Structural mutations that probe the interactions between the catalytic and dianion activation sites of triosephosphate isomerase

Article abstract of DOI:10.1021/bi401019h

Triosephosphate isomerase (TIM) catalyzes the isomerization of dihydroxyacetone phosphate to form d-glyceraldehyde 3-phosphate. The effects of two structural mutations in TIM on the kinetic parameters for catalysis of the reaction of the truncated substrate glycolaldehyde (GA) and the activation of this reaction by phosphite dianion are reported. The P168A mutation results in similar 50- and 80-fold decreases in (k_cat/K_m)_E and (k_cat/K_m)E·HP_i, respectively, for deprotonation of GA catalyzed by free TIM and by the TIM·HPO ₃^2- complex. The mutation has little effect on the observed and intrinsic phosphite dianion binding energy or the magnitude of phosphite dianion activation of TIM for catalysis of deprotonation of GA. A loop 7 replacement mutant (L7RM) of TIM from chicken muscle was prepared by substitution of the archaeal sequence 208-TGAG with 208-YGGS. L7RM exhibits a 25-fold decrease in (k_cat/K_m)_E and a larger 170-fold decrease in (k_cat/K_m)E·HP_i for reactions of GA. The mutation has little effect on the observed and intrinsic phosphodianion binding energy and only a modest effect on phosphite dianion activation of TIM. The observation that both the P168A and loop 7 replacement mutations affect mainly the kinetic parameters for TIM-catalyzed deprotonation but result in much smaller changes in the parameters for enzyme activation by phosphite dianion provides support for the conclusion that catalysis of proton transfer and dianion activation of TIM take place at separate, weakly interacting, sites in the protein catalyst.

Full text of DOI:10.1021/bi401019h

Article
pubs.acs.org/biochemistry
Structural Mutations That Probe the Interactions between the
Catalytic and Dianion Activation Sites of Triosephosphate Isomerase
Xiang Zhai,† Tina L. Amyes,† Rik K. Wierenga,‡ J. Patrick Loria,§ and John P. Richard*,†
†Department of Chemistry, University at Buffalo, Buffalo, New York 14260, United States
‡Department of Biochemistry and Biocenter Oulu, University of Oulu, P.O. Box 3000, FIN-90014 Oulu, Finland
§Department of Chemistry and Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut
06520, United States
S
* Supporting Information
ABSTRACT: Triosephosphate isomerase (TIM) catalyzes
the isomerization of dihydroxyacetone phosphate to form D-
glyceraldehyde 3-phosphate. The effects of two structural
mutations in TIM on the kinetic parameters for catalysis of the
reaction of the truncated substrate glycolaldehyde (GA) and
the activation of this reaction by phosphite dianion are
reported. The P168A mutation results in similar 50- and 80-
fold decreases in (kcat/Km)E and (kcat/Km)E·HP , respectively, for
i
deprotonation of GA catalyzed by free TIM and by the TIM·
2−
HPO3 complex. The mutation has little effect on the
observed and intrinsic phosphite dianion binding energy or the magnitude of phosphite dianion activation of TIM for catalysis of
deprotonation of GA. A loop 7 replacement mutant (L7RM) of TIM from chicken muscle was prepared by substitution of the
archaeal sequence 208-TGAG with 208-YGGS. L7RM exhibits a 25-fold decrease in (kcat/Km)E and a larger 170-fold decrease in
(kcat/Km)E·HP for reactions of GA. The mutation has little effect on the observed and intrinsic phosphodianion binding energy
i
and only a modest effect on phosphite dianion activation of TIM. The observation that both the P168A and loop 7 replacement
mutations affect mainly the kinetic parameters for TIM-catalyzed deprotonation but result in much smaller changes in the
parameters for enzyme activation by phosphite dianion provides support for the conclusion that catalysis of proton transfer and
dianion activation of TIM take place at separate, weakly interacting, sites in the protein catalyst.
allowing the substrate access to the enzyme active site. The
riosephosphate isomerase (TIM) catalyzes the stereo-
Tspecific and reversible conversion of dihydroxyacetone binding of substrate to TIM is followed by loop closure that
sequesters the ligand from interaction with solvent water and
strips several solvent molecules from the enzyme active site.27
The closure of loop 6 is accompanied by a conformational
change of loop 7, and this loop closure is driven energetically by
formation of hydrogen bonds between the backbone amides of
Gly171 of loop 6 and Ser211 of loop 7 to the ligand
phosphodianion, along with the formation of interloop
hydrogen bonds between loop 6 and the 208-YGGS motif of
loop 7.27−29
Two experiments provide reinforcing pieces of evidence that
interactions between loop 6 of TIM and the substrate
phosphodianion activate TIM for catalysis of isomerization.
(1) Truncation of residues Ile170−Gly173 at the tip of loop 6
of cTIM and the introduction of a peptide bond between
Ala169 and Lys174 disrupt loop−phosphodianion interactions
but should not affect the protein fold.22 The 105-fold decrease
in kcat and the much smaller 2.3-fold increase in Km determined
for isomerization of GAP catalyzed by this loop deletion mutant
phosphate (DHAP) to (R)-glyceraldehyde 3-phosphate
(GAP),1,2 by a proton transfer mechanism through enzyme-
bound cis-enediolate reaction intermediates (Scheme 1). The
a
carboxylate anion side chain of Glu165/167 functions as a
Brønsted base to abstract a proton from the α-carbonyl carbon
of the bound substrate,3−6 and the developing negative charge
at the carbonyl carbon is stabilized by hydrogen bonding to the
neutral imidazole side chain of His95.7−9 The isomerization
reaction is completed by reprotonation of the enediolate
intermediate at the adjacent carbon (Scheme 1).
The results of more than 50 years of studies of TIM have
served to define what is known about the mechanism for
enzymatic catalysis of deprotonation of carbon, and the
important problems that remain to be resolved.1,10−14 It was
suggested that catalysis by TIM is “not different, just better”
than catalysis by small molecules.15 TIM is “better” than small
molecules in the sense that the catalytic side chains of TIM are
activated for catalysis at the enzyme active site.16−21 The 11-
a
residue (166−176) flexible phosphodianion gripper loop 6 of
TIM from chicken muscle (cTIM) plays an important role in
Received: July 28, 2013
Published: August 2, 2013
enzyme activation.22−26 Loop 6 is open at unliganded TIM,
© 2013 American Chemical Society
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Scheme 1
of cTIM show that the full loop 6 is required to observe robust
turnover of the bound substrate. (2) Truncation of the
phosphodianion from GAP leads to a 109-fold reduction in
kcat/Km for TIM-catalyzed isomerization of the minimal neutral
substrate glycolaldehyde (GA).30 The binding of phosphite
dianion to TIM results in an ∼1000-fold increase in the
catalytic activity of TIM toward toward deprotonation of GA.30
A comparison of the TIM-catalyzed reactions of the whole
substrate GAP and the substrate pieces shows that 50% of the
total 11 kcal/mol intrinsic binding energy of the phosphodian-
ion of GAP may be recovered as phosphite dianion activation of
the reaction of GA.30
The determination of the mechanism for phosphite dianion
activation of TIM is a difficult and significant problem, whose
solution is broadly relevant to the mechanism of phosphite
dianion activation of several enzymatic reactions, including
enzyme-catalyzed decarboxylation,31 proton transfer,32,33 hy-
dride transfer,34 phosphoryl transfer,35 and reductoisomeriza-
tion reactions,36 and to the more general problem of the
mechanism for the utilization of the intrinsic substrate binding
energy in stabilizing the transition states for enzymatic
reactions.37,38 We report here the results of a study of the
effect of two structural mutations of TIM on enzyme activation
by phosphite dianion.
