Wildtype and engineered monomeric triosephosphate isomerase from Trypanosoma brucei: Partitioning of reaction intermediates in D2O and activation by phosphite dianion

Article abstract of DOI:10.1021/bi2005416

Product yields for the reactions of (R)-glyceraldehyde 3-phosphate (GAP) in D₂O at pD 7.9 catalyzed by wildtype triosephosphate isomerase from Trypanosoma brucei brucei (Tbb TIM) and a monomeric variant (monoTIM) of this wildtype enzyme were determined by ¹H NMR spectroscopy and were compared with the yields determined in earlier work for the reactions catalyzed by TIM from rabbit and chicken muscle [O'Donoghue, A. C., Amyes, T. L., and Richard, J. P. (2005), Biochemistry44, 2610 -2621]. Three products were observed from the reactions catalyzed by TIM: dihydroxyacetone phosphate (DHAP) from isomerization with intramolecular transfer of hydrogen, d-DHAP from isomerization with incorporation of deuterium from D₂O into C-1 of DHAP, and d-GAP from incorporation of deuterium from D₂O into C-2 of GAP. The yield of DHAP formed by intramolecular transfer of hydrogen decreases from 49% for the muscle enzymes to 40% for wildtype Tbb TIM to 34% for monoTIM. There is no significant difference in the ratio of the yields of d-DHAP and d-GAP for wildtype TIM from muscle sources and Trypanosoma brucei brucei, but partitioning of the enediolate intermediate of the monoTIM reaction to form d-DHAP is less favorable ((k_C1)_D/(k_C2) _D = 1.1) than for the wildtype enzyme ((k_C1) _D/(k_C2)_D = 1.7). Product yields for the wildtype Tbb TIM and monoTIM-catalyzed reactions of glycolaldehyde labeled with carbon-13 at the carbonyl carbon ([1-¹³C]-GA) at pD 7.0 in the presence of phosphite dianion and in its absence were determined by ¹H NMR spectroscopy [Go, M. K., Amyes, T. L., and Richard, J. P. (2009) Biochemistry48, 5769-5778]. There is no detectable difference in the yields of the products of wildtype muscle and Tbb TIM-catalyzed reactions of [1-¹³C]-GA in D₂O. The kinetic parameters for phosphite dianion activation of the reactions of [1-¹³C]-GA catalyzed by wildtype Tbb TIM are similar to those reported for the enzyme from rabbit muscle [Amyes, T. L. and Richard, J. P. (2007) Biochemistry46, 5841-5854], but there is no detectable dianion activation of the reaction catalyzed by monoTIM. The engineered disruption of subunit contacts at monoTIM causes movement of the essential side chains of Lys-13 and His-95 away from the catalytic active positions. We suggest that this places an increased demand that the intrinsic binding energy of phosphite dianion be utilized to drive the change in the conformation of monoTIM back to the active structure for wildtype TIM.

Full text of DOI:10.1021/bi2005416

ARTICLE
pubs.acs.org/biochemistry
Wildtype and Engineered Monomeric Triosephosphate Isomerase
from Trypanosoma brucei: Partitioning of Reaction Intermediates in
D2O and Activation by Phosphite Dianion
M. Merced Malabanan, Maybelle K. Go, Tina L. Amyes, and John P. Richard*
Department of Chemistry, University at Buffalo, The State University of New York (SUNY), Buffalo,
New York 14260-3000, United States
ABSTRACT: Product yields for the reactions of (R)-glyceraldehyde 3-phosphate
(GAP) in D2O at pD 7.9 catalyzed by wildtype triosephosphate isomerase from
Trypanosoma brucei brucei (Tbb TIM) and a monomeric variant (monoTIM) of this
1
wildtype enzyme were determined by H NMR spectroscopy and were compared
with the yields determined in earlier work for the reactions catalyzed by TIM from
rabbit and chicken muscle [O’Donoghue, A. C., Amyes, T. L., and Richard, J. P.
(2005), Biochemistry 44, 2610ꢀ2621]. Three products were observed from the
reactions catalyzed by TIM: dihydroxyacetone phosphate (DHAP) from isomerization with intramolecular transfer of hydrogen, d-
DHAP from isomerization with incorporation of deuterium from D2O into C-1 of DHAP, and d-GAP from incorporation of
deuterium from D2O into C-2 of GAP. The yield of DHAP formed by intramolecular transfer of hydrogen decreases from 49% for
the muscle enzymes to 40% for wildtype Tbb TIM to 34% for monoTIM. There is no significant difference in the ratio of the yields of
d-DHAP and d-GAP for wildtype TIM from muscle sources and Trypanosoma brucei brucei, but partitioning of the enediolate
intermediate of the monoTIM reaction to form d-DHAP is less favorable ((kC1)D/(kC2)D = 1.1) than for the wildtype enzyme
((kC1)D/(kC2)D = 1.7). Product yields for the wildtype Tbb TIM and monoTIM-catalyzed reactions of glycolaldehyde labeled with
carbon-13 at the carbonyl carbon ([1-13C]-GA) at pD 7.0 in the presence of phosphite dianion and in its absence were determined
by 1H NMR spectroscopy [Go, M. K., Amyes, T. L., and Richard, J. P. (2009) Biochemistry 48, 5769ꢀ5778]. There is no detectable
difference in the yields of the products of wildtype muscle and Tbb TIM-catalyzed reactions of [1-13C]-GA in D2O. The kinetic
parameters for phosphite dianion activation of the reactions of [1-13C]-GA catalyzed by wildtype Tbb TIM are similar to those
reported for the enzyme from rabbit muscle [Amyes, T. L., and Richard, J. P. (2007) Biochemistry 46, 5841ꢀ5854], but there is no
detectable dianion activation of the reaction catalyzed by monoTIM. The engineered disruption of subunit contacts at monoTIM
causes movement of the essential side chains of Lys-13 and His-95 away from the catalytic active positions. We suggest that this
places an increased demand that the intrinsic binding energy of phosphite dianion be utilized to drive the change in the conformation
of monoTIM back to the active structure for wildtype TIM.
riosephosphate isomerase (TIM) catalyzes the rapid inter-
conversion of the triosephosphates (R)-glyceraldehyde
and TIMs from other prokaryotes and eukaryotes.12 Despite the
limited sequence homology, the overall TIM barrel fold,13 the
essential active site residues,3 and the structure of the loop 6 that
closes over the active site14 are conserved; and similar kinetic
parameters are observed for catalysis by TIMs from throughout
the phylogenetic tree.
The mechanism of action of TIM has attracted the attention of
many prominent enzymologists.14ꢀ18 This is because proton
transfer at carbon is a fundamental reaction in organic chemistry
and cellular metabolic pathways, which is catalyzed by an
incredibly broad range of enzymes.19ꢀ22 Lessons on the mechan-
ism for proton transfer learned through studies on TIM, an
enzyme with the classic TIM barrel protein fold23ꢀ25 that
appeared early in evolution, might therefore be generalized to
enzymes descended from TIM.23,26
T
3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP)
by a stereospecific proton transfer mechanism through an
enzyme-bound cis-enediolate intermediate that uses the carbox-
ylate side chain of Glu-167a in acid/base catalysis (Scheme 1).1ꢀ3
More than 80% of the enzymatic rate acceleration for deprotona-
tion of GAP is due to the utilization of the intrinsic binding
energy of the remote, nonreacting phosphodianion group of the
substrate.4ꢀ6 A large fraction of this binding energy is used to
activate the enzyme for catalysis of deprotonation of bound
substrate.5 We have proposed that this activation is achieved by
using the binding interactions between TIM and the substrate
phosphodianion,4 or exogenous phosphite dianion5 to stabilize a
high-energy, catalytically active loop-closed form of TIM.7,8
TIM catalyzes an early reaction on the remarkably successful
glycolytic pathway for the metabolism of glucose. This pathway
was evident in the Archean period nearly 4 billion years ago,9ꢀ11
and the amino acid sequence of the proto-TIM has undergone
substantial changes during this time. There is now only ca. 32%
sequence homology between TIM from modern archaebacteria
Trypanosomes are parasitic unicellular protozoan homofla-
gellates that infect humans and cause a variety of diseases,
Received: April 11, 2011
Revised:
May 9, 2011
Published: May 09, 2011
r
2011 American Chemical Society
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|

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Scheme 1
successful effort at protein engineering.43,44,46,53 We have also
examined monoTIM using our new mechanistic probes. We
report that monoTIM does not catalyze any reactions of [1-13C]-
GA at the enzyme active site, either in the absence or in the
presence of phosphite dianion. These results show that there is a
correlation between the falloff in the catalytic activity of mono-
TIM compared with wildtype TIM and the loss in phosphite dianion
activation of the monoTIM-catalyzed reaction of [1-13C]-GA.
