Role of Lys-12 in catalysis by triosephosphate isomerase: A two-part substrate approach

Article abstract of DOI:10.1021/bi100538b

We report that the K12G mutation in triosephosphate isomerase (TIM) from Saccharomyces cerevisiae results in (1) a ～50-fold increase in K_m for the substrate glyceraldehyde 3-phosphate (GAP) and a 60-fold increase in K_i for competitive inhibition by the intermediate analogue 2-phosphoglycolate, resulting from the loss of stabilizing ground state interactions between the alkylammonium side chain of Lys-12 and the ligand phosphodianion group; (2) a 12000-fold decrease in k_cat for isomerization of GAP, suggesting a tightening of interactions between the side chain of Lys-12 and the substrate on proceeding from the Michaelis complex to the transition state; and (3) a 6 - 10⁵-fold decrease in k _cat/K_m, corresponding to a total 7.8 kcal/mol stabilization of the transition state by the cationic side chain of Lys-12. The yields of the four products of the K12G TIM-catalyzed isomerization of GAP in D₂O were quantified as dihydroxyacetone phosphate (DHAP) (27%), [1(R)-²H]DHAP (23%), [2(R)-²H]GAP (31%), and methylglyoxal (18%) from an enzyme-ca alyzed elimination reaction. The K12G mutation has only a small effect on the relative yields of the three products of the transfer of a proton to the TIM-bound enediol(ate) intermediate in D₂O, but it strongly favors catalysis of the elimination reaction to give methylglyoxal. The K12G mutation also results in a ≥14-fold decrease in k_cat/K _m for isomerization of bound glycolaldehyde (GA), although the dominant observed product of the mutant enzyme-catalyzed reaction of [1- ¹³C]GA in D₂O is [1-¹³C,2,2-di-²H]GA from a nonspecific protein-catalyzed reaction. The observation that the K12G mutation results in a large decrease in k_cat/K_m for the reactions of both GAP and the neutral truncated substrate [1-¹³C]GA provides evidence for a stabilizing interaction between the cationic side chain of Lys-12 and the negative charge that develops at the enolate-like oxygen in the transition state for deprotonation of the sugar substrate ";piece";.

Full text of DOI:10.1021/bi100538b

Biochemistry 2010, 49, 5377–5389 5377
DOI: 10.1021/bi100538b
Role of Lys-12 in Catalysis by Triosephosphate Isomerase:
A Two-Part Substrate Approach†
Maybelle K. Go, Astrid Koudelka, Tina L. Amyes, and John P. Richard*
Department of Chemistry, University at Buffalo, State University of New York, Buffalo, New York 14260-3000
Received April 9, 2010; Revised Manuscript Received May 17, 2010
ABSTRACT: We report that the K12G mutation in triosephosphate isomerase (TIM) from Saccharomyces
cerevisiae results in (1) a ∼50-fold increase in Km for the substrate glyceraldehyde 3-phosphate (GAP) and a
60-fold increase in Ki for competitive inhibition by the intermediate analogue 2-phosphoglycolate, resulting
from the loss of stabilizing ground state interactions between the alkylammonium side chain of Lys-12 and
the ligand phosphodianion group; (2) a 12000-fold decrease in kcat for isomerization of GAP, suggesting a
tightening of interactions between the side chain of Lys-12 and the substrate on proceeding from the Michaelis
complex to the transition state; and (3) a 6 ꢀ 105-fold decrease in kcat/Km, corresponding to a total 7.8 kcal/mol
stabilization of the transition state by the cationic side chain of Lys-12. The yields of the four products of the
K12G TIM-catalyzed isomerization of GAP in D2O were quantified as dihydroxyacetone phosphate (DHAP)
(27%), [1(R)-2H]DHAP (23%), [2(R)-2H]GAP (31%), and methylglyoxal (18%) from an enzyme-catalyzed
elimination reaction. The K12G mutation has only a small effect on the relative yields of the three products of
the transfer of a proton to the TIM-bound enediol(ate) intermediate in D2O, but it strongly favors catalysis of
the elimination reaction to give methylglyoxal. The K12G mutation also results in a g14-fold decrease in kcat/Km
for isomerization of bound glycolaldehyde (GA), although the dominant observed product of the mutant
enzyme-catalyzed reaction of [1-13C]GA in D2O is [1-13C,2,2-di-2H]GA from a nonspecific protein-catalyzed
reaction. The observation that the K12G mutation results in a large decrease in kcat/Km for the reactions of
both GAP and the neutral truncated substrate [1-13C]GA provides evidence for a stabilizing interaction
between the cationic side chain of Lys-12 and the negative charge that develops at the enolate-like oxygen in
the transition state for deprotonation of the sugar substrate “piece”.
Triosephosphate isomerase (TIM)1 catalyzes the stereospeci-
fic, reversible, 1,2-hydrogen shift in dihydroxyacetone phosphate
(DHAP) to give (R)-glyceraldehyde 3-phosphate (GAP) by a single-
base (Glu-165) proton transfer mechanism through an enzyme-
bound cis-enediol(ate) intermediate (Scheme 1) (1, 2). The enzyme’s
low molecular mass (dimer, 26 kDa/subunit), its high cellular
abundance (3), and the centrality of proton transfer at carbon in
metabolic processes (4-6) have made TIM a prominent target
for studies of the mechanism of enzyme action (1, 7-10).