We report here that the 208-TGAG for 208-YGGS loop 7
replacement mutation of cTIM and the P168A mutation of
TbbTIM result in decreases in the kinetic parameters for
enzyme-catalyzed deprotonation of [1-13C]GA in the absence
2−
and presence of HPO3 that are similar to the ∼100-fold
decreases in kcat for the reaction of the whole substrate GAP.
Both the P168A and loop 7 replacement mutant enzyme-
catalyzed reactions of [1-13C]GA are strongly activated by
phosphite dianion, and the activation is similar to that for the
corresponding wild-type enzyme. The observation that these
structural mutations cause a significant decrease in the reactivity
of TIM toward catalysis of deprotonation of [1-13C]GA but
have little effect on enzyme activation by phosphite dianion
shows that it is possible to modify the catalytic activity at the
site that conducts carbon deprotonation without severely
affecting enzyme activation at the dianion binding site.
EXPERIMENTAL PROCEDURES
■
Materials. Rabbit muscle α-glycerol-3-phosphate dehydro-
genase and glyceraldehyde-3-phosphate dehydrogenase were
purchased from Sigma. These enzymes were exhaustively
dialyzed against 20 mM triethanolamine buffer (pH 7.5) at 7
°C prior to being used in coupled enzyme assays. Bovine serum
albumin was purchased from Roche. DEAE Sepharose Fast
Flow was purchased from GE Healthcare. D,L-Glyceraldehyde-
3-phosphate diethyl acetal (barium salt), dihydroxyacetone
phosphate (lithium salt), NADH (disodium salt), Dowex
50WX4-200R, triethanolamine hydrochloride, and imidazole
were purchased from Sigma. Hydrogen arsenate heptahydrate
and sodium phosphite (dibasic, pentahydrate) were purchased
from Fluka and were dried under vacuum prior to use.30
[1-13C]Glycolaldehyde (99% enrichment of 13C at C-1, 0.09 M
in water) was purchased from Omicron Biochemicals.
Deuterium oxide (99% D) and deuterium chloride [35% (w/
w), 99.9% D] were purchased from Cambridge Isotope
Laboratories. Sodium 2-phosphoglycolate was synthesized by
following a published procedure.42 Imidazole was recrystallized
from benzene. All other chemicals were reagent grade or better
and were used without further purification.
The 208-YGGS sequence at residues 208−211 is generally
observed for loop 7 of TIM.29 The 208-TGAG sequence
commonly found at archaeal organisms has been substituted for
208-YGGS of cTIM, and the resulting loop 7 replacement
mutant (L7RM) of cTIM exhibits a 200-fold decrease in kcat/
Km for isomerization of GAP and DHAP.28 TROSY-Hahn
40
Echo39 and TROSY-selected R1p experiments indicate that
this mutation of loop 7 results in a doubling of the rate of
conformational exchange associated with active site loop
motion and a reduction in the coordinated motion of loop 6
relative to that of wild-type TIM.28 These results provide
evidence that interactions between loops 6 and 7 are necessary
to ensure the proper chemical environment for the enzymatic
reaction and that interloop interactions play a significant role in
modulating the chemical dynamics near the active site.28 The
P168A mutant of TIM from Trypanosoma brucei brucei
(TbbTIM) was targeted for study, because of the expectation
that the excellent high-resolution X-ray crystal structures for the
free and liganded P168A mutant enzyme would provide a
structure-based rationalization for the effect of the mutation on
the kinetic parameters for enzyme activation by phosphite
dianion.41
Preparation of Enzymes. Expression vectors (pET-15b)
containing genes encoding wild-type and L7RM cTIM were
available from a previous study.28 These plasmids were
introduced into the TIM-deficient tpiA− λDE3 lysogenic strain
of Escherichia coli, FB215471(DE3), which was a generous gift
from B. Miller.43 Cells were grown overnight at 37 °C in 200−
300 mL of Luria broth that contained 100 μg/mL ampicillin
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and 40 μg/mL kanamycin. These cultures were diluted into 6 L
of the same medium and incubated at 37 °C until an OD600 of
0.6 was reached. Protein expression was then induced by the
addition of 0.6 mM isopropyl 1-thio-D-galactoside (IPTG)
followed by incubation for an additional 6 h. The cells were
harvested, suspended in 25 mM Tris-HCl (pH 8.0, 25 mL), and
stored at −80 °C. Wild-type and L7RM cTIMs were purified
according to a published procedure.44 Fractions from the final
DEAE-Sepharose ion exchange column that were judged to be
homogeneous by gel electrophoresis were pooled, concen-
trated, and stored in 25 mM Tris-HCl (pH 8.0) at an ionic
strength of 0.1 (NaCl) containing 20% glycerol at −80 °C. The
protein concentration was determined from the absorbance at
280 nm using extinction coefficients of 33500 M−1 cm−1 for
wild-type cTIM and 32000 M−1 cm−1 for L7RM cTIM that
were calculated using the ProtParam tool available on the
Expasy server.45,46
The pET3a plasmid containing the gene encoding P168A
mutant TbbTIM was prepared in an earlier study.41 P168A
mutant TbbTIM was overexpressed in E. coli BL21 pLys S
grown in LB medium at 18 °C and purified by chromatography
over a CM Sepharose column, as described previously.47
Fractions that were judged to be homogeneous by gel
electrophoresis were pooled, concentrated, and stored at −80
°C in 25 mM TEA (pH 8.0) that contains 20% glycerol at an
ionic strength of 0.15 (NaCl). The protein concentration was
determined from the absorbance at 280 nm using an extinction
coefficient of 35000 M−1 cm−1 calculated using the ProtParam
tool available on the Expasy server.45,46
General Methods. The solution pH or pD was determined
at 25 °C using an Orion model 720A pH meter equipped with
a radiometer pHC4006-9 combination electrode that was
standardized at pH 7.0 and 10.0. Values of pD were calculated
by adding 0.40 to the observed reading of the pH meter.48
Stock solutions of D,L-glyceraldehyde 3-phosphate (D,L-GAP)
were prepared by hydrolysis of the corresponding diethyl acetal
(barium salt) using Dowex 50WX4-200R (H+ form) in boiling
H2O, as described previously.14 The resulting solutions were
stored at −20 °C and were adjusted to pH 7.5 by the addition
of 1 M NaOH prior to use in enzyme assays. Stock solutions of
buffers,30,49 phosphite, [1-13C]glycolaldehyde ([1-13C]GA),49
and 2-phosphoglycolate (PGA)16 were prepared as described in
previous work.
the initial velocity of TIM-catalyzed isomerization in the
presence of two fixed concentrations of PGA. In these assays,
the amount of α-glycerol-3-phosphate dehydrogenase coupling
enzyme was increased to overcome its inhibition by PGA.
Values of kcat and Km for TIM-catalyzed isomerization of DHAP
and the values of Ki for competitive inhibition by arsenate were
determined by examining the effect of increasing concen-
trations of DHAP on the initial velocity of TIM-catalyzed
isomerization in the presence of 2, 5, and 10 mM arsenate.
1
1H NMR Analyses. H NMR spectra at 500 MHz were
recorded in D2O at 25 °C using a Varian Unity Inova 500
spectrometer that was shimmed to give a line width of ≤0.5 Hz
for the most downfield peak of the double triplet due to the C-
1 proton of the hydrate of [1-13C]GA.49 Spectra (16−64
transients) were obtained using a sweep width of 6000 Hz, a
pulse angle of 90°, and an acquisition time of 6 s. A total
relaxation delay of 120 s (>8T1) between transients was used to
ensure that accurate integrals were obtained for the protons of
interest.51,52 Baselines were subjected to a first-order drift
correction before determination of integrated peak areas.