We suggest a structure-based explanation for the effect of the
engineered changes in protein structure on phosphite dianion
activation of monoTIM.
including sleeping sickness (Trypanosoma brucei rhodesiense and
Trypanosoma brucei gambiense) and Chagas disease
(Trypanosoma cruzi).27 Trypanosoma brucei brucei is morpholo-
gically and biochemically indistinguishable from the other sub-
species of T. brucei27 and is favored for laboratory study because it
causes infection in many mammalian species but not in humans.
Trypanosomes possess microsomal bodies called glycosomes,
which contain the enzymes that catalyze the first seven steps in
glycolysis.28ꢀ30 TIM from trypanosomes shows about 50%
sequence homology with the enzymes from humans, chicken
muscle, and yeast,31 but the protein from trypanosomes is
unusually basic, with an isoelectric point of 9.8 that is 3ꢀ4 units
higher than that for the widely studied TIMs from yeast,29
chicken muscle,32 and rabbit muscle.29 This unusually high pI
suggests that it might be possible to develop inhibitors selective
for trypanosomal TIM as therapeutic reagents. Consequently,
there have been extensive mechanistic33 and crystallographic31,34ꢀ37
studies of trypanosomal TIM in an effort to gain insight that may
help in guiding the design of Tbb TIM-specific tight-binding
enzyme inhibitors.35,38ꢀ40
There have been several high-resolution X-ray crystal struc-
tures reported by Wierenga and co-workers for Tbb TIM and
TIM from the protozoan parasite Leishmania mexicana.31,36,37,41ꢀ43
These structures were used to guide the engineering of an
interesting monomeric variant of TIM (monoTIM)44 and in
the design of mutagenesis studies to probe the mechanism of
monoTIM.45,46 In addition, the high quality of these struc-
tures has stimulated insightful mechanistic proposals, which
have been tested in mutagenesis experiments by Wierenga and
co-workers,47ꢀ49 and in ongoing investigation in our labora-
tory. It is tempting to generalize the results and conclusions
from studies on trypanosomal TIM to TIMs from other
organisms. However, there are relatively few results from
classical mechanistic studies to support these generalizations,
because Tbb TIM has not yet been subject to analyses using
sensitive mechanistic probes, such as those pioneered by
Knowles and co-workers.1,16,17
’ EXPERIMENTAL SECTION
Materials. Rabbit muscle glycerol 3-phosphate dehydrogen-
ase (GPDH) and glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) were purchased from MP Biomedicals or Sigma.
Bovine serum albumin (BSA) was from Roche. CM Sepharose
Fast Flow was from GE Healthcare. D,L-Glyceraldehyde 3-phos-
phate diethyl acetal (barium salt), DHAP (dilithium salt),
NADH (disodium salt), dithiothreitol (DTT), Dowex 50WX4-
200R, TEA HCl, and imidazole were from Sigma. Hydrogen
3
arsenate heptahydrate (disodium salt) and sodium phosphite
(dibasic, pentahydrate) were from Fluka. NAD (free acid, oxidized
form) was from MP Biomedicals. [1-13C]-GA (99% enriched with
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 from Cambridge Isotope Laboratories.
Imidazole was recrystallized from benzene. The barium salt of
D-glyceraldehyde 3-phosphate diethyl acetal was prepared according
to a literature procedure.54 All other chemicals were reagent
grade or better and were used without further purification.
The plasmid pTIM containing the wildtype gene for TIM
from Trypanosoma brucei brucei (Tbb)55 and the pTIM plasmid
containing the subcloned gene for monoTIM44 were gener-
ous gifts from Professor Rik Wierenga. Both wildtype and
monoTIM were overexpressed in Escherichia coli BL21 pLys S
grown in LB medium at 18 °C and purified by ammonium sulfate
precipitation followed by gradient elution on ion exchanger CM-
Sepharose.44,55 The enzymes obtained showed a single band by
gel electrophoresis. The concentration of TIM was determined
from the absorbance at 280 nm using the extinction coefficient of
3.50 ꢁ 104 Mꢀ1 cmꢀ1 calculated using the ProtParam tool available
on the Expasy server.32,56 The concentration of Tbb TIM
obtained using this tool is within 2% of the concentration
obtained using a published value of A280 = 1.32 for a solution
that contains 1 mg/mL of protein.55
We have reported, in studies on TIM from yeast, chicken
muscle, and rabbit muscle, three experimental protocols that
provide a wealth of detailed mechanistic information: (i) experi-
ments to determine the yields of the three products of the
TIM-catalyzed reactions of GAP50,51 or DHAP52 in D2O; (ii)
experiments to determine the kinetic parameters for activation of
TIM-catalyzed deprotonation of glycolaldehyde (GA) by phos-
phite dianion;5 (iii) experiments to determine the yields of the
three products of the unactivated and phosphite dianion-acti-
vated TIM-catalyzed reactions of [1-13C]-GA in D2O.8 We now
extend these probes to this study on the mechanism of action
of Tbb TIM. The results reported here are consistent with a
high conservation of the catalytic properties of TIMs from
across the phylogenetic tree.
Preparation of Solutions. Solutions buffered by TEA at pH
7.5 were prepared by neutralization of the hydrochloride salt with
1 M NaOH, and solutions buffered by imidazole and phosphite
were prepared as described in previous work.5,8,57 Solution pH 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, 4.0, or 10.0 at 25 °C. Values of pD were
obtained by adding 0.40 to the observed reading of the pH meter.58
Stock solutions of D,L-glyceraldehyde 3-phosphate (D,L-GAP) and
GAP at pH 7.5 were prepared by hydrolysis of the diethyl acetal
using Dowex 50WX4-200R (Hþ form) in water at 90ꢀ100 °C as
described in previous work.50 The concentration of GAP in these
solutions was determined by coupled enzymatic assay.50 Solu-
tions of [1-13C]-GA were prepared and the concentration of this
compound was determined using published procedures.8,57
The design of a monomeric variant of TIM (monoTIM) that
shows significant enzyme activity represents an early and largely
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Biochemistry
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Enzyme Assays. All enzyme assays were carried out at 25 °C.
The change in [NADH] was calculated from the change in the
absorbance at 340 nm using an extinction coefficient of 6200
Mꢀ1 cmꢀ1. GPDH and GAPDH were exhaustively dialyzed at
7 °C against 20 mM TEA buffer (pH 7.5). GPDH was assayed by
monitoring the oxidation of NADH by DHAP at 340 nm. The
assay mixture contained 100 mM TEA (pH 7.5, I = 0.1), 0.01%
BSA, 0.2 mM NADH, and 1 mM DHAP. GAPDH was assayed by
monitoring the enzyme-catalyzed reduction of NADþ by GAP in
an assay mixture containing 30 mM TEA (pH 7.5, I = 0.1, NaCl),
1 mM NAD, 2 mM GAP, 5 mM disodium hydrogen arsenate,
3 mM DTT, and 0.01% BSA.
(16 transients) periodically until 70ꢀ80% of GAP was converted
to products. The reactions of GAP (10 mM or 19 mM) catalyzed
by monoTIM in D2O at 25 °C were monitored by 1H NMR in a
reaction mixture that contained GAP, 14 mM imidazole (70%
free base, I = 0.1 (NaCl)), and 600 or 660 nM monoTIM. Each
reaction was monitored by 1H NMR spectroscopy. Spectra were
recorded at hourly intervals for a period of 8 h, during which time
∼80% of GAP was converted to products. In all experiments, the
fraction of the remaining substrate GAP (fGAP) and the fraction
of GAP converted to DHAP (fDHAP), d-DHAP (fd-DHAP), d-GAP
(fd-GAP), and methylglyoxal (MG, fMG) at time t were determined
1
from the integrated areas of the appropriate H NMR signals.