Deprotonation of the truncated neutral substrate (R)-glycer-
aldehyde by TIM is ∼109-fold slower than the partly diffusion-
controlled (11) turnover of the natural phosphorylated substrate
GAP (12). We showed previously that more than 80% of the
4 ꢀ 1010-fold enzymatic rate acceleration for carbon deprotona-
tion of GAP is derived from the 12 kcal/mol “intrinsic phosphate
binding energy” (13) of the small nonreacting phosphodianion
group of the substrate (12, 14). Similar intrinsic phosphate
binding energies of 12 kcal/mol are observed for the decarboxy-
lation reaction catalyzed by orotidine 50-monophosphate decar-
boxylase (15) and the hydride transfer reaction catalyzed by
glycerol-3-phosphate dehydrogenase (16).
Scheme 1
†This work was supported by Grant GM39754 from the National
Institutes of Health.
*To whom correspondence should be addressed. Telephone: (716)
645-4232. Fax: (716) 645-6963. E-mail: jrichard@buffalo.edu.
1Abbreviations: TIM, triosephosphate isomerase; DHAP, dihydroxy-
acetone phosphate; GAP, (R)-glyceraldehyde 3-phosphate; PGA, 2-phos-
phoglycolate; GA, glycolaldehyde; GPDH, glycerol-3-phosphate dehydro-
genase; BSA, bovine serum albumin; MES, 2-(N-morpholino)ethanesulfonic
acid; CAPSO, 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid;
NADH, nicotinamide adenine dinucleotide, reduced form; TEA, trietha-
nolamine; MOPS, 3-(N-morpholino)propanesulfonic acid; Pfu, DNA
polymerase from hyperthermophilic Pyrococcus furiosus; PCR, polymerase
chain reaction; DpnI, restriction endonuclease from Diplococcus pneumo-
We want to understand the source of the large 12 kcal/mol
intrinsic phosphate binding energy for TIM. X-ray crystallographic
analyses of TIM in a complex with DHAP (7) or 2-phosphogly-
colate (PGA) (10, 17) reveal interactions between the ligand
phosphodianion group and the backbone amide NH groups of
Ser-211 in loop 7, Gly-232 and Gly-233 in loop 8, and Gly-171 in
the flexible “phosphate gripper” loop 6 (18).2 There should be
little or no enthalpic advantage to stabilization of a TIM-bound
ligand by hydrogen bonds between backbone amide NH groups
niae G41; NMR, nuclear magnetic resonance; D,L-GAP, D,L-glyceralde-
hyde 3-phosphate; d-DHAP, [1(R)-2H]-dihydroxyacetone phosphate;
d-GAP, [2(R)-2H]glyceraldehyde 3-phosphate; MG, methylglyoxal; PGH,
2-phosphoglycolohydroxamate.
2Unless noted otherwise, residues are numbered according to the
sequence for the enzyme from yeast.
r
2010 American Chemical Society
Published on Web 05/18/2010
pubs.acs.org/Biochemistry

5378 Biochemistry, Vol. 49, No. 25, 2010
Go et al.
and the ligand phosphodianion group, relative to its stabilization
in aqueous solution by hydrogen bonding to water (19, 20).
However, there is probably a significant entropic advantage to
formation of a network of four effectively intramolecular hydro-
gen bonds to a TIM-bound ligand relative to formation of four
intermolecular hydrogen bonds in aqueous solution, because the
latter is accompanied by the loss of translational and rotational
entropy of four water molecules (19, 20). Furthermore, hydrogen
bonding to the backbone amide NH groups of Gly-232 and Gly-
233 may be enhanced because these residues lie at the N-terminal
end of a short R-helix that has its positive dipole directed toward
the substrate phosphodianion group (18). We note, however, that
it is difficult to examine by experiment the contribution of
individual hydrogen bonds between substrate and backbone
amide NH groups to the rate acceleration for TIM.
that develops at the enolate-like oxygen in the transition state for
deprotonation of this sugar substrate “piece”.
MATERIALS AND METHODS
Materials. Glycerol-3-phosphate dehydrogenase from rabbit
muscle (GPDH) and glycylglycine were from United States
Biochemical. Bovine serum albumin (BSA) was from Roche.
D,L-Glyceraldehyde 3-phosphate diethyl acetal (barium salt), dihy-
droxyacetone phosphate (lithium salt), 2-(N-morpholino)ethane-
sulfonic acid (MES), 3-(cyclohexylamino)-2-hydroxy-1-propane-
sulfonic acid (CAPSO), NADH (disodium salt), and Dowex
50WX4-200R were from Sigma. Triethanolamine hydrochloride
(TEA HCl) and imidazole were from Aldrich. 3-(N-Morpholino)-
3
propanesulfonic acid (MOPS) and ethylamine hydrochloride were
from Fluka. Sodium phosphite (dibasic, pentahydrate) was from
13
€
Riedel-de Haen (Fluka). [1- C]Glycolaldehyde (99% enriched
A major source of the intrinsic phosphate binding energy for
TIM is the interaction between the substrate phosphodianion
and the cationic alkylammonium side chain of Lys-12. The
K12M mutant of yeast TIM was prepared in earlier work, but
the effect of the mutation on the kinetic parameters could not be
determined because the observed activity was due to contaminat-
ing wild-type TIM (21). This wild-type contaminant was pre-
sumed (21) to form as a result of rare errors in the translation of
the methionine codon (AUG) to give the product of translation of
the closely related lysine codon (AAG). The observation of respecta-
ble activities for both the K12R and K12H mutants of yeast TIM,
along with a distinctive pH-rate profile for the K12H mutant,
showed that a positively charged side chain at position 12 of TIM is
required for the observation of robust enzymatic activity (21).
Electrostatic potential maps calculated from X-ray crystal-
lographic data showed that the surface of the active site pocket
for wild-type yeast TIM is cationic, while the corresponding
surface for the K12M mutant is almost entirely anionic (22).