Chemical shifts are reported relative to that for HOD at 4.67
ppm.
TIM-Catalyzed Reactions of [1-13C]GA in D2O Moni-
1
tored by H NMR. The unactivated and phosphite dianion-
activated reactions of [1-13C]GA catalyzed by wild-type cTIM,
L7RM cTIM, and P168A mutant TbbTIM in D2O at 25 °C
were monitored by 1H NMR spectroscopy, as described
previously.49 The enzymes were exhaustively dialyzed against
30 mM imidazole buffer (20% free base) in D2O at pD 7.0
[ionic strength of 0.1 (NaCl)] for reactions in the absence of
phosphite dianion, or against 30 mM imidazole buffer (20%
free base) in D2O at pD 7.0 (ionic strength of 0.024) for
reactions in the presence of phosphite dianion. The unactivated
TIM-catalyzed reactions of [1-13C]GA in the absence of
phosphite dianion were initiated by the addition of enzyme to
give reaction mixtures (850 μL) containing 20 mM [1-13C]GA,
20 mM imidazole (20% free base) in D2O at pD 7.0 and an
ionic strength of 0.1, and 0.34 mM wild-type cTIM, 0.48 mM
L7RM cTIM, or 0.22−0.49 mM P168A TbbTIM. The TIM-
catalyzed reactions of [1-13C]GA in the presence of phosphite
dianion were initiated by the addition of enzyme to give
reaction mixtures (850 μL) containing 20 mM [1-13C]GA, 20
2−
mM imidazole (20% free base), up to 20 mM HPO3 (1:1
Enzyme Assays. All enzyme assays were conducted at pH
7.5, 25 °C, and an ionic strength of 0.1 (NaCl) according to
procedures described in our previous work. α-Glycerol-3-
phosphate dehydrogenase was assayed by monitoring the
oxidation of NADH by DHAP,14 and glyceraldehyde-3-
phosphate dehydrogenase was assayed by monitoring the
enzyme-catalyzed reduction of NAD+ by GAP.20 The TIM-
catalyzed isomerization of GAP was monitored by coupling the
formation of the product DHAP to the oxidation of NADH
catalyzed by α-glycerol-3-phosphate dehydrogenase.14,49 The
TIM-catalyzed isomerization of DHAP was monitored by
coupling the formation of the product GAP to the reduction of
NAD+ catalyzed by glyceraldehyde-3-phosphate dehydrogenase
in the presence of 2−10 mM arsenate.20,50
Values of kcat and Km for TIM-catalyzed isomerization of
GAP were determined from the nonlinear least-squares fit of
the initial velocity data to the Michaelis−Menten equation.
Values of Ki for competitive inhibition of TIM by PGA at pH
7.5 [ionic strength of 0.1 (NaCl)] were determined by
examining the effect of increasing concentrations of GAP on
dianion:monoanion) in D2O at pD 7.0 and an ionic strength of
0.1 (NaCl), and 9−30 μM wild-type cTIM, 90−520 μM L7RM
cTIM, or 60−350 μM P168A TbbTIM. In each case, 750 μL of
1
the reaction mixture was transferred to an NMR tube, the H
NMR spectrum was recorded immediately, and spectra were
then recorded at regular intervals. The remaining reaction
mixture was incubated at 25 °C and was used to conduct
periodic assays of the activity of TIM toward catalysis of
isomerization of GAP. No significant loss of TIM activity was
observed during any of these reactions that were followed for
up to 7 days. The reactions in the absence of phosphite dianion
were followed until they were 70−80% complete, and the
reactions in the presence of phosphite dianion were typically
followed until they were 40% complete, after which the protein
was removed by ultrafiltration and the solution pD determined.
There was no significant change in pD during any of these
reactions.
1H NMR analyses of the products of these reactions were
conducted by monitoring the signals for the hydrates of the
products [2-13C]GA, [2-13C,2-2H]GA, [1-13C,2-2H]GA, and
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Table 1. Kinetic Parameters for Isomerization of GAP and DHAP Catalyzed by Wild-Type and Mutant Forms of
a
Triosephosphate Isomerase from Chicken Muscle and T. brucei brucei at pH 7.5 and 25 °C
b
c
GAP
DHAP
kcat/Km
kcat/Km
Ki (mM)
e
d
TIM
kcat (s−1
3200 100
16
2100
24
)
Km (mM)
(M−1 s−1
)
Ki (mM) PGA
kcat (s−1
340
)
Km (mM)
(M−1 s−1
)
arsenate
wild-type cTIM
L7RM cTIM
0.29 0.02
0.27 0.02
0.25
1.1 × 107
5.9 × 104
8.4 × 106
2.6 × 105
0.019 0.001
2.3 0.1
5
0.59 0.05
4.0 0.2
0.70
5.8 × 105
2.0 × 103
4.3 × 105
1.3 × 104
9.6 1.6
3.8 0.3
4.6
1
8.0 0.5
300
f
g
wild-type TbbTIM
P168A TbbTIM
0.055
1
0.091 0.005
0.14 0.01
6.5 0.1
0.49 0.02
10
1
a
Under standard assay conditions of 30 mM triethanolamine buffer at pH 7.5, 25 °C, and an ionic strength of 0.1 (NaCl). The kinetic parameters
b
have been calculated using the total concentration of the free carbonyl and hydrated forms of GAP or DHAP. The errors for TIM-catalyzed
isomerization of GAP were determined from the average of kinetic parameters determined in two experiments. The errors for TIM-catalyzed
isomerization of DHAP are the standard deviations determined from the nonlinear least-squares fits of the kinetic data. The initial velocity of the
c
d
isomerization of several concentrations of GAP was determined in the presence of 0.021 and 0.053 mM PGA for wild-type cTIM, 1.9 and 4.9 mM
e
PGA for L7RM cTIM, and 0.13 and 0.39 mM PGA for P168A TbbTIM. The initial velocity of the isomerization of several concentrations of DHAP
f
g
was determined in the presence of 2, 5, and 10 mM arsenate. Data from ref 54, unless noted otherwise. From ref 19.
Scheme 2
[1-13C,2,2-di-2H]GA, and the fractional yields of these
products, f P, were determined at several times during the first
20−40% of the reaction, as described previously.49 Observed
first-order rate constants for the disappearance of [1-13C]GA,
kobs (s−1), were determined from the slopes of linear
semilogarithmic plots of reaction progress against time covering
the first 30−40% of the reaction, according to eq 1
inhibition of these TIMs by 2-phosphoglycolate (PGA) were
determined from the dependence of the initial velocity of
isomerization of GAP at several concentrations of GAP at two
different fixed concentrations of PGA. These values of Ki
(Table 1) were obtained from the nonlinear least-squares fit
of the initial velocity data to eq 3, where [I] = [PGA], and using
the values of Km listed in Table 1. Arsenate is an activator of
glyceraldehyde-3-phosphate dehydrogenase, which was used as
the coupling enzyme in assays of the TIM-catalyzed isomer-
ization of DHAP to give GAP.20,50 The initial velocity of the
isomerization of several concentrations of DHAP catalyzed by
wild-type cTIM, L7RM cTIM, and P168A TbbTIM at pH 7.5,
25 °C, and an ionic strength of 0.1 (NaCl) was determined in
the presence of 2, 5, and 10 mM arsenate. Values of kcat and Km
for the isomerization of DHAP, along with values of Ki for
arsenate, were obtained from the nonlinear least-squares fit of
the initial velocity data to a modified form of eq 3
ln fS = − kobs
t
(1)
where f S is the fraction of [1-13C]GA remaining at time t. The
observed second-order rate constants for the disappearance of
[1-13C]GA, (kcat/Km)obs (M−1 s−1), were calculated from the
values of kobs using eq 2
kobs
(1 − fhyd )[E]
(kcat/Km)obs
=
(2)
where fhyd (=0.94) is the fraction of [1-13C]GA present in the
unreactive hydrate form and [E] is the concentration of TIM.30
vi
[E]
kcat[GAP]
[GAP] + Km(1 + [I]/Ki)
=
(3)
RESULTS
■
where [I] = [arsenate] for the substrate DHAP, and are
reported in Table 1.