The TIM-catalyzed isomerization of GAP was monitored by
coupling the isomerization of GAP to the oxidation of NADH
catalyzed by GPDH.50 Dilute solutions of TIM were prepared to
contain 0.01% BSA in order to minimize the adsorption of
enzyme to the container walls. The assay mixtures (1.0 mL)
contained 30 or 100 mM TEA (pH 7.5, I = 0.1), 0.2 mM NADH,
3 mM D,L-GAP (1.5 mM GAP ≈ 6 Km), 0.01% BSA, 1 unit of
GPDH, and 0.05ꢀ0.1 nM wildtype TIM. A low background
velocity, vo, due to the nonenzymatic isomerization of GAP and
the isomerization catalyzed by TIM that was present in the
commercial preparation of GPDH, was determined over a period
of 2ꢀ4 min. An aliquot of TIM was then added, and the initial
velocity, vobs, was determined by monitoring the reaction for an
additional 5ꢀ10 min. The initial velocity of the TIM-catalyzed
reaction was then calculated as vi = vobs ꢀ vo, where vo was e3%
of the observed initial velocity, vobs. The TIM-catalyzed isomer-
ization of DHAP to form GAP was monitored by coupling the
isomerization of DHAP (0.15ꢀ5 mM) to the reduction of NADþ
catalyzed by GAPDH59 and following the increase in absorbance
at 340 nm. The assay mixtures (1.0 mL) contained 30 mM TEA
(pH 7.5, I = 0.1, NaCl), 1 mM NAD, 5 mM arsenate, 3 mM DTT,
0.01% BSA, 1 unit of GAPDH, and 0.8 nM wildtype TIM.
1H NMR Analyses. 1H 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 e0.7 Hz
for each peak of the doublet due to the C-1 proton of GAP hydrate,
or e0.5 Hz for the most downfield peak of the double triplet due
to the C-1 proton of [1-13C]-GA hydrate. Spectra (16ꢀ64
transients) were obtained using a sweep width of 6000 Hz, a
pulse angle of 90°, an acquisition time of 6 s, and a relaxation
delay of 60 s (4T1) for experiments on the TIM-catalyzed
isomerization of GAP in D2O or 120 s (>8T1) for experiments
on the TIM-catalyzed reactions of [1-13C]-GA in D2O.8,50
Baselines were subjected to a first-order drift correction before
determination of peak areas. Chemical shifts are reported relative
to HOD at 4.67 ppm.
The peak areas were normalized using the invariant signal for the
C-(4,5) protons of imidazole as an internal standard.50
Reactions of [1-13C]-GA in D2O. The Tbb TIM-catalyzed reac-
tions of [1-13C]-GA in D2O in the presence and absence of
HPO32ꢀ at 25 °C and I = 0.1 (NaCl) were followed by 1H NMR
spectroscopy.8,60 The reaction in the absence of phosphite was
initiated by addition of enzyme to the reaction mixture, which
contains [1-13C]-GA, imidazole and NaCl in D2O to give final
concentrations of 20 mM [1-13C]-GA, 20 mM imidazole (20%
free base, pD 7.0, I = 0.1 (NaCl)), and 0.27ꢀ0.47 mM TIM in a
volume of 850 μL. The reactions in the presence of HPO32ꢀ were
initiated by addition of enzyme to the reaction mixture, which
contains [1-13C]-GA, HPO32ꢀ, imidazole, and NaCl in D2O to
give final concentrations of 20 mM [1-13C]-GA, 18ꢀ20 mM
imidazole (20% free base, pD 7.0), 5ꢀ40 mM HPO3Dꢀ/
2ꢀ
HPO3 (50% dianion, pD 7.0), and 4ꢀ140 μM TIM in a
volume of 850 μL (I = 0.1). In every case 750 μL of the
reaction mixture was transferred to an NMR tube, and the
NMR spectrum was recorded immediately and then at regular
2ꢀ
intervals. The reaction in the absence of HPO3 was mon-
itored over a period of 6 days (ca. 60% reaction of [1-13C]-
2ꢀ
GA), and the reactions in the presence of HPO3 were
monitored over a period of 2ꢀ3 h (ca. 75% reaction of
[1-13C]-GA). After collection of the last spectrum, the protein
was removed by ultrafiltration and the pD was determined.
There was no significant change in pD (e0.03 unit) during
these reactions. The remaining reaction mixture was incu-
bated at 25 °C and used to monitor the activity of TIM toward
GAP. The activity of wildtype Tbb TIM was found to remain
unchanged during the time for these experiments.
The monoTIM-catalyzed reaction of [1-13C]-GA in D2O in the
absence of HPO32ꢀ was monitored in a 750 μL reaction mixture
that contained 20 mM [1-13C]-GA, 20 mM imidazole, and 0.21 mM
monoTIM at pD 7.0 (I = 0.1, NaCl). The monoTIM-catalyzed
reaction of [1-13C]-GA in D2O in the presence of phosphite was
monitored in a reaction mixture that contained 20 mM [1-13C]-
GA, 40 mM HPO3Dꢀ/HPO32ꢀ (50% dianion), 20 mM imidazole,
and 0.092 mM monoTIM at pD 7.0 (I = 0.12). These reactions
were monitored for the disappearance of [1-13C]-GA and for
the formation of reaction products. The activity of monoTIM
was monitored during the course of this experiment using a
solution set aside for this purpose. Unlike wildtype Tbb TIM, a
substantial falloff in activity toward GAP was observed.
Isomerization of GAP in D2O. Wildtype TIM from Tbb was
exhaustively dialyzed at 7 °C against 40 mM imidazole (70% free
base) in D2O at pD 7.9 and I = 0.1 (NaCl) or 30 mM imidazole
(20% free base) in D2O at pD 7.0 and I = 0.1 (NaCl) or I = 0.024,
and the resulting enzyme was used in experiments to determine
the product yields for reactions of GAP and [1-13C]-GA in D2O,
respectively. The turnover of GAP catalyzed by Tbb TIM in D2O
at 25 °C and I = 0.1 (NaCl) was followed by 1H NMR spectro-
scopy.8,50 The reaction in a volume of 750 μL was initiated by
addition of enzyme to the reaction mixture containing GAP,
imidazole buffer (pD 7.9), and NaCl in D2O to give final
concentrations of 5ꢀ10 mM GAP, 14 mM imidazole (70% free
base, I= 0.1 (NaCl)), and 0.3 or 0.6 nM TIM. The reaction at 25 °C
was monitored by 1H NMR spectroscopy by recording spectra
The fraction of the remaining substrate [1-13C]-GA (fS, eq 1)
and the fraction of [1-13C]-GA converted to the identifiable
products [2-13C]-GA, [2-13C, 2-2H]-GA, [1-13C, 2-2H]-GA, and
[1-13C, 2,2-di-2H]-GA were determined from the integrated
areas of the relevant 1H NMR signals as described in previous
work.5,8,61 The observed peak areas were normalized using the
signal due to the C-(4,5) protons of imidazole or the upfield
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Table 1. Kinetic Parameters for Catalysis by TIM, from Different Organisms, of the Isomerization Reactions of GAP and DHAP
and the Phosphite-Activated and Unactivated Reactions of [1-13C]-GAa
GAP
DHAP
[1-13C]-GA
k
cat/Km
k
cat/Km
(kcat/Km)E (kcat/Km)E 3 HPi (kcat/Km)E 3 Hpi/Kd
organism
Km (M)
kcat (sꢀ1
)
(Mꢀ1 sꢀ1
)
Km (M)
kcat (sꢀ1
)
(Mꢀ1 sꢀ1
)
(Mꢀ1 sꢀ1
)
(Mꢀ1 sꢀ1
)
(Mꢀ2 sꢀ1
)
Kd (M)
T. brucei (wildtype)
T. brucei (monoTIM) 5.2 ꢁ 10ꢀ3
2.5 ꢁ 10ꢀ4
2100
5.7
8.4 ꢁ 106 7.0 ꢁ 10ꢀ4
300
4.3 ꢁ 105
0.07d
64
3400
0.019
910
no detectable
reaction
190e
rabbit muscle
4.5 ꢁ 10ꢀ4b 4300 b
9.6 ꢁ 106 6.2 ꢁ 10ꢀ4c
870 c
1.4 ꢁ 106
0.26e,f
4900e
5500g
0.038e
210g
a Under standard assay conditions: 30 mM TEA, pH 7.5, and 25 °C (I = 0.1, NaCl). b Ref 5. c Ref 84. d Second-order rate constant for the TIM-catalyzed
reaction of [1-13C]-GA at the enzyme active site calculated using eq 7. e Observed kinetic parameters for the reaction of unlabeled GA catalyzed by rabbit
muscle TIM.5 f Observed second-order rate constant for the unactivated TIM-catalyzed reaction of unlabeled GA determined by monitoring the
disappearance of GA using eqs 1 and 2. g Kinetic parameters for the reaction of [1-13C]-GA catalyzed by rabbit muscle TIM calculated from the observed
kinetic parameters for the reaction of unlabeled GA. The TIM-catalyzed reaction of [1-13C]-GA is estimated to be 14% faster than GA because there is an
additional pathway for isomerization to form [2-13C]-GA in a yield of 12% (Table 4).
peak of the doublet due to the PꢀH proton of phosphite as an
Km = 0.70 mM. The value of kcat = (300 ( 30) sꢀ1 determined
for the Tbb TIM-catalyzed reaction of DHAP is also smaller than
internal standard.
an earlier published value of (730 ( 200) s .