These maps provided evidence that the cationic side chain of Lys-
12 stabilizes the enzyme-bound substrate phosphodianion. How-
ever, it is not clear whether this interaction is expressed entirely
at the Michaelis complex (Km effect) or if it strengthens on pro-
ceeding to the transition state for deprotonation of the bound
substrate (kcat effect). We therefore aimed to characterize and
quantify the effect of removal of the cationic side chain of Lys-12
on the activity of TIM, by substituting glycine for lysine at
position 12. We have constructed the K12G mutant of yeast TIM
by changing the native AAA lysine codon at position 12 to
the GGC codon encoding glycine. This substitution effectively
eliminates the possibility of contamination by the wild-type
enzyme.
We report here the preparation of K12G mutant yeast TIM
and the determination of the effect of this mutation on both the
kinetic parameters and the product distribution for the turnover
of the natural substrate GAP and of the truncated neutral
substrate [1-13C]glycolaldehyde ([1-13C]GA). A comparison of
the kinetic parameters for the wild-type and K12G TIM-cata-
lyzed reactions of GAP shows that the alkylammonium side
chain of Lys-12 stabilizes the transition state for isomerization
of GAP by 7.8 kcal/mol. Approximately 30% of this interac-
tion is expressed at the Michaelis complex with GAP, but ∼70%
(5.6 kcal/mol) is expressed specifically at the transition state for
deprotonation of GAP (kcat effect). We also observe a sizable
effect of the K12G mutation on the kinetic parameters for isomeri-
zation of the truncated substrate [1-13C]GA to give [2-13C]GA,
which provides strong evidence for a stabilizing interaction
between the cationic side chain of Lys-12 and the negative charge
with 13C at C-1, 0.09 M in water) was from Omicron Biochem-
icals. Deuterium oxide (99.9% D) and deuterium chloride [35%
(w/w), 99.9% D] were from Cambridge Isotope Laboratories.
DEAE Sepharose (fast flow) was from GE Healthcare. Water
was obtained from a Milli-Q Academic purification system.
Imidazole was recrystallized from benzene. All other commer-
cially available chemicals were reagent grade or better and were
used without further purification.
2-Phosphoglycolic acid was prepared according to a literature
procedure (23). The barium salt of D-glyceraldehyde 3-phosphate
diethyl acetal was prepared by J. Tait according to a literature
procedure (24).
Preparation of Wild-Type and K12G Mutant Yeast
TIM. The plasmid containing the gene for wild-type TIM from
Saccharomyces cerevisiae (25) and Escherichia coli strain DF502
(strepR, tpi-, and his-) whose DNA lacks the gene for TIM (26)
were generous gifts from N. Sampson.
Site-directed mutagenesis of yeast TIM to introduce the K12G
mutation was conducted using Pfu Ultra High Fidelity DNA
polymerase following the Stratagene protocol. The starting
plasmid DNA (30 ng) was placed into a PCR mixture containing
5 μL of 10ꢀ Pfu ultra buffer, 125 ng each of the forward and
reverse mutagenesis primers, 5 μL of a 2 mM dNTP mixture, and
2.5 units of Pfu Ultra High Fidelity DNA polymerase in a final
volume of 50 μL. The parameters for the PCR were as follows: 45 s
at 95 °C followed by 17 cycles of 45 s at 95 °C, 1.5 min at 55 °C,
and 10 min at 68 °C. The primer used to introduce the K12G
mutation, in which the altered codon is underlined, was 50-CT-
TTC-TTT-GTC-GGT-GGT-AAC-TTT-GGC-TTA-AAC-GGT-
TCC-AAA-CAA-TCC-30. After completion of the PCR, 30 units
of DpnI was added to 30 μL of the reaction mixture followed by
incubation for 1 h at 37 °C, to degrade the methylated template
DNA. E. coli strain K802 was transformed with 1 μL of the DpnI-
digested PCR product. Several colonies containing possible
mutants were picked, and the plasmid DNA was purified using
the QIAprep Miniprep Kit from Qiagen. The presence of the gene
for K12G mutant TIM was verified by sequencing at the Roswell
Park Cancer Institute (Buffalo, NY).
E. coli strain DF502 was transformed with the plasmid con-
taining the gene for wild-type or K12G mutant yeast TIM, and
the proteins were expressed and purified according to published
procedures, with ion exchange chromatography performed using
DEAE Sepharose (27, 28). The purity of the protein in the indi-
vidual column fractions was determined by gel electrophoresis.
Fractions containing the desired TIM at >95% purity were
pooled and dialyzed against 20 mM TEA (pH 7.5).

Article
Preparation of Solutions. 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 7.00, and 4.00 or 10.00 at 25 °C.
Values of pD were obtained by adding 0.40 to the observed
reading of the pH meter (29).
Biochemistry, Vol. 49, No. 25, 2010 5379
isomerization of GAP to form DHAP that was coupled to the
oxidation of NADH using GPDH.
The activities of wild-type and K12G mutant yeast TIM were
determined by coupling the isomerization of GAP to form
DHAP to the oxidation of NADH using GPDH (35). The stan-
dard assay mixture (1.0 mL) contained 30 mM TEA (pH 7.5),
The volume of [1-13C]glycolaldehyde (1 mL of a 90 mM
solution in H2O) was reduced to ∼100 μL by rotary evaporation.