Kinetic Parameters and Inhibition Constants. Kinetic
parameters kcat and Km for the isomerization of GAP catalyzed
by wild-type cTIM, L7RM cTIM, and P168A TbbTIM at pH
7.5, 25 °C, and an ionic strength of 0.1 (NaCl) were
determined by Michaelis−Menten analyses of initial velocities
and are reported in Table 1. Values of Ki for competitive
TIM-Catalyzed Reactions of [1-13C]GA in D2O. The
disappearance of the substrate and the formation of the four
identifiable products, shown in the boxes in Scheme 2, from the
reaction of the truncated neutral substrate [1-13C]GA catalyzed
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by wild-type cTIM, L7RM cTIM, and P168A TbbTIM in D2O
buffered by 20 mM imidazole at pD 7.0, 25 °C, and an ionic
strength of 0.1 (NaCl) were monitored by 1H NMR
spectroscopy for up to 7 days, as described previously.20,49
The fractional yields of products [2-13C]GA, [2-13C,2-2H]GA,
[1-13C,2-2H]GA, and [1-13C,2,2-di-2H]GA, f P, were determined
by 1H NMR analysis at five different times during the
disappearance of the first 20−40% of [1-13C]GA, as described
previously.20,49 The observed first-order rate constants for the
disappearance of [1-13C]GA in these reactions, kobs (s−1), were
determined as the slopes of semilogarithmic plots of reaction
progress versus time, according to eq 1. The observed second-
order rate constants for the disappearance of [1-13C]GA, (kcat/
Km)obs (M−1 s−1), were calculated from the values of kobs using
eq 2.
these were calculated from the observed second-order rate
constants (kcat/Km)obs using eq 4
⎛
⎞
kcat
Km
kcat
=
(f )
E
P
⎜
⎝
⎟
⎠
∑
K
m
(4)
obs
with ∑(f P)E = 1.0, where ∑(f P)E is the sum of the fractional
yields of these specific products. A second-order rate constant
kcat/Km of 0.1 M−1 s−1 for the unactivated reaction in the
absence of phosphite dianion was calculated using eq 4 with a
(kcat/Km)obs of 0.19 M−1 s−1 and a ∑(f P)E of 0.5 reported
previously.49
Figure 1A shows the dependence of kcat/Km (M−1 s−1) for
wild-type cTIM-catalyzed reactions of [1-13C]GA on the
Wild-Type cTIM-Catalyzed Reactions of [1-13C]GA in
D2O. The relatively rapid phosphite-activated reactions of
[1-13C]GA catalyzed by wild-type cTIM in D2O in the presence
2−
of ≥2.0 mM HPO3
result in essentially quantitative
conversion of the substrate to the three products shown in
Scheme 2A: [2-13C]GA, [2-13C,2-2H]GA, and [1-13C,2-2H]GA
(Table S1 of the Supporting Information).49 By contrast, the
very slow unactivated reaction of [1-13C]GA in the presence of
wild-type cTIM gives the three products observed for the
phosphite-activated reaction (Scheme 2A), along with a
significant 19% yield of [1-13C,2,2-di-2H]GA that forms in a
nonspecific protein-catalyzed reaction that occurs outside the
active site of TIM (Scheme 2B), and the total yield of the four
identifiable products is only ∼60% (Table S1 of the Supporting
Information).49 We proposed previously that [1-13C,2,2-
di-2H]GA forms by a slow protein-catalyzed reaction that
involves deuterium exchange into [1-13C]GA catalyzed by the
amine side chains of surface lysine residues (Scheme 2B).53
The absolute fractional yields of the products of the unactivated
and phosphite dianion-activated wild-type cTIM-catalyzed
reactions, fP, are reported in Table S1 of the Supporting
Information. There is good agreement between the fractional
product yields from the phosphite dianion-activated reactions
determined here and those reported previously.49 However, the
normalized yields of 45% [2-13C,2-2H]GA and 42%
[1-13C,2-2H]GA (Table S1 of the Supporting Information)
from the specific reaction of [1-13C]GA at the active site of
wild-type cTIM determined here are smaller and larger,
respectively, than those of 60 and 30% reported previously.49
We conclude that, in our hands, the uncertainty in the yields of
the products of the unactivated wild-type cTIM-catalyzed
reactions of [1-13C]GA is larger than that for the phosphite
dianion-activated reactions. These unactivated reactions of
[1-13C]GA in the presence of TIM are very slow, were
monitored for several days, and give only low yields of
[2-13C,2-2H]GA and [1-13C,2-2H]GA (Table S1 of the
Supporting Information). In addition, the signals of the
multiplet due to the C-2 proton of [1-13C,2-2H]GA are not
fully resolved from the very large doublet due to the C-2 proton
of the substrate [1-13C]GA, which complicates the reliable
determination of the yield of this product.49
Figure 1. Dependence of the second-order rate constants kcat/Km for
the TIM-catalyzed turnover of the free carbonyl form of [1-13C]GA in
D2O on [HPO32−] at pD 7.0, 25 °C, and an ionic strength of 0.1
(NaCl). The data were fit to eq 5 derived for the model shown in
Scheme 3: (A) wild-type cTIM, (B) L7RM cTIM, and (C) P168A
TbbTIM.
concentration of added phosphite dianion (HPO32−).30 The
solid line shows the nonlinear least-squares fit of these data to
eq 5, derived for Scheme 3 (S = [1-13C]GA), with (kcat/Km)E =
0.1 M−1 s−1 for the unactivated reaction in the absence of
Table S1 of the Supporting Information gives the true
second-order rate constants, kcat/Km (M−1 s−1), for the wild-
type cTIM-catalyzed reaction of the carbonyl form of
[1-13C]GA to give the specific products of the TIM-catalyzed
reaction [2-13C]GA, [2-13C,2-2H]GA, and [1-13C,2-2H]GA
(Scheme 2A). For the reactions in the presence of phosphite,
phosphite. The data give a (kcat/Km)E·HP of 65 M−1 s−1 for
i
turnover of [1-13C]GA by the phosphite-liganded enzyme and a
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[2-13C,2-2H]GA and ∼0.2% [2-13C]GA show that L7RM
cTIM maintains only a low activity for catalysis of the reactions
of [1-13C]GA at the enzyme active site (Scheme 2A).53 The
observed yield of 3.3% [1-13C,2-2H]GA is formed by both the
specific reaction shown in Scheme 2A and the nonspecific
reaction shown in Scheme 2B. A yield of ∼1.1%
[1-13C,2-2H]GA from only the specific reaction was estimated
with the assumption that the ratio of the yields of
[1-13C,2-2H]GA and [2-13C,2-2H]GA from the unactivated
reaction is the same as that for the phosphite-activated
reactions (see Table S2 of the Supporting Information).
Table 2 gives the observed second-order rate constants (kcat/
Km)obs for the L7RM cTIM-catalyzed reactions of [1-13C]GA
and the true second-order rate constants kcat/Km for the specific
reactions at the active site of L7RM cTIM that were calculated
from the values of (kcat/Km)obs using eq 4 with the values of
∑(fP) listed in Table 2, where ∑(f P)E is the sum of the
fractional yields of these specific products (Table S2 of the
Supporting Information).