ꢀ1 49 However, the
ln fS ¼ ꢀ kobs
t
ð1Þ
ð2Þ
ratio of the values of kcat/Km for the Tbb TIM-catalyzed reactions
of GAP and DHAP is (8.4 ꢁ 106 Mꢀ1 sꢀ1)/(4.3 ꢁ 105 Mꢀ1 sꢀ1) =
20 is in good agreement with the value of the equilibrium
constant for the conversion of GAP to DHAP, Keq = 22,
determined at 38 °C,62 as required by the Haldane relationship.
Table 1 also lists kinetic parameters for the isomerization reactions
catalyzed by TIM from rabbit muscle.
kobs
ðkcat=KmÞobs
¼
ð1 ꢀ fhydÞ½Eꢂ
Observed first-order rate constants, kobs (sꢀ1), for the disap-
pearance of [1-13C]-GA were determined from the slopes of
linear semilogarithmic plots of reaction progress against time
that covered the first 70ꢀ80% of the reaction using eq 1,
where fS is the fraction of [1-13C]-GA that remains at time t.
Observed second-order rate constants, (kcat/Km)obs, for the
TIM-catalyzed reaction of [1-13C]-GA were determined from
the values of kobs using eq 2, where fhyd = 0.94 is the fraction of
[1-13C]-GA present as the hydrate form and [E] is the enzyme
concentration.5,8
1
TIM-Catalyzed Isomerization of GAP in D2O. H NMR
spectroscopy is a powerful analytical method for monitoring
proton transfer reactions in D2O at simple63ꢀ65 and complex66ꢀ68
carbon acids. The disappearance of substrate GAP and the
appearance of the products of the nonenzymatic and TIM-
catalyzed reactions of GAP in D2O was monitored by 1H NMR
spectroscopy, as described in previous work.50 Figure 1A shows the
decrease with time in the fraction of remaining substrate
during the reaction of 10 mM GAP catalyzed by 0.6 nM Tbb
TIM in D2O buffered by 14 mM imidazole at pD 7.9 and 25 °C
(I = 0.1, NaCl). The yields of the four products of this
reaction, (fP)obs (P = DHAP, d-DHAP, d-GAP or methylglyox-
al (MG),69 Scheme 2), were calculated from the normalized
1H NMR peak area of a single proton for the particular
product and the sum of the peak areas of single protons for
all of the reaction products, as shown in eq 3.50 The decrease in
the normalized peak area of the signal for the substrate GAP
agrees with the sum of the normalized peak areas of the signals
for the four products to within 10% during the TIM-catalyzed
reaction of 70% of GAP.
The fractional yields (fP)E of the products of the TIM-
catalyzed reactions of GAP were calculated by correcting the
observed product yields for the MG that forms by a non-
enzymatic reaction69 using eqs 4ꢀ6.50 Figure 1B shows the
change in the product yields with time. A small increase in
fd-DHAP and a decrease in fd-GAP is observed due to the TIM-
catalyzed isomerization of d-GAP to form the thermodyna-
mically favored product d-DHAP.50 The initial product yields,
fE, were determined by extrapolation of the values of (fP)E
(Figure 1B) to t = 0. Table 2 reports the average of the
initial product yields determined in separate experiments for
the reaction of 10 mM GAP catalyzed by 0.6 nM Tbb TIM
’ RESULTS
The gene for wildtype TIM from Trypanosoma brucei brucei
(Tbb TIM) was expressed in E. coli and the enzyme was purified
to homogeneity by following a published procedure.55 The
kinetic parameters for Tbb TIM-catalyzed isomerization of
GAP and DHAP at pH 7.5 and 25 °C (I = 0.1) determined in
this work are reported in Table 1. The values of kcat = (2100 (
300) sꢀ1 determined for several preparations of Tbb TIM is
smaller than the value of kcat = (3600 ( 400) sꢀ1 reported by
Wierenga and co-workers, but there is excellent agreement
between the values of Km = 0.25 mM for GAP determined in
our two laboratories.49 Table 1 also reports the kinetic param-
eters for monoTIM-catalyzed isomerization of GAP, which
are in good agreement with previously published kinetic
parameters.44
The coupled enzyme assay for the Tbb TIM-catalyzed reaction
of DHAP was carried out in the presence of 5 mM sodium arsenate,
in order to ensure the formation of an unstable acyl arsenate ester
as the product of the GAPDH-catalyzed reaction. The apparent
Km = 1.45 mM determined for the Tbb TIM-catalyzed reaction of
DHAP under these conditions was corrected using Ki = 4.6 mM
for competitive inhibition of Tbb TIM by arsenate33 to give
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Table 2. Yields of the Products from the Reaction of GAP in
D2O in the Presence of Triosephosphate Isomerase from
Different Organismsa
fractional product yield
TIM
T. brucei
DHAP
d-DHAP
d-GAP
MG
b
fT
0.42c
0.36d
0.35c
0.37d
0.22c
0.21d
0.01c
0.06d
e,f
fE
0.40 ( 0.02 0.37 ( 0.02 0.22 ( 0.002
0.31g
0.34g
0.32g
0.03g
0.09h
b
T. brucei
(monoTIM)
fT
0.33h
0.33h
0.26h
e,f
fE
fE
0.34 ( 0.02 0.35 ( 0.01 0.31 ( 0.02
rabbit muscle50
chicken muscle50 fE
0.49
0.31
0.20
e
e
0.50
0.31
0.19
Figure 1. Rate and product data for the reaction of GAP (10 mM)
catalyzed by Tbb TIM (0.60 nM) in D2O buffered by 14 mM imidazole
at pD 7.9 and 25 °C (I = 0.1, NaCl), determined by 1H NMR
spectroscopy. (A) The decrease with time in the fraction of remaining
GAP. (B) The change with time in the fractional yields of only the
products of enzymatic reaction of GAP, normalized using the sum of the
observed fractions of d-GAP, DHAP, and d-DHAP according to eqs 4ꢀ6.
The initial product yields, fE, reported in Table 2 were obtained by making
a short linear extrapolation of product yields to zero reaction time. Key:
(4) yield of DHAP; (9) yield of d-DHAP; (b) yield of d-GAP.
a For the reaction of GAP at pD 7.9, 25 °C and I = 0.1 (NaCl). b Yield of
the product of the enzymatic or nonenzymatic reactions of GAP
(Scheme 2) determined by extrapolation to zero time of linear plots
of product yield, ( fP)obsd, against time (data not shown). c Reaction of
10 mM GAP catalyzed by 0.60 nM TIM (Figure 1). d Reaction of 5 mM
GAP catalyzed by 0.3 nM TIM (data not shown). e Initial product yields
determined by extrapolation of the normalized product yields for the
TIM-catalyzed reaction, ( fP)E (eqs 4ꢀ6), to zero time (see Figure 1B).
f The quoted errors are the average of product yields determined in two
reactions catalyzed by 0.3 nM or 0.6 nM Tbb TIM. g Reaction of 10 mM
GAP catalyzed by 660 nM monoTIM in D2O (I = 0.1, NaCl). h Reaction
of 19 mM GAP catalyzed by 600 nM monoTIM in D2O (I = 0.06).