Five milliliters of D2O was added, and the volume was again
reduced to ∼100 μL by rotary evaporation. This procedure was
repeated twice more, and 900 μL of D2O was added to the final
solution to give a volume of ∼1 mL. The stock solution of
[1-13C]GA in D2O was stored at room temperature to minimize
the content of glycolaldehyde dimer (30). The concentration of
0.2 mM NADH, 5 mM
D
,
L
-GAP (2.5 mM GAP), and ∼1 unit of
GPDH at an ionic strength of 0.10 (NaCl). A low background
velocity vo that is due mainly to the isomerization of GAP cata-
lyzed by TIM that was present as an impurity in the commer-
cial preparation of GPDH was determined over a period of
2-4 min. An aliquot of wild-type or K12G TIM was then added,
and the total initial velocity vobs was determined by monitor-
ing the reaction for an additional 5-10 min. The initial velocity
of the TIM-catalyzed reaction (vi) was then calculated with the
relationship vi = vobs - vo, where vo generally represented e2%
1
[1-13C]GA in the stock solution was determined by H NMR
spectroscopy, as described previously (28, 30, 31).
Solutions of GAP in D2O and of
D,L-glyceraldehyde 3-phos-
of vobs.
phate ( -GAP) in H2O were prepared by hydrolysis of the cor-
D
,
L
Values of Ki for competitive inhibition of wild-type and K12G
mutant yeast TIM by PGA at pH 7.5 (30 mM TEA), 25 °C, and
an ionic strength of 0.10 (NaCl) were determined using several con-
centrations of PGA up to 130 μM for wild-type TIM or 2.1 mM
for K12G mutant TIM.
pH-Rate Profile for Turnover of GAP by K12G TIM.
The pH dependence of kcat/Km for isomerization of GAP cata-
lyzed by K12G mutant yeast TIM at 25 °C was determined using
the following buffers: acetate for pH 5.1, MES for pH 5.6 and 6.3,
MOPS for pH 7.1, TEA for pH 7.5, glycylglycine for pH 8.3,
CAPSO for pH 8.9, and carbonate for pH 9.9. The standard
assay mixture (1.0 mL) contained 30 mM buffer, 0.2 mM
responding diethyl acetals, as described previously (28, 32). The
resulting solutions were stored at -20 °C. These solutions were
adjusted to the appropriate pD or pH using 1 M NaOD or 1 M
NaOH before use, after which they were again stored at -20 °C.
The stock solution of PGA was prepared in D2O and was
adjusted to pD 6.9 with 1 M NaOD before use. The concentra-
tion of PGA in the stock solution was determined by 1H NMR as
follows. An aliquot (50 μL) of the stock solution of PGA was
added to 700 μL of 30 mM imidazole buffer (pD 7.0, 20% free
base). Comparison of the integrated areas of the signals due to the
C-4 and C-5 protons of imidazole and the C-2 protons of PGA
gave a concentration of PGA in the stock solution of 42 mM.
Buffered solutions of TEA, MES, MOPS, glycylglycine, and car-
bonate in H2O were prepared by neutralization of the acidic form
with sufficient 1 M NaOH to give the desired pH. Buffered solu-
tions of acetate and CAPSO in H2O were prepared via addition of
sufficient 1 M HCl to the sodium salt to yield the desired pH.
Before the preparation of solutions in D2O, the bulk of the
NADH, 0.8-6 mM
D,L-GAP (0.4-3 mM GAP), and ∼1 unit
of GPDH. The relative specific activity of the coupling enzyme
GPDH was determined at each pH, and the amount used was
adjusted so that the velocity of consumption of NADH was inde-
pendent of the concentration of the coupling enzyme (35).
1H NMR Analyses. 1H NMR spectra at 500 MHz were
recorded in D2O at 25 °C using a Varian Unity Inova 500 spectro-
meter 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 downfield peaks of the double triplet due to the
C-1 proton of [1-13C]GA hydrate. Spectra (16-64 transients)
were recorded using a sweep width of 6000 Hz, a pulse angle of
90°, and an acquisition time of 4-6 s, with zero-filling of the data
to 128 K. To ensure accurate integrals for the protons of interest,
a relaxation delay between pulses of 120 s (>8T1) was used.
Baselines were subjected to a first-order drift correction before
determination of integrated peak areas. Chemical shifts are
reported relative to HOD at 4.67 ppm.
water of crystallization of Na2HPO3 5H2O was removed by in
3
vacuo drying as described previously (30). The acidic protons of
ethylamine hydrochloride were exchanged for deuterium by
repeated dissolution in D2O followed by removal of the solvent
under reduced pressure and in vacuo drying. The stock solution
of EtND3þCl- in D2O was adjusted to pD 6.7 using 1 M NaOD.
Buffered solutions of imidazole and phosphite in D2O were
prepared by dissolving the basic form, and where appropriate
NaCl, followed by the addition of a measured amount of a stock
solution of DCl to give the desired buffer ratio.
Enzyme Assays. All enzyme assays were conducted at 25 °C.
One unit is the amount of enzyme that converts 1 μmol of
substrate to product in 1 min under the specified conditions.
Changes in the concentration of NADH were calculated using
an extinction coefficient of 6220 M-1 cm-1 at 340 nm. We dialy-
zed GPDH at 4 °C against 20 mM TEA (pH 7.5) and assayed
GPDH by monitoring the oxidation of NADH by DHAP at
340 nm, as described previously (28). Dilute solutions of TIM
were stabilized by the inclusion of 0.01% (0.1 mg/mL) BSA. The
subunit concentration of wild-type or K12G yeast TIM in stock
solutions was determined from the absorbance at 280 nm using
an extinction coefficient of 2.55 ꢀ 104 M-1 cm-1 that was cal-
culated using the ProtParam tool available on the ExPASy
server (33, 34). The concentration of GAP in stock solutions of
K12G TIM-Catalyzed Reaction of GAP in D2O Monitored
1
by H NMR. K12G TIM was exhaustively dialyzed at 4 °C
against 10 mM imidazole (pD 7.9, 70% free base) in D2O at an
ionic strength of 0.10 (NaCl). The reaction of GAP (10 mM) in
the presence of K12G TIM in D2O at pD 7.9 (10 mM imidazole),
25 °C, and an ionic strength of 0.15 (NaCl) was monitored by 1H
NMR spectroscopy, as described previously (32). The fraction of
the remaining substrate GAP (fGAP) and the fraction of GAP
converted to products DHAP (fDHAP), d-DHAP (fd-DHAP),
d-GAP (fd-GAP), and methylglyoxal (fMG) at time t were deter-
mined from the integrated areas of the relevant 1H NMR signals
(normalized using the signal due to the C-4 and C-5 protons of
imidazole as an internal standard), as described previously (32).