Scheme 3
Kd of 11 mM for binding of phosphite dianion to the free
enzyme (Table 3).30 Table 3 also gives the corresponding
kinetic parameters for the reactions of [1-13C]GA catalyzed by
wild-type TbbTIM that were determined previously.54
⎛
⎜
⎝
⎞
⎟
⎠
Kd
kcat/Km
=
(kcat/Km)E
2−
K + [HPO
]
d
3
2−
⎛
⎞
⎟
⎠
[HPO3
]
⎜
⎝
+
(kcat/Km)E·HP
i
2−
K + [HPO
]
(5)
d
3
Figure 1B shows the dependence of kcat/Km (M−1 s−1) for the
L7RM cTIM-catalyzed reactions of [1-13C]GA on the
concentration of added phosphite dianion (HPO32−).30 The
solid line shows the nonlinear least-squares fit of these data to
eq 5, with a (kcat/Km)E of 0.0045 M−1 s−1 for the unactivated
reaction in the absence of phosphite (Table 2). The data give a
L7RM cTIM-Catalyzed Reactions of [1-13C]GA in D2O.
The 208-TGAG for 208-YGGS loop 7 replacement mutation of
cTIM results in a decrease in the midpoint for thermal
denaturation from 60 °C for the wild type to 49 °C for L7RM
cTIM.28 However, despite this, we find that L7RM cTIM
maintains full catalytic activity over a period of 7 days during
the reaction of 20 mM [1-13C]GA at pD 7.0, 25 °C, and an
ionic strength of 0.1 (NaCl).
(kcat/Km)E·HP of 0.39 M−1 s−1 for turnover of [1-13C]GA by the
i
phosphite-liganded enzyme, and Kd = 4.1 mM for binding of
phosphite dianion to the free enzyme (Table 3).30 The upper
limit for (kcat/Km)E, calculated using eq 4 with the assumption
that [1-13C,2-2H]GA forms exclusively by the specific pathway
shown in Scheme 2A, is ≤0.008 M−1 s−1.
Table S2 of the Supporting Information gives the fractional
yields of the four identifiable products from the unactivated and
phosphite dianion-activated L7RM cTIM-catalyzed reactions of
[1-13C]GA that are shown in Scheme 2. The major product of
the unactivated reaction of [1-13C]GA in the absence of
phosphite dianion is [1-13C,2,2-di-2H]GA, which is formed in a
yield of 27% by the nonspecific protein-catalyzed reaction
shown in Scheme 2B. The very small yields of 1.5%
P168A TbbTIM-Catalyzed Reactions of [1-13C]GA in
D2O. Table S3 of the Supporting Information gives the
fractional yields of the four identifiable products from the
unactivated and phosphite dianion-activated P168A TbbTIM-
Table 2. Second-Order Rate Constants for the Reactions of [1-13C]GA in D2O Catalyzed by L7RM cTIM and P168A TbbTIM in
a
the Absence and Presence of Phosphite Dianion in D2O at 25 °C
L7RM cTIM
P168A TbbTIM
c
d
c
d
2−
2−
[HPO3
]
[TIM]
(mM)
(kcat/Km)obs
(kcat/Km)
[HPO3
]
[TIM]
(mM)
(kcat/Km)obs
(kcat/Km)
b
b
(mM)
∑(f P)E
(M−1 s−1
)
(M−1 s−1
)
(mM)
∑(f P)E
(M−1 s−1
)
(M−1 s−1
)
e
e
0
1.0
2.0
3.0
4.0
5.0
7.5
10
13
15
18
20
20
0.48
0.34
0.52
0.37
0.24
0.21
0.30
0.17
0.14
0.15
0.10
0.090
0.10
0.03
0.15
0.24
0.24
0.28
0.32
0.39
0.40
0.40
0.47
0.46
0.54
0.56
0.58
0.0045
0.089
0.15
0.17
0.19
0.23
0.24
0.26
0.29
0.29
0.34
0.34
0.34
0
0
0.22
0.49
0.35
0.30
0.25
0.20
0.12
0.12
0.080
0.070
0.060
0.060
0.14
0.02
0.07
0.06
0.11
0.15
0.21
0.34
0.38
0.50
0.57
0.62
0.77
0.75
0.68
0.0014
0.0012
0.047
0.090
0.15
e
0.37
0.64
0.59
0.59
0.59
0.61
0.65
0.61
0.62
0.63
0.61
0.59
0.02
0.5
1.0
2.0
4.0
5.0
7.5
10
13
15
18
20
0.43
0.60
0.70
0.65
0.71
0.70
0.71
0.66
0.67
0.70
0.80
0.22
0.27
0.35
0.40
0.41
0.52
0.53
0.54
a
Determined by 1H NMR analysis of the reaction of 20 mM [1-13C]GA in D2O at pD 7.0 (20 mM imidazole), 25 °C, and an ionic strength of 0.1
b
(NaCl). The sum of the yields of the specific products [2-13C]GA, [2-13C,2-2H]GA, and [1-13C,2-2H]GA from the reaction of [1-13C]GA at the
c
enzyme active site (Scheme 2A), taken from Tables S2 and S3 of the Supporting Information. Observed second-order rate constant for the TIM-
d
catalyzed reactions of [1-13C]GA, calculated from the observed first-order rate constant using eq 2. Second-order rate constant for the specific TIM-
catalyzed reaction of [1-13C]GA to give the products shown in Scheme 2A, calculated from (kcat/Km)obs for the disappearance of [1-13C]GA using eq
e
4. Calculated using the estimated yield of [1-13C,2-2H]GA formed by the specific reaction shown in Scheme 2A listed in Tables S2 and S3 of the
Supporting Information.
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Table 3. Kinetic Parameters for the Unactivated and Phosphite-Activated Reactions of the Free Carbonyl Form of [1-13C]GA
Catalyzed by Wild-Type and Mutant Forms of Triosephosphate Isomerase from Chicken Muscle and T. brucei brucei in D2O at
a
25 °C (Scheme 3)
b
c
i
d
e
f
(kcat/Km)E (M−1 s−1
)
(kcat/Km)E·HP (M−1 s−1
)
Kd (mM) Kd (mM) (kcat/Km)E·HP /(kcat/Km)E (kcat/Km)E·HP /Kd (M−2 s−1
)
‡
TIM
i
i
g
wild-type cTIM
L7RM cTIM
0.1
65
4
11 1.3
4.1 0.5
19
0.017
0.042
0.021
0.016
650
100
900
600
5900
95
h
0.0045 (<0.008)
0.39 0.02
64
i
wild-type TbbTIM
P168A TbbTIM
0.07
3400
h
0.0013 (<0.01)
0.83 0.05
10 1.3
83
a
Determined by 1H NMR analysis of the reaction of 20 mM [1-13C]GA in D2O at pD 7.0 (20 mM imidazole), 25 °C, and an ionic strength of 0.1
b
c
(NaCl). Second-order rate constant for the unactivated TIM-catalyzed reaction of [1-13C]GA in the absence of phosphite dianion. Second-order
d
rate constant for the reaction of [1-13C]GA catalyzed by the phosphite-liganded enzyme. Dissociation constant for binding of phosphite dianion to
e
the free enzyme. Dissociation constant for release of phosphite dianion from the transition state complex, calculated using eq 6 derived for Scheme
f
g
4. Third-order rate constant for the phosphite-activated TIM-catalyzed reaction of [1-13C]GA. Calculated from data reported in ref 49 (see Table
h
S1 of the Supporting Information). Upper limit for (kcat/Km)E, calculated using eq 4 with the assumption that [1-13C,2-2H]GA forms exclusively by
i
the specific pathway shown in Scheme 2A. Data from ref 54.
catalyzed reactions of [1-13C]GA that are shown in Scheme 2.