Scheme 2
The reactions of GAP (10 or 19 mM) catalyzed by 0.6 μM
monoTIM in D2O at pD 8.0 (I = 0.1, NaCl) were monitored by
1H NMR for a period of 8 h, during which time the concentration
of GAP decreased by ca. 80%. The product yields were deter-
mined at hourly intervals as described above, and the initial
product yields were determined by extrapolation of plots of (fP)E
against time to t = 0, as shown in Figure 1B for the reaction
catalyzed by wildtype Tbb TIM. The average of the product
yields (DHAP, d-DHAP, and d-GAP) determined in two sepa-
rate experiments are reported in Table 2.
TIM-Catalyzed Reactions of [1-13C]-GA in D2O. The Tbb
1
TIM-catalyzed reactions of [1-13C]-GA were monitored by H
NMR spectroscopy and the product yields were determined
from the normalized areas of the relevant product peaks, as
described previously.8 Figure 2A shows the decrease in the
fraction of the remaining substrate (fS) with time during the
reaction of 20 mM [1-13C]-GA catalyzed by Tbb TIM (82 μM)
in the presence of 15 mM phosphite dianion at pD 7.0, 25 °C,
I = 0.1 (NaCl). The product yields were determined over the first
ca. 20ꢀ30% reaction, and the disappearance of [1-13C]-GA was
monitored for ca. 70ꢀ80% reaction.
(Figure 1B) and for the reaction of 5 mM GAP catalyzed by
0.3 nM TIM (data not shown).
AP
The Tbb TIM-catalyzed reactions of [1-13C]-GA gave the
same three isotopomers of GA as reported in earlier work for the
chicken muscle TIM-catalyzed reaction of [1-13C]-GA: [2-13C]-
GA, [2-13C, 2-2H]-GA, [1-13C, 2-2H]-GA (Chart 1).8 The yield
of a fourth product [1-13C, 2,2-di-2H]-GA, that forms by a
protein-catalyzed reaction outside of the enzyme active site,8,57,70
is too low to be detected for the phosphite dianion-activated
reactions. The fractional yields of products, fP, were calculated
from the normalized 1H NMR peak area of a single proton for the
particular product and the sum of the peak areas of single protons
for all of the reaction products.8 Figure 2B shows the fractional
yields, fP, of the products obtained during the first 30% of the
ðfPÞobsd
¼
ð3Þ
ð4Þ
ð5Þ
ð6Þ
ADHAP þ Ad-DHAP þ Ad-GAP þ AMG
fd-GAP
ðfd-GAPÞE ¼
ðfDHAPÞE ¼
ðfd-DHAPÞE ¼
fd-GAP þ fDHAP þ fd-DHAP
fDHAP
fd-GAP þ fDHAP þ fd-DHAP
fd-DHAP
fd-GAP þ fDHAP þ fd-DHAP
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monitored for 25 h, during which time there was 12% reaction of
[1-13C]-GA. A 65% falloff of the initial monoTIM activity toward
GAP was observed during the first 12 h of the reaction. The
reaction in the presence of phosphite dianion (0.09 mM mono-
TIM) was monitored for 14 h during which time there was 10%
reaction of [1-13C]-GA. A 90% loss in the activity of monoTIM
was observed during the first 9 h of the reaction. In both cases, the
reaction remained first-order in the concentration of [1-13C]-GA
during this substantial loss of enzymatic activity. Apparent
second-order rate constants (kcat/Km)obs = 0.1 and 0.4 Mꢀ1 sꢀ1
were determined, respectively, for the monoTIM-catalyzed
reactions of [1-13C]-GA in the absence and presence of phos-
phite by monitoring the disappearance of [1-13C]-GA using
eqs 1 and 2.
1
The H NMR analysis of the products of the monoTIM-
catalyzed reaction of [1-13C]-GA showed no detectable [2-13C]-
GA or [2-13C, 2-2H]-GA (<1%) from reactions at a functioning
enzyme active site. The major product was [1-13C, 2,2-di-2H]-GA
from the direct deuterium exchange reaction, which is formed in
a yield of ca. 75%.
Figure 2. Rate and product data for the reaction of [1-13C]-GA
catalyzed by Tbb TIM in the presence of 15 mM phosphite dianion in
D2O buffered by 20 mM imidazole at pD 7.0 and 25 °C (I = 0.1, NaCl),
determined by 1H NMR spectroscopy. (A) The decrease with time in
the fraction of remaining [1-13C]-GA. (B) The change with time in the
fractional yields of the products, fP, of the phosphite-activated TIM-
catalyzed reaction of [1-13C]-GA. The initial product yields reported in
Table 4 were obtained by making a short linear extrapolation of product
yields to zero reaction time. Key: (3) yield of [2-13C]-GA; ()) yield of
[2-13C, 2-2H]-GA; (9) yield of [1-13C, 2-2H]-GA.
’ DISCUSSION
Reactions of GAP in D2O. DHAP binds at a cavity of TIM that
is exposed to solvent. The carbon acid fragment of DHAP
projects toward the cavity bottom, and the phosphodianion lies
at the protein surface, where it is covered by the flexible phos-
phate-gripper loop 6.3,7 This loop closure shields the bound
carbon acid from interaction with the bulk solvent. Proton
transfer from bound triosephosphate to the carboxylate side
chain of Glu-167 in a solvent of D2O gives an enediolate inter-
mediate57 and a protonated acid side chain of Glu-167. The
substrate-derived hydrogen partitions between intramolecular
transfer and effectively irreversible exchange with solvent D2O to
form the D-labeled carboxylic acid (Scheme 3). This D-labeled
side chain next partitions between protonation of the enediolate
intermediate at C-1 and C-2 (Scheme 3) to form d-DHAP and
d-GAP, respectively.
reaction of 20 mM [1-13C]-GA catalyzed by Tbb TIM (82 μM)
in the presence of 15 mM phosphite dianion at pD 7.0, 25 °C,
I = 0.1 (NaCl). The initial fractional product yields for the
phosphite-activated reactions reported in Table 4 were determined
by making short extrapolations of the normalized fractional yields, fP,
to zero reaction time as shown in Figure 2B. A similar protocol was
followed in determining the initial product yields of the unactivated
TIM-catalyzed reactions of [1-13C]-GA (Table 4).
The observed first-order rate constants, kobs (sꢀ1), for the
disappearance of [1-13C]-GA and the apparent second-order rate
constants, (kcat/Km)obs (Mꢀ1 sꢀ1), for the TIM-catalyzed reac-
tions of the reactive carbonyl form of [1-13C]-GA were deter-
mined using eq 1 and 2. The kinetic data for these TIM-catalyzed
reactions in the presence and absence of phosphite dianion are
summarized in Table 3. There is fair mass balance observed for
the phosphite dianion-activated TIM-catalyzed reactions of
[1-13C]-GA, so that (kcat/Km)obs is equal to the second-order
rate constant for the Tbb-TIM catalyzed reaction at the function-
ing enzyme active site.
Table 2 compares the product yields of the Tbb and chicken
TIM-catalyzed isomerization reactions of GAP in D2O deter-
1
mined by H NMR analyses. Two rate constant ratios can be
determined from these product yields:
(A) The ratio kex/(kC1 H for partitioning of hydrogen-
)
labeled TIM between protonation of the enediolate
at carbon-1 and exchange with -D from solvent was
determined as the ratio of the yields of H-labeled
(DHAP) and D-labeled (d-DHAP and d-GAP) pro-
ducts. The 40 ( 2% yield of DHAP for Tbb TIM is
detectably smaller than the 50% yield of DHAP from
the chicken and rabbit muscle TIM-catalyzed reactions
(Table 2); and the derived rate constant ratio for Tbb
Only ca. 70% of [1-13C]-GA is converted to [2-13C]-GA,
[2-13C, 2-2H]-GA, [1-13C, 2-2H]-GA, or [1-13C, 2,2-di-2H]-GA
for reactions in the absence of phosphite dianion. The other
products formed were not determined. Combining the 20% yield
of [1-13C, 2,2-di-2H]-GA with (kcat/Km)obs = 0.13 Mꢀ1 sꢀ1 gives
(kcat/Km) = (0.13 Mꢀ1 ꢀ1)(0.20) = 0.03 Mꢀ1 sꢀ1 for the
s
unactivated Tbb TIM-catalyzed reaction of [1-13C]-GA to form
[1-13C, 2,2-di-2H]-GA. The second-order rate constant for the
unactivated TIM-catalyzed reaction at the enzyme active site was
TIM, kex/(kC1)H = 1.48 is larger than for the muscle
enzymes, kex/(kC1)H = 1.04.