A control experiment was conducted to monitor the rate of
the nonenzymatic elimination reaction of GAP (10 mM) to give
GAP in D2O or of
D,L-GAP in H2O was determined from the
amount of NADH consumed during quantitative TIM-catalyzed

5380 Biochemistry, Vol. 49, No. 25, 2010
Go et al.
methylglyoxal (14) in D2O at pD 7.9 (10 mM imidazole), 25 °C,
and an ionic strength of 0.15 (NaCl).
K12G TIM-Catalyzed Reactions of [1-13C]GA in D2O
Monitored by 1H NMR. K12G TIM was exhaustively dialyzed
at 4 °C against 30 mM imidazole (pD 7.0, 20% free base) in D2O
at an ionic strength of 0.10 (NaCl) for reactions in the absence of
phosphite and/or EtND3þ, or at an ionic strength of 0.024 for
reactions in their presence.
The K12G TIM-catalyzed reaction of [1-13C]GA in D2O at pD
7.0 (reaction 1) was initiated by addition of 650 μL of enzyme
(∼13 mg/mL) to 350 μL of a solution containing [1-13C]GA and
NaCl to give final concentrations of 20 mM [1-13C]GA, 20 mM
imidazole, and 240 μM K12G TIM in D2O at pD 7.0 and an ionic
strength of 0.10 (NaCl). The K12G TIM-catalyzed reaction of
þ
[1-13C]GA in D2O at pD 7.0 in the presence of 100 mM EtND3
(reaction 2) was initiated by addition of 650 μL of enzyme
(∼13 mg/mL) to 350 μL of a solution containing [1-13C]GA and
EtND3 to give final concentrations of 20 mM [1-13C]GA,
þ
20 mM imidazole, 100 mM EtND3þ, and 310 μM K12G TIM
in D2O at pD 7.0 and an ionic strength of 0.12. The K12G TIM-
catalyzed reaction of [1-13C]GA in D2O at pD 7.0 in the presence
of 50 mM EtND3þ and 10 mM phosphite dianion (20 mM total
phosphite) (reaction 3) was initiated by addition of 600 μL
of enzyme (∼9 mg/mL) to 250 μL of a solution containing
F
IGURE 1: Data for the reaction of GAP (10 mM) in the presence of
85 μM K12G TIM in D2O at pD 7.9 (10 mM imidazole), 25 °C, and
an ionic strength of 0.15 (NaCl) monitored by 1H NMR spectrosco-
py. (A) Time course for the first-order disappearance of the substrate
GAP. (B) Dependence of the observed fractional yields of the pro-
ducts on time. Extrapolation of these data to zero time (;) gave the
initial fractional yields of the products of the enzyme-catalyzed and
nonenzymatic reactions of GAP, (fP)o or (fMG tot
(fMG)E, reported in Table 1: ([) methylglyoxal, (9) d-DHAP, (2)
DHAP, and (b) d-GAP.
[1-13C]GA, phosphite buffer (50% free base), and EtND3 to
þ
give final concentrations of 20 mM [1-13C]GA, 20 mM imidazole,
50 mM EtND3þ, 10 mM phosphite dianion (20 mM total
phosphite), and 290 μM K12G TIM in D2O at pD 7.0 and an
ionic strength of 0.12.
)
= (fMG N
)
þ
described previously (32). Figure 1A shows the time course for
the disappearance of GAP (10 mM) catalyzed by 85 μM K12G
TIM in D2O at pD 7.9 (10 mM imidazole), 25 °C, and an ionic
strength of 0.15 (NaCl). The solid line shows the nonlinear least-
squares fit of the experimental data to a single exponential which
gave a kobs of 3.6ꢀ10-4 s-1 as the observed rate constant for the
disappearance of GAP (Table 1). Figure 1B shows the time
dependence of the observed fractional yields of the four products
of this reaction, (fP)obs (P = DHAP, d-DHAP, d-GAP, or MG).
These fractional yields were calculated from the fraction of the
particular product P (see Materials and Methods) and the sum of
the fractions of all the products of both the enzymatic and
nonenzymatic reactions of GAP using eq 3 (Scheme 2). There
is no significant change in (fP)obs for DHAP with time, but
the changes with time in the observed fractional yields of
d-DHAP (increasing) and d-GAP (decreasing) result from
In each case, 750 μL of the reaction mixture was transferred to
an NMR tube and 1H NMR spectra (32 transients) at 25 °C were
recorded over a period of 4-7 days. The remaining portion of the
reaction mixture was incubated at 25 °C, and the activity of
K12G TIM was monitored by a periodic standard assay (see
above). For reaction 1, there was an ∼20% drop in the activity of
K12G TIM over 78 h at 25 °C. For reactions 2 and 3, there was an
∼40% drop in the activity of K12G TIM over 7 days at 25 °C.
The fraction of the remaining substrate [1-13C]GA (fS) and
the fraction of [1-13C]GA converted to identifiable products
[1-13C,2-2H]GA and [1-13C,2,2-di-2H2]GA were determined from
the integrated areas of the relevant 1H NMR signals (normalized
using the signal due to the C-4 and C-5 protons of imidazole as an
internal standard), as described previously (28).