DISCUSSION
■
The major product of the unactivated reaction of [1-13C]GA in
The TIMs from T. brucei brucei (TbbTIM) and chicken muscle
the absence of phosphite dianion is [1-13C,2,2-di-2H]GA, which
is formed in a yield of 33% by the nonspecific protein-catalyzed
pathway shown in Scheme 2B. There is only a small yield of
1.0% [2-13C,2-2H]GA and no detectable formation of
[2-13C]GA. The observed yield of 11% [1-13C,2-2H]GA from
both the specific reaction shown in Scheme 2A and the
nonspecific reaction shown in Scheme 2B is substantially larger
than the 3.3% yield of this product from the corresponding
L7RM cTIM-catalyzed reaction, but similar to the 10% yield
observed for the reaction catalyzed by a monomeric variant of
TbbTIM, for which there is no detectable activation by
phosphite dianion.54 These results are consistent with a
relatively high activity of TbbTIM for catalysis of deuterium
exchange by the nonspecific pathway shown in Scheme 2B.
TbbTIM has an isoelectric point of 9.8,55 which is 3−4 units
higher than that for the TIMs from yeast55 and rabbit muscle.55
We suggest that the greater reactivity of TbbTIM in the
pathway shown in Scheme 2B reflects a relatively large surface
density of the amine side chains of lysine residues. A yield of
∼1% [1-13C,2-2H]GA from only the specific reaction was
estimated with the assumption that the ratio of the yields of
[1-13C,2-2H]GA and [2-13C,2-2H]GA from the unactivated
reaction is the same as that for the phosphite-activated
reactions (see Table S3 of the Supporting Information).
Table 2 gives the observed second-order rate constants, (kcat/
Km)obs, for the P168A TbbTIM-catalyzed reactions of
[1-13C]GA and the true second-order rate constants, kcat/Km,
for the specific reactions at the active site of P168A TbbTIM
that were calculated from the values of (kcat/Km)obs using eq 4
with the values of ∑(fP) listed in Table 2, where ∑(f P)E is the
sum of the fractional yields of these specific products (Table S3
of the Supporting Information).
(cTIM) exhibit 50% sequence indentity,56 and the active site
structures determined for complexes of these two TIMs with
phosphoglycolohydroxamate (PGH) are nearly superimpos-
able.54,57,58 No significant differences in the mechanism of
action of TIM from these two sources have been observed,59
and the mechanistic conclusions from studies of TIM from
different organisms have been broadly generalized to all TIMs,
except perhaps the enzymes from archaea.28,29
The kinetic parameters for wild-type and mutant TIMs
determined here (Table 1) are in agreement with those
reported previously.28,41,60 The P168A mutation of TbbTIM
and the loop 7 replacement mutation of cTIM result in 30- and
190-fold decreases, respectively, in kcat/Km for catalysis of
isomerization of GAP, which are due largely to the effects of
these mutations on kcat (Table 1). The loop 7 replacement
mutant of cTIM results in a 120-fold increase in Ki for
competitive inhibition by 2-phosphoglycolate (PGA) at pH 7.5.
PGA is an early example of a tight-binding enzyme inhibitor
that was proposed to be an analogue of the enediolate-like
transition state for the catalyzed reaction (Chart 1).61−63 The
Chart 1
observation that the loop 7 replacement mutation results in
similar large changes in kcat/Km for isomerization of GAP and
DHAP and in Ki for inhibition by PGA shows that this
mutation weakens, to a similar extent, interactions that stabilize
the TIM·PGA complex and the transition state for isomer-
ization. By contrast, the P168A mutation of TbbTIM results in
only a 2.5-fold increase in Ki for inhibition by PGA, which is
significantly smaller than the 30-fold effect of this mutation on
kcat/Km for the TbbTIM-catalyzed isomerization of GAP. This
shows that the P168A mutation affects interactions that control
the barrier for formation of the enzyme-bound transition state
but do not strongly stabilize the TIM·PGA complex.
Figure 1C shows the dependence of kcat/Km (M−1 s−1) for
the P168A TbbTIM-catalyzed reactions of [1-13C]GA on the
concentration of added phosphite dianion (HPO32−).30 The
solid line shows the nonlinear least-squares fit of these data to
eq 5, with a (kcat/Km)E of 0.0013 M−1 s−1 for the unactivated
reaction in the absence of phosphite (Table 2). The data give a
(kcat/Km)E·HP of 0.83 M−1 s−1 for turnover of [1-13C]GA by the
i
phosphite-liganded enzyme, and Kd = 10 mM for binding of
phosphite dianion to the free enzyme (Table 3).30 The upper
limit for (kcat/Km)E, calculated using eq 4 with the assumption
that [1-13C,2-2H]GA forms exclusively by the specific pathway
shown in Scheme 2A, is ≤0.01 M−1 s−1.
Reactions of Substrate Pieces. We have characterized the
activation of wild-type and mutant TIM-catalyzed reactions of
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the truncated neutral substrate glycolaldehyde (GA) by
phosphite dianion in terms of three kinetic parameters (Scheme
4): (1) the second-order rate constant, (kcat/Km)E, for the
Scheme 5
Scheme 4
affinity of HPO32− and of the transition state GA‡ for free TIM
is relatively weak, because a substantial portion of the ligand
binding energy is used to drive the transformation from inactive
unactivated reaction catalyzed by the free enzyme in the
absence of phosphite, (2) the second-order rate constant, (kca2t−/
EO to active EC in forming the binary EC·HPO3 or EC·GA‡
2−
Km)E·HP , for the reaction catalyzed by the binary E·HPO3
i
complex (Scheme 5). By contrast, the full intrinsic binding
energy of the second ligand is observed upon conversion of
these binary complexes of the ternary EC·HPO32−·GA‡
complex. We use Scheme 5 as the framework for our discussion
of structure−reactivity relationships for the P168A and loop 7
replacement mutations of TIM.
complex, and (3) the dissociation constant, Kd, for breakdown
of the E·HPO3 complex, which measures the affinity of the
2−
free enzyme for phosphite dianion. Additionally, the dissocia-
tion constant for release of phosphite from the transition state,
‡
Kd (Scheme 4), which is a measure of the intrinsic phosphite
binding energy, may then be calculated from these three
experimental parameters according to eq 6. Table 3 summarizes
these parameters for the wild-type and mutant TIM studied
P168A Mutation. Figure 2 shows the X-ray crystal
structures in the active site region of unliganded wild-type
here, along with the third-order rate constants, (kcat/Km)E·HP
/
i
Kd (M−2 s−1), for the reactions of the two-part substrate GA
with phosphite.
Kd(kcat/Km)E
Kd‡ =
(kcat/Km)E·HP
(6)
i
The P168A mutation of TbbTIM results in similar 30- and
40-fold decreases, respectively, in second-order rate constant
kcat/Km (Table 1) for the reaction of the whole substrate GAP
and third-order rate constant (kcat/Km)E·HP /Kd (Table 3) for
i
the reaction of the substrate pieces GA and phosphite, while the
loop 7 replacement mutation of cTIM results in a slightly larger
190-decrease in kcat/Km for GAP and a 60-fold decrease in (kcat/
Figure 2. Superposition of models, from X-ray crystal structures,
which show the active sites of unliganded wild-type TbbTIM (gold,
PDB entry 5TIM), PGA-liganded TIM from L. mexicana (cyan, PDB
entry 1N55), and PGA-liganded P168A mutant TbbTIM (green, PDB
entry 2J27). The ligand-induced enzyme conformational changes
observed for wild-type and P168A TbbTIM are similar, except that the
carboxylate side chain of Glu167 remains in an open swung-out
conformation in the P168A mutant.