(B) The ratio (kC1)D/(kC2)D for partitioning of D-labeled
TIM between D-transfer to carbon-1 and to carbon-2 is
determined as the ratio of the yields of d-DHAP and
d-GAP. The rate constant ratio (kC1)D/(kC2)D = 1.7 (
0.2 is the same, within experimental error, as the values of
(kC1)D/(kC2)D = 1.5 for the reactions catalyzed by TIM
from muscle sources.50
calculated as (kcat/Km) = (0.13 Mꢀ1 sꢀ1)(0.54) = 0.07 Mꢀ1 sꢀ1
,
where 0.54 is the fractional yield of [2-13C]-GA, [2-13C, 2-2H]-
GA, and [1-13C, 2-2H]-GA from the active-site reactions.
The reaction of [1-13C]-GA catalyzed by monoTIM at 25 °C
was monitored in both the absence and presence of 40 mM
phosphite (50% dianion) in solutions that contain 20 mM
imidazole (20% free base) at pD 7.0 and I = 0.1 (NaCl). The
reaction in the absence of phosphite (0.21 mM monoTIM) was
Previous studies on TIM have shown the following: (i) The
exchange reaction of the protonated carboxylic acid side chain of
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Chart 1
Table 3. Kinetic Data for the Reaction of [1-13C]-GA Cata-
lyzed by Wildtype Tbb TIM in D2O in the Absence and
Presence of Phosphite Dianiona
(kcat/Km)obs
(Mꢀ1 sꢀ1
)
b
c
[HPO32ꢀ] (M)
0
[TIM] (M)
kobs (sꢀ1
)
2.72 ꢁ 10ꢀ4
2.15 ꢁ 10ꢀ6
0.13d
0.07e
8.6
2.6 ꢁ 10ꢀ3
5.3 ꢁ 10ꢀ3
7.9 ꢁ 10ꢀ3
9.9 ꢁ 10ꢀ3
12.6 ꢁ 10ꢀ3
15.3 ꢁ 10ꢀ3
17.3 ꢁ 10ꢀ3
1.36 ꢁ 10ꢀ4
1.15 ꢁ 10ꢀ4
1.02 ꢁ 10ꢀ4
9.50 ꢁ 10ꢀ5
9.50 ꢁ 10ꢀ5
8.20 ꢁ 10ꢀ5
6.82 ꢁ 10ꢀ5
7.08 ꢁ 10ꢀ5
1.01 ꢁ 10ꢀ4
1.13 ꢁ 10ꢀ4
1.28 ꢁ 10ꢀ4
1.56 ꢁ 10ꢀ4
1.48 ꢁ 10ꢀ4
1.26 ꢁ 10ꢀ4
14.3
18.2
22.0
26.9
29.6
30.3
Figure 3. Dependence of the observed second-order rate constant
(kcat/Km)obs (Mꢀ1 ꢀ1) for the turnover of the free carbonyl form of
s
[1-13C]-GA by Tbb TIM and of unlabeled GA by rabbit muscle TIM in
D2O on the concentration of added phosphite dianion at pD 7 and 25 °C
(I = 0.10). Key: (Red circles) reaction of 20 mM [1-13C]-GA catalyzed
by Tbb TIM; (green upward triangles) reaction of 20 mM unlabeled GA
catalyzed by rabbit muscle TIM;5 (green downward triangles) reaction
of 10 mM unlabeled GA catalyzed by rabbit muscle TIM.5
a For reactions of 20 mM [1-13C]-GA in 20 mM imidazole (20% free
base), pD 7.0, 25 °C, and I = 0.1 (NaCl). b Observed first-order rate
constant for the reaction of [1-13C]-GA calculated using eq 1. c Observed
second-order rate constant for the TIM-catalyzed reaction of [1-13C]-
GA calculated using eq 2, unless noted otherwise. d Second-order rate
constant determined by monitoring the disappearance of total [1-13C]-
GA. e Second-order rate constant for the reactions of [1-13C]-GA at the
TIM active site, calculated from the overall reaction rate constant of 0.13
M
ꢀ1 sꢀ1 (determined from two independent experiments) and the 54%
product yield (Table 4).
Scheme 3
Figure 4. A comparison of the amino acid sequences for wildtype Tbb TIM
and the engineered monoTIM. The first amino acid residue in loop 3 that
follows β-strand 3 is Gln-65. Residues 69ꢀ79 (red) from loop 3 were replaced
by four amino acids (N A D A) that extend R-helix 3 by one turn. Additional
amino acid substitutions for the wildtype enzyme are shown in blue.
The similarity in the product yields for the reactions catalyzed
by TIM from several sources shows that the dynamics and
mechanism for these exchange reactions are unaffected by the
relatively large differences in the sequences of these different
TIMs. The small differences in the very large rate constants for
the fast exchange reactions between the electronegative car-
boxylic acid oxygen72 and basic atoms at the TIM-active site
correspond to a small 0.2 kcal/mol difference in relative barriers
to (kC1)H and kex, that we are unable to rationalize.
The monomers of wildtype TIM are held together in the dimer
by interactions between loop 3 (residues 65ꢀ79) and residues at
a deep crevice of the second subunit near loops 1 and 4, which
contain the active site residues Lys-13 and His-95, respectively.
The monomeric form of TIM was engineered as shown in
Figure 4 by shortening the long fifteen amino acid-residue loop
3 by seven residues and making judicious substitutions at the
remaining residues to reduce hydrophobicity and extend R-helix
3 by one turn.44 These sequence changes reduce the interactions
between the TIM subunits, and produce a monomeric form of
TIM with lower, but still significant, enzymatic activity.44,47
Glu-167 involves deuterons that are sequestered from interac-
tions with bulk solvent at the enzyme active site.51 (ii) The O-1
and O-2 enediolate intermediates of deprotonation of GAP and
DHAP, respectively, do not achieve chemical equilibrium with
one another during the isomerization reaction.52 (iii) The
substrate-derived -H undergoes exchange between the carboxylic
acid side chain of Glu-167 and a relatively large pool of -D that are
sequestered from solvent at the enzyme active site.71 In other
words, these exchange reactions are fast and involve an unde-
termined number of hydrons at an active site that is inaccessible
to solvent on the time scale for the exchange reaction.
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The monoTIM-catalyzed reaction of GAP in D2O gives a
lower 34 ( 2% yield of DHAP from the reaction with intramo-
lecular transfer of -H than the 40 ( 2% yield observed for
wildtype Tbb TIM. A decrease in (kC1)D/(kC2)D for partitioning
of the D-labeled enzyme is also observed, from (kC1)D/(kC2)D =
1.7 for the wildtype enzyme to 1.1 for monoTIM. The relatively
larger yield of D-labeled products observed here for the reaction
catalyzed by monoTIM might, nominally, reflect the increased
exposure of the active site to solvent due to the elimination of
hydrophobic interactions at the subunit interface. We do not
have an explanation for the difference in the rate constant ratios
for partitioning of deuterium-labeled enzyme between proton-
ation of the enediolate reaction intermediate at C-1 and C-2 at
the active sites for wildtype and monoTIM.
chicken muscle (Table 4). Note that these products may form
with phosphite bound in two different conformations [not shown
in Scheme 4].8 The dianion may bind next to 2-12C, to mimic
the binding of GAP, or next to 1-13C to mimic the binding
of DHAP.