Observed first-order rate constants, kobs, for the disappearance
of [1-13C]GA were determined from the slopes of linear semi-
logarithmic plots of reaction progress versus time according to eq 1
Scheme 2
ln fS ¼ - kobs
t
ð1Þ
Observed second-order rate constants for the total protein-
catalyzed reactions of [1-13C]GA were calculated using eq 2
ꢀ
ꢁ
kcat
Km
kobs
¼
ð2Þ
fcar½Eꢁ
obs
where fcar (0.061) is the fraction of GA present in the reactive
carbonyl form (30, 31) and [E] is the concentration of K12G TIM.
RESULTS
K12G TIM-Catalyzed Reaction of GAP in D2O. The
disappearance of the substrate and the appearance of the pro-
ducts of the nonenzymatic and K12G TIM-catalyzed reactions
1
of GAP in D2O were monitored by H NMR spectroscopy, as

Article
Biochemistry, Vol. 49, No. 25, 2010 5381
Table 1: Fractional Product Yields for the Reaction of (R)-Glyceraldehyde 3-Phosphate in the Presence of K12G Mutant Yeast Triosephosphate Isomerase
in D2Oa
kobs (s-1 b
)
MG (fMG tot
)
MG (fMG N
)
MG (fMG E
)
d-GAP (fP)o
DHAP (fP)o
d-DHAP (fP)o
c
d
e
f
f
f
[K12G TIM] (μM)
85
3.6 ꢀ 10-4
8.2 ꢀ 10-5
0.25
0.34
0.05
0.21
0.20
0.21
0.27
0.28
0.35
0.26
0.33
0.40
0.25
0.26
0.33
0.22
0.28
0.33
0.21
0.22
0.28
0.19
0.24
0.29
g
fE
h
h
h
(fE)PT
12
0.13
0.16
g
fE
(fE)PT
g
fE
average values
0.18 ( 0.02
0.31 ( 0.03
0.27 ( 0.01
0.23 ( 0.01
(fE)PT
0.38 (0.21)i
0.33 (0.49)i
0.29 (0.31)i
aProduct distributions for the reaction of GAP (10 mM) at pD 7.9 (10 mM imidazole), 25 °C, and an ionic strength of 0.15 (NaCl) were determined by 1H
NMR spectroscopy as described previously (32). bObserved first-order rate constant for the disappearance of GAP in the presence of the indicated con-
centration of K12G TIM. cTotal initial fractional yield of methylglyoxal determined by extrapolation of (fP)obs to zero time (intercept in Figure 1B). dInitial
fractional yield of methylglyoxal from the competing nonenzymatic reaction of GAP, calculated using eq 4. eInitial fractional yield of methylglyoxal from the
enzymatic reaction of GAP, calculated using eq 5. fInitial fractional product yields determined by extrapolation of (fP)obs to zero time (intercepts in
Figure 1B). gNormalized fractional yields of the products of the enzymatic reaction of GAP, calculated using eq 9. hNormalized fractional yields of the three
products of the transfer of a proton to the enzyme-bound enediolate intermediate, calculated using eq 10. iData for the wild-type enzyme from chicken muscle
taken from previous work (32).
enzyme-catalyzed isomerization of d-GAP to give the thermodyna-
mically favored product d-DHAP (32). Table 1 reports the initial
fractional product yields, (fP)o, forDHAP, d-DHAP, and d-GAP, or
(fMG tot for MG, that were determined by extrapolation of the ob-
)
served product yields (fP)obs to zero time (intercepts in Figure 1B).
fP
ðfPÞobs
¼
ð3Þ
fDHAP þ fd-DHAP þ fd-GAP þ fMG
kN
ðfMGÞN
¼
ð4Þ
ð5Þ
kobs
ðfMGÞE ¼ ðfMGÞtot - ðfMGÞN
In a control experiment, the nonenzymatic elimination reac-
tion of GAP (10 mM) to form methylglyoxal (14) in D2O at pD
7.9 (10 mM imidazole), 25 °C, and an ionic strength of 0.15
(NaCl) was monitored by 1H NMR spectroscopy (32). The fit of
the data to a single exponential gave an observed first-order rate
F
IGURE 2: Semilogarithmic plots of the fraction of the remaining
substrate vs time for the reaction of [1-13C]glycolaldehyde in the pre-
sence of K12G TIM in D2O at pD 7.0 (20 mM imidazole) and 25 °C.
The observed rate constants were determined from the slopes accord-
ing to eq 1: (2) 240 μM K12G TIM at an ionic strength of 0.10, (9)
310μMK12GTIMand 100mMEtND3þ atanionic strengthof0.12,
and (b) 290 μM K12GTIM, 50 mMEtND3þ, and 10mMHPO32- at
an ionic strength of 0.12.
constant for the disappearance of GAP (kN) of 1.7 ꢀ 10-5 s-1
.
This was combined with a kobs of 3.6ꢀ10-4 s-1 for the reaction of
GAP in the presence of 85 μM K12G TIM, according to eq 4, to
give the calculated fractional yield of methylglyoxal from the
nonenzymatic elimination reaction in this experiment [(fMG)N =
0.05 (Table 1)]. The total initial fractional yield of methylglyoxal
[(fMG)tot] of 0.25 (Table 1) is 5-fold larger than (fMG)N because,
for 78 h in the presence of 240 μM K12G TIM in D2O at pD 7.0
and 25 °C. Under our reaction conditions, glycolaldehyde exists
as 93.9% hydrate and 6.1% free carbonyl form (30, 31), and the
following chemical shifts refer to the hydrates of the isotopomers
shown in Chart 1. The signal due to the C-1 proton of [1-13C]GA
unlike wild-type TIM, K12G TIM also catalyzes the elimination
reaction of GAP to give methylglyoxal. The initial fractional
yield of methylglyoxal from the K12G TIM-catalyzed reaction
[(fMG)E] of 0.20 (Table 1) was calculated using eq 5.