Km)E·HP /Kd for the pieces. We conclude that these quite
i
different structural mutations result in a similar loss of
stabilizing interactions between the protein and the transition
state for the enzyme-catalyzed reaction of both the whole
substrate GAP and the substrate pieces GA and HPO32−. These
results add to a large body of data that are consistent with the
conclusion that the primary effect of covalent connections
between substrate pieces,20,30−32,34,64−66 or in some cases
enzyme pieces,17,67 is to reduce the unfavorable entropic barrier
associated with the reaction of the pieces, relative to that for the
reaction of the whole substrate or enzyme.68
TbbTIM (gold, 2.1 Å resolution),69 PGA-liganded wild-type
TIM from Leishmania mexicana (cyan, 0.83 Å resolution),70
and PGA-liganded P168A mutant TbbTIM (green, 1.15 Å
resolution).41 A comparison of the X-ray crystal structures for
unliganded wild-type and P168A mutant TbbTIM shows that
the mutation causes only small changes in protein structure.41
The binding of PGA to wild-type TIM (Figure 2, gold) triggers
the large conformational change to give the loop-closed enzyme
(Figure 2, green).41 This movement of loop 6 from the open to
the closed conformation is accompanied by 90° and 180°
rotations in the planes defined by the peptide bonds of Gly211
and Gly212, respectively, which results in a steric clash between
the carbonyl oxygen of Gly211 and the pyrolidine side chain of
Pro168.69 This strain is relieved by movement of the pyrolidine
ring of Pro168 that “swings” the neighboring carboxylate side
The well-documented phosphodianion-driven conforma-
tional change of TIM has been incorporated into the model
shown in Scheme 5, which rationalizes the activation of the
20,21,49
2−
enzyme by the binding of HPO3
.
In this model, TIM
exists in a dominant loop open enzyme form EO that is inactive
and a rare, higher-energy but active, loop-closed enzyme form
EC [Kc ≪ 1 (Scheme 5)]. A large fraction of the free energy
barrier for the conversion of EO to EC may represent the barrier
to desolvation of the enzyme active site that accompanies ligand
binding and loop closure.18,37 The loop-closed form shows
2−
specificity for binding of both HPO3 and the transition state
for the reaction of the substrate piece, GA‡. The overall binding
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2−
Figure 3. Free energy profiles for turnover of GA by free TIM (EO) and by TIM that is saturated with HPO3 (EC·HPO32−), constructed for
Scheme 5 using the kinetic parameters listed in Table 3. The profiles show the activation free energy changes calculated using the Eyring equation at
298 K for reactions catalyzed by wild-type and P168A mutant TbbTIM. (A) Reactions catalyzed by wild-type cTIM. The difference between the total
intrinsic phosphite dianion binding energy of −6.4 kcal/mol and the ΔG° of −2.4 kcal/mol for binding of HPO32− to inactive open enzyme EO to
give active closed liganded enzyme EC·HPO32− is attributed to the ΔGC of 4.0 kcal/mol for the conformational change that converts EO to EC. (B)
Reactions catalyzed by the P168A mutant. The observed barriers for conversion of EO to the transition state for the unactivated and phosphite
dianion-activated reaction are 2.6 kcal/mol higher than for the wild-type cTIM-catalyzed reaction (green bars).
chain of the active site base Glu167 toward the PGA ligand.41,69
Figure 2 shows that the binding of PGA to the P168A mutant
of TbbTIM triggers nearly the same conformational change as
that observed for wild-type TIM. However, the effect of
replacement of the cyclic pyrolidine side chain with the smaller
methyl group at position 168 is to remove the steric clash with
the mobile carbonyl oxygen of Gly211, so that the side chain of
Glu167 remains in the “swung out” position that is observed for
unliganded open wild-type TIM.41 PGA binds, formally, as the
trianion to TIM, and this binding is accompanied by the uptake
of a proton by Glu167 that results in the formation of a
hydrogen bond between the carboxyl groups of bound PGA
and Glu167.19 The P168A mutation of TbbTIM results in
movement of the PGA ligand toward the nearly stationary side
chain of Glu167. This preserves the ligand−side chain
hydrogen bond (Figure 2) and so may provide a rationalization
for the small effect of the mutation on Ki for inhibition by PGA
(Table 1).
position, compared with the “swung in” position for wild-type
TbbTIM (Figure 2). This structural change results in a 30-fold
fold decrease in kcat/Km for the enzyme-catalyzed isomerization
of GAP (Table 1), and 50- and 80-fold decreases in (kcat/Km)E
and (kcat/Km)E·HP , respectively, for the deprotonation of GA by
i
TIM and by the TIM·HPO32− complex (Table 3). There is also
almost no effect of the P168A mutation on the ratio of the
second-order rate constants for the reactions of GA catalyzed
by the free enzyme and the phosphite-liganded enzyme, (kcat/
Km)E·HP/(kcat/Km)E. This is consistent with an only small effect
i
of this mutation on the equilibrium constant for the
interconversion of the open inactive and closed active forms
of the enzyme, KC (eq 7), so that the change in (kcat/Km)E is
due mainly to the change in (kcat/Km)E′ ≈ (kcat/Km)E·HP
.
i
Finally, there is almost no effect of the mutation on Kd‡ for the
binding of phosphite dianion to the transition state EC·GA‡
(Table 3), resulting in essentially identical intrinsic phosphite
dianion binding energies of 6.4 and 6.5 kcal/mol for wild-type
and P168A mutant TbbTIM, respectively (Figure 3).
Panels A and B of Figure 3 show free energy diagrams,
constructed for the model shown in Scheme 5, illustrating the
effect of the P168A mutation of TbbTIM on the unactivated
and phosphite-activated TIM-catalyzed reactions of GA. The
free energies of activation were calculated from the kinetic
parameters listed in Table 3 using the Eyring equation at 298 K,
with the assumption that the phosphite dianion functions solely
as a “spectator” to hold TIM in the active closed EC form, so
The decreases in (kcat/Km)E and (kcat/Km)E·HP observed for
i
P168A mutant TbbTIM reflect the increases in the reaction
barrier associated with the shift in the side chain of Glu167
away from its optimally aligned swung in position in the wild-
type enzyme.71 The P168A mutation is not expected to alter
protein−phosphodianion interactions, and we observe no effect
of this mutation on the intrinsic phosphite binding energy
(Figure 3). Calculations are consistent with the conclusion that
the five-membered pyrolidine ring of the side chain of Pro168
adopts a strained planar configuration at the complex between
PGA and TIM from L. mexicana.70,72 The relief of this strain by
excision of the pyrolidine ring at the P168A mutant would be
expected to stabilize active enzyme EC relative to inactive
enzyme EO and result in an increase in KC. However, our
results can be rationalized by an only small effect of the
mutation on KC, as discussed above. The small effect of the
P168A mutation on the relative barriers to (kcat/Km)E and (kcat/
that (kcat/Km)E′ = (kcat/Km)E·HP (Scheme 5).20,30 The free
i
energy barrier to the unactivated wild-type enzyme-catalyzed
reaction of GA, calculated from (kcat/Km)E, can be partitioned
into the barrier for the conformational change that converts
inactive enzyme EO to active enzyme EC (given by −RT ln KC,
eq 7), and the barrier to catalysis by the active closed enzyme
(kcat/Km)E′ = (kcat/Km)E·HP (Scheme 5).
i
(kcat/Km)E HP
1
KC
·
i
=
(kcat/Km)E
(7)
The carboxylate anion side chain of Glu167 at the P168A
mutant enzyme·PGA complex remains in the swung out
Km)E·HP might reflect a compensating increase in KC and a
i
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Biochemistry
Article
decrease in (kcat/Km)E′, but we can provide no simple
rationalization for such compensating changes.