A ca. 54% total yield of [2-13C]-GA, [2-13C, 2-2H]-GA and
[1-13C, 2-2H]-GA was observed from the slow Tbb TIM-
catalyzed reactions of [1-13C]-GA in the absence of exogenous
phosphite dianion in a ratio of 0.10:0.61:0.29, respectively
(Table 4). These products form by the reactions of [1-13C]-
GA at the active site of TIM. A similar overall product yield in a
ratio of 0.10:0.60:0.30 for [2-13C]-GA, [2-13C, 2-2H]-GA, and
[1-13C, 2-2H]-GA, respectively, was determined for the unac-
tivated chicken muscle TIM-catalyzed reaction of [1-13C]-GA.8
Similar ca. 20% yields of the dideuterium-labeled [1-13C, 2,2-
di-2H]-GA were determined for the unactivated Tbb and chicken
muscle TIM-catalyzed reactions.8 [1-13C, 2,2-di-2H]-GA is the
only product detected from the reaction of [1-13C]-GA catalyzed
by the severely crippled K12G mutant of TIM from yeast,57
and bovine serum albumin (BSA) was also found to catalyze
the conversion of [1-13C]-GA to [1-13C, 2,2-di-2H]-GA
in D2O.70 These results show that this activity is intrinsic
to some globular proteins. We have proposed that [1-13C, 2,
2-di-2H]-GA forms by the reaction of [1-13C]-GA with the
alkylamino side chain of surface lysine residues to form a
Schiff base that undergoes base-catalyzed deuterium ex-
change of the C-2 protons through an enamine intermediate
(Scheme 5).8,70
Wildtype Tbb TIM-Catalyzed Reactions of [1-13C]-GA.
Three products form from the phosphite-activated TIM-catalyzed
reactions of [1-13C]-GA in D2O (Scheme 4): (a) [2-13C]-GA
from intramolecular transfer of the substrate-derived hydrogen to
the 13C-labeled carbon of the enediolate reaction intermediate;
(b) [1-13C, 2-2H]-GA from transfer of deuterium from solvent to
the 12C-labeled carbon of the enediolate; and (c) [2-13C, 2-2H]-
GA from transfer of deuterium from solvent to the 13C-labeled
carbon of the enediolate. We observe the same yields of [2-13C]-
GA, [1-13C, 2-2H]-GA, and [2-13C, 2-2H]-GA from the reactions
of [1-13C]-GA catalyzed by TIM from trypanosomes and from
Scheme 4
Phosphite Activation of Tbb TIM-Catalyzed Reaction of
[1-13C]-GA in D2O. The observed second-order rate constant
(kcat/Km)obs = 0.13 Mꢀ1 sꢀ1 for the Tbb TIM-catalyzed reaction
of [1-13C]-GA in the absence of exogenous phosphite is similar
to the values of (kcat/Km)obs = 0.26 Mꢀ1 sꢀ1 and 0.19 Mꢀ1 sꢀ1
reported for rabbit muscle TIM5 and chicken muscle TIM,8
respectively. These observed rate constants were corrected using
eq 7 to obtain the second-order rate constant (kcat/Km)E for the
reactions to form [2-13C]-GA, [1-13C, 2-2H]-GA, and [2-13C,
2-2H]-GA that occur at the enzyme active site. The sum of the
fractional yields of these products, (fE)T = 0.54, determined for
Table 4. Yields of the Products from the Reaction of [1-13C]-GA in D2O Catalyzed by Tbb TIM and from the Phosphite Dianion-
Activated TIM-Catalyzed Reactiona
fractional product yieldb
[HPO32ꢀ] mM
[2-13C]-GA
[2-13C, 2-2H]-GA
[1-13C, 2-2H]-GA
[1-13C, 2,2-di-2H]-GA
0
0.05 ( 0.01c
0.10d
0.32 ( 0.04c
0.61d
0.17 ( 0.03c
0.29d
0.21 ( 0.09c
0
5
0.13 ( 0.01
0.13 ( 0.003
0.14 ( 0.004
0.13 ( 0.01
0.13 ( 0.004
0.13
0.63 ( 0.03
0.64 ( 0.01
0.63 ( 0.01
0.63 ( 0.04
0.65 ( 0.02
0.63
0.24 ( 0.04
0.23 ( 0.01
0.24 ( 0.01
0.24 ( 0.03
0.21 ( 0.03
0.24
n.d
n.d
n.d
n.d
10
16
20
20
average product yielde
chicken muscle TIM8
0.12
0.64
0.23
a For reactions of 20 mM [1-13C]-GA in 20 mM imidazole (20% free base), pD 7.0, 25 °C, and I = 0.1 b The initial fractional yields of the products of the
reaction of [1-13C]-GA determined as described in previous work.8 The quoted error in the initial product yields is the standard deviation in the y-
intercept of plots of fractional product yield against time (Figure 2). c Fractional product yields for the unactivated reaction of [1-13C]-GA catalyzed by
Tbb TIM, determined as the average of the data for three experiments in the range [TIM] = 270ꢀ470 μM. d Average yield calculated for products
[2-13C]-GA, [2-13C, 2-2H]-GA, and [1-13C, 2-2H]-GA that form from reactions at the enzyme active site, determined for several experiments at different
concentrations of Tbb TIM (270ꢀ470 μM, data not shown). e Average of the experiments at four different concentrations of phosphite dianion.
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Scheme 5
Scheme 6
compared to rabbit muscle TIM) and kcat/Km (similar for Tbb
and rabbit muscle TIM) for enzyme-catalyzed isomerization of
GAP (Table 1). These trends show that the total catalytic
efficiency for TIM catalysis at low substrate or activator con-
centration is similar, but that the rabbit muscle enzyme shows a
larger activity for catalysis of the reaction at saturating concen-
trations of substrate or activator. The results are consistent with
the notion that a slightly greater fraction of the total substrate
or ligand binding energy is expressed at the ground-state for the
Tbb TIM-catalyzed reaction, where it is expressed in the para-
meter kcat/Km and Km, while a larger fraction of this binding
energy is expressed at the transition state for the rabbit muscle
TIM-catalyzed reaction, where it is expressed specifically in kcat
but not in Km.
wildtype Tbb TIM gives (kcat/Km)E = 0.07 Mꢀ1 ꢀ1, which is
s
similar to (kcat/Km)E = 0.1 Mꢀ1 sꢀ1 determined for TIM from
chicken muscle.8
ꢀ
ꢁ
ꢀ
ꢁ
kcat
Km
kcat
Km
¼
ðfEÞT
obs
ð7Þ
E
Figure 3 shows the dependence on the concentration of
exogenous HPO32ꢀ of (kcat/Km)obs for turnover of the carbonyl
form of [1-13C]-GA by Tbb TIM in D2O at pD 7.0 and 25 °C (I =
0.1, NaCl). The phosphite-activated TIM-catalyzed reactions of
[1-13C]-GA are very fast relative to the nonspecific protein-
catalyzed reaction, and [2-13C]-GA, [1-13C, 2-2H]-GA, and
[2-13C, 2-2H]-GA account for 100% of the observed reaction
products. Figure 3 includes earlier data for the rabbit muscle
TIM-catalyzed deuterium exchange reaction of GA under the
same reaction conditions.
MonoTIM-Catalyzed Reactions of [1-13C]-GA. It is possible
to monitor the very slow wildtype TIM-catalyzed reactions of
[1-13C]-GA at 25 °C, because the enzyme remains fully active for
at least one week. By contrast, monoTIM loses substantial
activity over a period of several hours. For example, there was
90% loss in enzyme activity during a 9-h room temperature
reaction of 20 mM [1-13C]-GA catalyzed by monoTIM
(0.09 mM) in the presence of 20 mM phosphite dianion at pD
7.0. The value of (kcat/Km)obs = 0.4 Mꢀ1 sꢀ1 for this monoTIM-
catalyzed reaction in the presence of 20 mM phosphite dianion is
80-fold smaller than the calculated value of (kcat/Km)obs = 33
!
ꢀ
ꢁ
ꢀ
ꢀ
ꢁ
kcat
Km
Kd
kcat
Km
¼
Kd þ ½HPO23ꢀꢂ
obs
E
!