K12G TIM-Catalyzed Reaction of [1-13C]GA in D2O.
Figure 2 shows semilogarithmic plots according to eq 1 of the
time course for the disappearance of [1-13C]GA in D2O at pD 7.0
(20 mM imidazole) and 25 °C, monitored by 1H NMR spectro-
scopy (28), in the presence of K12G TIM, K12G TIM and 100 mM
EtND3þ, or K12G TIM, 50 mM EtND3þ, and 10 mM phosphite
dianion (20 mM total phosphite). Table 2 gives the observed first-
order rate constants for these reactions that were calculated
from the slopes of these linear correlations that covered one to
two half-times.
appears as a double triplet at 4.945 ppm (1JHC=163 Hz; 3JHH
=
5 Hz) (Figure 3B). The signal due to the C-1 proton of [1-13C,2,2-
di-2H]GA appears as a broad doublet (1JHC = 163 Hz) at 4.930
ppm that is shifted 0.015 ppm upfield from the double triplet due
to the C-1 proton of [1-13C]GA as a result of the two β-deuteriums
(Figure 3B). The signal due to the C-2 protons of [1-13C]GA app-
ears as a double doublet at 3.410 ppm (2JHC=3 Hz; 3JHH=5 Hz)
(Figure 3A). The signals due to the C-2 protons of [2-13C]GA and
[1,2-di-13C]GA, present initially at 0.8 and 0.9%, respectively,
in our commercial [1-13C]GA, appear at 3.410 ppm as a double
Figure 3 shows portions of the 1H NMR spectrum at 500 MHz
of the reaction mixture obtained from the reaction of [1-13C]GA
3
doublet (1JHC = 142 Hz; JHH = 5 Hz) and a double double

5382 Biochemistry, Vol. 49, No. 25, 2010
Go et al.
Table 2: Rate and Product Data for the Reactions of [1-13C]GA in the Presence of K12G Mutant Yeast Triosephosphate Isomerase and the Potential
Activators EtND3þ and Phosphite Dianion in D2Oa
fractional product yield
c
d
[K12G TIM]
(kcat/Km)obs
(kcat/Km)iso
(M-1 s-1
(μM)
activator
kobsb (s-1
)
(M-1 s-1
)
)
[2-13C]GA
[1-13C,2-2H]GA
[1-13C,2,2-di-2H2]GA
totale
240
310
290
nonef
1.6 ꢀ 10-6
2.3 ꢀ 10-6
3.8 ꢀ 10-6
0.11
0.12
0.22
e7 ꢀ 10-4
e0.006g
small
0.10
0.14
0.56
0.20
0.19
0.56
0.30
0.32
þh
100 mM EtND3
50 mM EtND3þ and
none detected
none detected
2-h
10 mM HPO3
aProduct distributions for the reaction of [1-13C]GA (20 mM) at pD 7.0 (20 mM imidazole) and 25 °C were determined by 1H NMR spectroscopy as
described previously (28). bObserved first-order rate constant for the disappearance of [1-13C]GA. cTotal observed second-order rate constant for the specific
and nonspecific protein-catalyzed reactions. dSecond-order rate constant for the specific K12G TIM-catalyzed isomerization of [1-13C]GA to give [2-13C]GA,
calculated as the product of the (kcat/Km)obs (0.11 M-1 s-1) and 0.006 as the upper limit on the fractional yield of [2-13C]GA. eTotal fractional yield of the
identifiable products. fAt an ionic strength of 0.10 (NaCl). gUpper limit on the fractional yield of [2-13C]GA (see the text). hAt an ionic strength of 0.12
(NaCl).
Chart 1
product of isomerization with deuterium exchange, [2-13C,2-2H]GA,
would appear as a double double triplet at 3.389 ppm (1JHC
=
142 Hz; 3JHH =5 Hz; 2JHD ≈ 2 Hz) shifted 0.021 ppm upfield of
the double doublet due to the C-2 protons of [2-13C]GA as a
result of the R-deuterium (28). However, this signal was not obser-
ved in the spectrum shown in Figure 3A.
AP
fP
¼
ð6Þ
ðASÞo - AS
During the reaction of [1-13C]GA catalyzed by wild-type TIM
in D2O, we observed that enzyme-catalyzed isomerization of
the substrate results in an increase in the peak area of the signals
due to [2-13C]GA (28). By contrast, we observe here that the
peak area of the signal due to the C-2 protons of [2-13C]GA
(initially present as 0.8% of total GA) decreases during the
reaction of [1-13C]GA for 78 h in the presence of 240 μM K12G
mutant TIM in D2O. However, during this time, there is a small
increase in the ratio of the peak areas of the signals due to the
C-2 protons of [2-13C]GA and [1-13C]GA from 0.008 to 0.010,
but no change in the ratio (0.009) of the peak areas of the sig-
nals due to the C-2 protons of [1,2-di-13C]GA and [1-13C]GA.
These changes in relative peak areas are consistent with essen-
tially equal velocities of conversion of the three starting isotopo-
mers to their corresponding C-2 doubly deuteriated analogues,
for which there are no 1H NMR signals in the C-2 region, along
with a very slow enzyme-catalyzed isomerization of [1-13C]GA
to give [2-13C]GA. These data are consistent with conversion of
e0.2% of the [1-13C]GA to [2-13C]GA that accompanies the
much faster conversion of 20% of the [1-13C]GA to [1-13C,2,2-
di-2H]GA during this 78 h reaction. Therefore, the fractional
yield of [2-13C]GA [e0.006 (Table 2)] from the K12G TIM-
catalyzed isomerization of [1-13C]GA is estimated to be at least
100-fold lower than the fractional yield of [1-13C,2,2-di-2H]GA
(0.56).