Loop 7 Replacement Mutation. The binding of the
enediolate intermediate analogue PGH to cTIM results in the
formation of a stunning array of hydrogen bonding interactions
with loop 7 (Figure 4).27 These include (1) interloop H-bonds
loop interactions that stabilize the active closed form of the
enzyme.
Mechanism for Dianion Activation. The primary
2−
imperative for models for enzyme activation by HPO3 is to
provide a rationale for the specificity of phosphite dianion in
binding to the transition state complex E·GA‡ [Kd (Scheme
‡
4)]. The specificity of closed enzyme EC for binding both GA‡
2−
and HPO3 (Scheme 5) provides for the tighter binding of
2−
HPO3 to the transition state complex E·GA‡ compared with
the binding to the free enzyme that exists mainly as EO (Figure
3). Selectivity in the binding of HPO32− to the E·GA‡ transition
state complex might be the result of a direct stabilizing
interaction between GA‡ and phosphite dianion. We discount
this possibility for the following reasons. First, the electrostatic
interaction between phosphite dianion and the enolate−anion-
like transition state for deprotonation of carbon is destabilizing.
Second, only weak stabilizing intermolecular van der Waals
interactions are expected for these small polar molecules. Third,
only a single intermolecular hydrogen bond between the C-2
hydroxyl of GA and phosphite dianion is possible. However,
inspection of X-ray crystal structures shows that the ligands
adopt an extended conformation when bound to TIM (for
example, see Figure 2). A stabilizing intermolecular hydrogen
bond is unlikely to form at a transition state ternary complex E·
Figure 4. Representation of the structure of the closed form of TIM
from chicken muscle in the region of the active site. This figure shows
the important interactions between flexible loop 6 (Pro166−Ala176)
and loop 7 (Tyr208−Ser211), which form upon binding of PGH, an
analogue of the enediolate reaction intermediate (PDB entry
1TPH).57,58 The 208-YGGS sequence was replaced by the 208-
TGAG sequence in the loop 7 mutant of cTIM. Reprinted from ref 27.
Copyright 2010 Elsevier.
2−
GA·HPO3 that adopts a similar conformation.
The P168A and loop 7 replacement mutations of TIM result
in an increase in the activation barriers to TIM-catalyzed
deprotonation of GA, but little change in the intrinsic
phosphite dianion binding energy. This result is consistent
with a catalytic site at TIM that operates in a manner
independent of the dianion activation site. The catalytic and
activating sites must interact to the extent that dianion binding
at the activator site triggers a conformational change that
extends to the catalytic site. We propose for TIM (Scheme 5)
between (a) the backbone amide NH group of Gly173 from
loop 6 and γ-O of Ser211 from loop 7, (b) the backbone amide
NH group of Ala176 from loop 6 and the phenol oxygen of
Try208 from loop 7, and (c) the carbonyl oxygen of Ala169
from loop 6 and the γ-OH group of Ser211 from loop 7 and
(2) hydrogen bonds between the phosphodianion group of
PGH and the backbone amide NH groups of Ser211.58 This
hydrogen bonding pattern has been perturbed by substitution
of the YGGS motif at residues 208−211 with the 208-TGAG
sequence commonly found in archaeal organisms.28 Like the
P168A mutation, this structural mutation results in significant
2−
that GA and HPO3 bind essentially independently at the
2−
protein and that (a) the binding of HPO3 at the dianion site
plays the passive role of stabilizing the preexisting active
enzyme EC but does not affect the structure or intrinsic catalytic
activity of EC and (b) the binding of GA to EC has little or no
2−
effect on the affinity of HPO3 at the catalytic site. The result
of the independent binding of the substrate pieces is to
sequester GA at the catalytically active closed form of TIM. The
binding loci for GA and HPO32− lie essentially adjacent to one
another at the active site for TIM, so that some structural
mutations may affect the enzyme structure and function at both
sites.
Finally, we note that mutations that affect the relative
stability of EC and EO [KC (Scheme 5)] may show complex
effects on the kinetic parameters for catalysis of substrate
deprotonation and on the magnitude of enzyme activation by
phosphite dianion, as has been discussed in an earlier study of
the L232A mutation of TbbTIM.20,21 The data reported in this
paper are consistent with a small 6.5-fold effect of L7RM on KC
that is reflected by the 6.5-fold decrease in the magnitude of the
enzyme activation by phosphite dianion (Table 3).
decreases in (kcat/Km)E·HP and (kcat/Km)E. Inspection of Figure
i
4 suggests that L7RM should have no effect on the important
protein−phosphodianion interactions, so that no change in the
intrinsic phosphite dianion binding energy is expected; an only
‡
small 2.5-fold increase in Kd is observed (Table 3).
The values of (kcat/Km)E·HP and (kcat/Km)E for L7RM cTIM
i
are 2-fold smaller and 3-fold larger, respectively, than the
corresponding kinetic parameters for P168A mutant TbbTIM
(Table 3). This difference reflects the similar effects of the
P168A mutation on (kcat/Km)E·HP and (kcat/Km)E discussed
i
above, compared with the larger decrease in (kcat/Km)E·HP
i
relative to (kcat/Km)E determined for L7RM of cTIM. We
propose that the P168A mutation has little effect on the
magnitude of phosphite dianion activation of TIM-catalyzed
reactions of GA, while the loop 7 replacement mutation results
in a significant reduction in the level of dianion activation.
These results are consistent with a 6.5-fold decrease in the value
of 1/KC (eq 7), from 650 for wild-type cTIM to 100 for L7RM
cTIM (Table 3). This is consistent with the conclusion that the
loop 7 replacement mutation results in a weakening of intra-
ASSOCIATED CONTENT
* Supporting Information
■
S
Rate constants and fractional product yields for the reaction of
[1-13C]GA catalyzed by wild-type cTIM (Table S1) and
fractional product yields for the reaction of [1-13C]GA
catalyzed by the loop 7 replacement mutant of TIM from
chicken muscle and the P168A mutant of TbbTIM (Tables S2
5937
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Biochemistry
Article
(11) Richard, J. P., and Amyes, T. L. (2001) Proton transfer at
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AUTHOR INFORMATION
Corresponding Author
*E-mail: jrichard@buffalo.edu. Phone: (716) 645-4232. Fax:
(716) 645-6963.
■
(14) O’Donoghue, A. C., Amyes, T. L., and Richard, J. P. (2005)
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Funding
This work was supported by Grant GM 039754 from the
National Institutes of Health.
Notes
The authors declare no competing financial interest.
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Mechanism for Activation of Triosephosphate Isomerase by Phosphite
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(22) Pompliano, D. L., Peyman, A., and Knowles, J. R. (1990)
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ABBREVIATIONS
■
TIM, triosephosphate isomerase; TbbTIM, TIM from T. brucei
brucei; cTIM, TIM from chicken muscle; DHAP, dihydrox-
yacetone phosphate; GAP, (R)-glyceraldehyde 3-phosphate;
GA, glycolaldehyde; PGA, 2-phosphoglycolate; PGH, phos-
phoglycolohydroxamate; L7RM, loop 7 replacement mutant;
NADH, nicotinamide adenine dinucleotide, reduced form;
NAD+, nicotinamide adenine dinucleotide, oxidized form;
NMR, nuclear magnetic resonance; PDB, Protein Data Bank.
ADDITIONAL NOTE
We note the following small differences in the numbering of
amino acid residues at TIM from chicken muscle and TIM
from T. brucei brucei (cTIM and TbbTIM): Glu165 and
Glu167, Pro166 and Pro168, Pro166−Ala176 and Pro168−
Ala178 (loop 6), and Tyr208−Ser211 and Tyr210−Ser213
(loop 7), respectively.
■
a
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