ꢁ
½HPO23ꢀꢂ
ꢀ1 sꢀ1 for the wildtype Tbb TIM-catalyzed reaction of [1-13C]-
kcat
Km
M
þ
ð8Þ
GA under the same reaction conditions.b
Kd þ ½HPO23ꢀꢂ
E•HPi
The major product detected of monoTIM catalyzed reactions
is [1-13C, 2,2-di-2H]-GA. The observation that these reactions
remain first-order in the concentration of [1-13C]-GA as mono-
TIM loses more than 90% of its activity toward GAP for catalysis
of isomerization at a functioning active site confirms that [1-13C, 2,
2-di-2H]-GA is formed in a protein-catalyzed reaction,8,70 with a
velocity that is independent of enzymatic activity. Therefore, the
value of kcat/Km for the monoTIM-catalyzed reaction of [1-13C]-
GA at the enzyme active site must be smaller than
The fit of the data for the Tbb TIM-catalyzed reaction to eq 8,
derived for Scheme 6, gave Kd = 19 ( 3 mM for binding of
HPO32ꢀ to the free enzyme and (kcat/Km)E•HPi = 64 ( 6 Mꢀ1 sꢀ1
as the second-order rate constant for turnover of [1-13C]-GA by
enzyme that is saturated by HPO32ꢀ (Table 1). By comparison,
values of Kd = 38 mM and (kcat/Km)E•HPi = 185 Mꢀ1 sꢀ1 were
reported in an earlier study5 of the rabbit muscle TIM-catalyzed
deuterium exchange reaction of unlabeled GA (Table 1). We
estimate that the TIM-catalyzed reaction of [1-13C]-GA in D2O
is ca. 14% faster than the reaction of GA, because the 12% yield of
[2-13C]-GA from the reaction of the carbon-13 labeled substrate
cannot be detected in the degenerate reaction of the unlabeled
substrate. This small correction in the previously reported kinetic
parameters for the rabbit muscle TIM-catalyzed reaction of GA is
given in Table 1.
(kcat/Km)obs = 0.4 Mꢀ1 ꢀ1. We conclude that the structural
s
differences between wildtype Tbb TIM and monoTIM result in
a > 80-fold decrease in the second-order rate constant for the reac-
tion of [1-13C]-GA in the presence of 20 mM phosphite dianion.
A comparison of the X-ray crystal structures for wildtype and
monoTIM shows that elimination of the monomer contacts
results in large movements of loop 1 and loop 4, which lie at the
monomer interface.43 Loop 1 has much larger B-factors for
monoTIM compared with the wildtype enzyme, and there is
no detectable electron density for Lys-13, Cys-14, and Asn-15 for
monoTIM, consistent with a high degree of loop mobility. The
shift in the position of loop 4 results in a 12 Å displacement of the
imidazole side chain of His-95.43
In comparison to rabbit muscle TIM, there is a smaller Kd
(19 compared to 38 mM) and limiting second order rate
constant ((kcat/Km)E•HPi = 64 compared to 210 Mꢀ1 ꢀ1) for
s
phosphite dianion activation of the wildtype Tbb TIM-catalyzed
reactions of [1-13C]-GA, but more similar third order rate
constants for activation by low concentrations of phosphite
dianion ((kcat/Km)E•HPi/Kd = 3400 compared to 5500 Mꢀ2 sꢀ1).
There is a similar trend for values of Km and kcat (smaller for Tbb
Both Lys-1357,73ꢀ75 and His-9576,77 play critical roles in the
wildtype TIM-catalyzed isomerization reaction. It is notable and
surprising that such large changes in the position of these
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ARTICLE
Scheme 7
essential side chains do not eliminate the enzymatic activity for
engineered monoTIM. The observation that K13A and H95A
mutations of monoTIM46 cause further falloffs in catalytic
activity shows that these side chains play an important role in
the reaction catalyzed by the engineered protein. This suggests
that the position of these side chains moves toward that for active
wildtype Tbb TIM on proceeding to the transition state for the
monoTIM catalyzed reaction. Indeed, X-ray crystal structures of
complexes between point mutation variants of monoTIM [kinetic
parameters nearly identical to parent monoTIM studied here]
and 2-phosphoglycolohydroxamate (PGH) or 2-phosphoglyco-
late (PGA) provide direct evidence that ligand binding causes the
side chains of Lys-13 and His-95 to adopt conformations similar
to those observed in wildtype Tbb TIM and very different from
the position at unliganded monoTIM.45
We have proposed that phosphite activation of the TIM-
catalyzed reaction of [1-13C]-GA is due to the utilization of the
intrinsic binding energy78 of the exogenous dianion to stabilize a
rare (Kc , 1, Scheme 7), but catalytically active, closed form of
TIM.5,7,8 This change in conformation from inactive Eo to active
Ec is a complex process that involves the dramatic movement of
loop 6, smaller changes in the positions of loop 7 and 8, and
movement of catalytic side chains at the enzyme active site.42,79ꢀ81
The elimination of contacts between the subunits of TIM in
monoTIM causes additional large movements observed for
loops 1 and 4 away from the catalytically active conformation.
We propose that the effect of these engineered changes in the
conformation of free monoTIM is to cause an increase in the
thermodynamic barrier for the change in conformation from
inactive Eo to active Ec (decrease in Kc, Scheme 7). This places an
additional requirement that the phosphodianion binding energy
be used to drive the enzyme conformational change.
Figure 5. Superimposed crystal structures of the loop-closed Tbb TIM-
PGH complex, shown in gold ribbons and in atoms colored by element
(PDB entry 1TRD) and chicken muscle TIM-PGH complex, shown in
sea green atoms and ribbons (PDB entry 1TPH).
for the mechanism of action of Tbb TIM reported in this work
show a similar high degree of similarity to results from studies on
TIM from yeast, rabbit muscle, and chicken muscle. Taken as a
whole, these data demonstrate a high degree of structural and
mechanistic homology between TIMs from different organisms,
so that the results of studies on triosephosphate isomerase from
one organism may be readily generalized to all TIMs.
By comparison, the engineering of monoTIM causes large
changes in the structure of the unliganded enzyme. However,
there is sufficient conformational mobility in monoTIM to allow
ligand binding to induce a change to the conformation of the
active wildtype structure, which shows optimal interactions with
bound ligands.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: (716) 645 4232. Fax: (716) 645 6963. E-mail: jrichard@
buffalo.edu.
We propose that the binding of phosphite dianion to mono-
TIM drives a change in enzyme conformation similar to that
observed for the binding of phosphate dianion,82 PGH,83
Funding Sources
This work was supported by Grant GM39754 from the National
Institutes of Health.
80
and PGA to wildtype TIM. The failure to detect a specific
monoTIM-catalyzed reaction of [1-13C]-GA or of activation of
this reaction by phosphite dianion would then reflect the overall
larger barrier to the conformational change from Eo to Ec and the
resulting reduction in the concentration of the catalytically active
closed form of the enzyme. We suggest that the concentration of
Ec is too low to give a monoTIM-catalyzed reaction of [1-13C]-
GA or of phosphite activation of this reaction that is detectable by
our analytical methods.
’ ABBREVIATIONS USED
TIM, triosephosphate isomerase;DHAP, dihydroxyacetone
phosphate; DTT, dithiothreitol; GAP, (R)-glyceraldehyde 3-phos-
phate; GA, glycolaldehyde; GPDH, glycerol 3-phosphate dehydro-
genase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase;
BSA, bovine serum albumin; NMR, nuclear magnetic resonance;
D,L-GAP, D,L-glyceraldehyde 3-phosphate; d-DHAP, [1(R)-2H]-di-
hydroxyacetone phosphate; d-GAP, [2(R)-2H]-glyceraldehyde
3-phosphate; MG, methylglyoxal; NADH, nicotinamide adenine
dinucleotide, reduced form; NAD, nicotinamide adenine dinucleo-
tide, oxidized form; PGH, 2-phosphoglycolohydroxamate; PGA,
2-phosphoglycolate; TEA, triethanolamine; Tbb TIM, triosepho-
sphate isomerase from Trypanosoma brucei brucei
Summary and Conclusions. Figure 5 shows the X-ray crystal
structure in the region of the active site of Tbb TIM complexed to
the inhibitor phosphoglycolohydroxamate (PGH) (PDB entry
37
1TRD) superimposed on the X-ray crystal structure of the
chicken TIM-PGH complex (PDB entry 1TPH).81 This com-
parison shows that, while the sequences of Tbb and chicken TIM
are only 50% homologous, the active site architecture for the two
enzymes is nearly identical. The results of several sensitive probes
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ARTICLE
’ ADDITIONAL NOTE
a Unless noted otherwise, residues are numbered according to
the sequence for the enzyme from Trypanosoma brucei.
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b The value of (kcat/Km)obs for the Tbb TIM-catalyzed reaction in
the presence of 20 mM HPO32ꢀ was calculated using eq 8 and
the kinetic parameters for Tbb TIM reported in this paper.
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