P ligated chicken TIM were assigned by
solution state NMR (BMRB entries 15064 and 16065 respectively) (75). The
TROSY-detected longitudinal and transverse 15N spin relaxation experiments were
used to measure the rates R1 and R2, and to conform the significance of Pro166 on
loop6 by a quintuple mutant (PGG/GGG) (76). The TROSY-selected (TS) offresonance R1ρ experiment was performed and an exchange rate constant of 9000 s-1 at
25 °C was determined (75).
All of the studies went to the same conclusion that the rate of loop 6 motion is 103 –
104 s-1 and is highly dependent on the temperature; loop opening is the rate-limiting
step for the TIM catalysis in the direction from DHAP to GAP.
Simplified Two-Step Model for the TIM Motion and Brief
Definitions for the Chemical Exchange Time Scale Observed by
NMR
Optimally the catalysis of the enzyme with the substrate is of great interest, however
at the functional temperature or below this point, the rapid elimination of phosphate
from the substrate and the enzyme distortion introduced by the highly reactive
25
product methylglyoxal (61) largely limit the applicable temperature in the solid state
NMR experiments. This phenomenon has actually been observed in solid state NMR
by Rozovsky and McDermott (77) over long signal averaging or temperature increase,
which largely added to the difficulties in the sample preparation. Therefore attempts
have been made using substrate analogues to study the transition state (12, 31, 78-80).
A simplified two-step model was proposed for the enzyme in the presence of the
substrate analogue (72):
kclose
kon
E+S
koff
ESopen
kopen
ESclose
Scheme 1
In this model three forms of the enzyme are at equilibrium: the unligated free enzyme
(E), the encounter complex with the ligand bound to the enzyme while the loop 6
remains open (ESopen), and the complex with the ligand bound to the enzyme while
the loop 6 remains closed (ESclosed). The encounter complex ESopen is actually an
intermediate that is completely exposed to the bulk solvent. As confirmed by various
experimental techniques, one of the important functions of the closure of loop 6 is
suppressing the phosphate elimination, and a solvent-exposed intermediate is highly
vulnerable to the phosphate elimination (59, 81, 82). Therefore the population of
ESopen is minimal, and kopen/kclosed could be estimated to be less than 0.01 (kopen «
kclosed).
26
The relationship between the dissociation constant Kd and the elementary reaction
constants in the Scheme 1 can be described as:
Kd  (
k off
kon
)(
kopen
k closed
),
in which Kd ~ 1 x 10-3 M, while the kon value for the ligand association step ranges
between 109 M-1 s-1 and 1010 M-1 s-1 due to the diffusion limit under the experimental
conditions (9). The above equation can then be rearranged into the form:
k
koff  K d  kon  ( close ) .
kopen
Since kopen « kclosed as discussed, koff » Kd · kon ~ 106 s-1. As a result, the typical
experimental rate constant at the order of 103 – 104 s-1 can only be attributed to kopen
and the loop opening process was assigned to be the rate-determining step in the
simplified Scheme 1.
Solid state NMR is sensitive to chemical exchange on the time scales ranging from
microseconds to milliseconds. The TIM motion at temperatures near the
physiological range is exactly within this region. Assuming the enzyme is undergoing
chemical exchange between A conformer and B conformer at a certain temperature
with a chemical exchange rate constant kex (83-85), there is:
k1
A
k-1
B
.
For some specific spin, one expects an NMR detection at ω1 in units of angular
frequency when it’s in the A conformer, and at ω2 when it’s in the B conformer. The
chemical shift difference of them is Δω = ω1 – ω2.
27
kex = k1 + k-1
The populations of the spin in the two conformers are given by
pA 
k 1
k
 1
k1  k 1 kex
pB 
k1
k
 1
k1  k 1 kex
As a convention, the NMR time scales for two-site chemical exchange are defined by
the relative magnitude of kex and Δω. At the slow exchange range, which is when kex
< Δω, two resolved (depending on the linewidth) but broadened peaks should be
observed in NMR. At the intermediate exchange range, when the exchange rate is of
the order of the chemical shift separation between the two sites (kex ≈ Δω), one very
broad coalesced peak would appear. As it goes up to the fast exchange range (kex >
Δω), it would show one single peak at the position
ω = p1ω1 + p2ω2.
Motivation, Strategy and Goal
Although there have been a vast number of studies on the TIM reaction mechanisms,
kinetics, and dynamics, it still remains as a large-sized protein for solid state NMR
studies. Under in vivo conditions, TIM is surrounded by highly concentrated
glycolytic enzymes. Even in the viscous cellular environments TIM keeps its full
catalysis power. The previous solid state NMR approaches have been focused on one
single residue on the active site loop 6. Up to date, the protein-wide site specific
28
conformational change and dynamic studies on TIM were carried out by solution state
NMR. However in the solution environment, slow micro- to millisecond motions are
easily hidden by the fast Brownian overall tumbling. In addition, the relationship
between the behavior of the enzyme under solid-like environment and the one in
solution is still an open question. The dynamic studies on systems with high
molecular weight by solid state NMR are still very limited. However, it doesn’t make
it any less significant. In this work we aim to site specifically probe the TIM
conformational change upon ligand binding and to observe the chemical exchange
phenomena through SSNMR experiments.
29
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