1
F
IGURE 3: Portions of the H NMR spectrum at 500 MHz of the
reaction mixture obtained from the reaction of [1-13C]GA (20 mM)
for 78 h in the presence of 240 μM K12G TIM in D2O at pD 7.0, 25 °C,
and an ionic strength of 0.10 (NaCl). (A) Spectrum in the region of
the C-2 hydron(s) of the isotopomers of GA. (B) Spectrum in the
region of the C-1 hydron of the isotopomers of GA.
doublet (1JHC=142 Hz; 2JHC=3 Hz; 3JHH=5 Hz), respectively.
Figure 3A (inset) shows the upfield peaks of these signals; the cor-
responding downfield peaks appear along with other small peaks
for unidentified reaction products.
We observe that the major identifiable product of the reaction
of [1-13C]GA in the presence of K12G mutant TIM in D2O is the
doubly deuteriated isotopomer [1-13C,2,2-di-2H]GA which is
formed in a nonspecific reaction occurring outside the active
site (28). The fractional yield of this product [fP=0.56 (Table 2)]
was calculated from the normalized peak area for its C-1 proton,
AP, and the difference in the normalized peak area for the C-1
proton of the substrate [1-13C]GA at time zero, (AS)o, and at time t,
AS, according to eq 6. The signal due to the C-2 proton of the

Article
Biochemistry, Vol. 49, No. 25, 2010 5383
nonlinear least-squares fit of these data to eq 7 with a kcat/Km
of 12 M-1 s-1 in the absence of PGA.
kcat=Km
ðkcat=KmÞapp
¼
ð7Þ
1 þ ½PGAꢁ=Ki
Figure 6 shows pH-rate profiles of the observed second-order
rate constants (kcat/Km)obs for the isomerization of GAP cata-
lyzed by wild-type TIM from chicken muscle [data of Plaut and
Knowles (35)] and by K12G mutant yeast TIM (data from this
work). The literature data for wild-type TIM were fit to eq 8,
derived for Scheme 3, using a (kcat/Km)* of 0 and a pKSH of 6.0 for
the ionization of the phosphodianion group of GAP and a pKEH
of 9.2 for the ionization of an essential residue at TIM (35). The
solid and dashed lines through the data for K12G yeast TIM
compare the fit obtained using a kcat/Km of 12 M-1 s-1 (Table 3),
a pKSH of 6.0, a pKEH of 9.2, and a (kcat/Km)* of 0 (solid line) with
that using a (kcat/Km)* of 1.1 M-1 s-1 (dashed line) that was
obtained from least-squares analysis that included (kcat/Km)* as
an additional parameter.
"
#
ꢂꢀ
ꢁ
½Hþꢁ
kcat
Km
ðkcat=KmÞobs
¼
KSH
ðKEH þ ½HþꢁÞðKSH þ ½HþꢁÞ
ꢀ
ꢁ
ꢃ
ꢂ
kcat
Km
þ
½Hþꢁ
ð8Þ
Scheme 3
F
IGURE 4: Michaelis-Menten plot of initial velocity data for
the isomerization of GAP catalyzed by K12G TIM at pH
7.5 (30 mM TEA), 25 °C, and an ionic strength of 0.10 (NaCl).
The solid line is the fit of the data to the Michaelis-Menten
equation, and the dashed line is the linear relationship for the
case in which [GAP] , Km. The inset shows the linear correlation
of the data for e3 mM GAP, the slope of which gives a kcat/Km of
12 M-1 s-1
.
Table 2 also gives the fractional yields of the identifiable
products of the K12G TIM-catalyzþed reactions of [1-13C]GA
þ
in the presence of 100 mM EtND3 or 50 mM EtND3 and
10 mM phosphite dianion. These yields were calculated from
the normalized peak areas of the signals due to these products
using eq 6. In all cases, the sum of the fractional yields of the
products of these slow reactions of [1-13C]GA is well under
100% (28). No attempt was made to identify the other
pathways for the slow reactions of [1-13C]GA in the presence
of K12G TIM.
Kinetic Parameters and pH-Rate Profile for Isomeriza-
tion of GAP by K12G TIM. Figure 4 shows the Michaelis-
Menten plot for the isomerization of GAP catalyzed by
K12G mutant yeast TIM at pH 7.5 (30 mM TEA), 25 °C,
and an ionic strength of 0.10 (NaCl). The solid line shows the
nonlinear least-squares fit of these data to the Michaelis-
Menten equation which gave a kcat of 0.6 ( 0.2 s-1 and a Km of
50 ( 20 mM (Table 3); the dashed line is the linear relationship for
the case in which [GAP] , Km. Table 3 also gives the much more
reliable value of kcat/Km (12 ( 0.4 M-1 s-1) that was determined
as the slope of the linear correlation of the data at e3 mM GAP
(Figure 4, inset).
DISCUSSION
The X-ray crystal structure of yeast TIM shows that the
cationic side chain of Lys-12 runs along the enzyme surface (7).
Closure of phosphate gripper loop 6 over the substrate sequesters
the carbon acid fragment from interaction with solvent, but the
tip of the substrate phosphodianion group lies at the protein sur-
face where it forms a solvent-separated ion pair with the cationic
side chain of Lys-12 (7). X-ray crystallographic analysis of
K12M/G15A TIM crystallized in the presence of the enediol(ate)
intermediate analogue 2-phosphoglycolohydroxamate (PGH)
revealed no bound ligand and showed that the structure of this
enzyme was nearly identical to that of unliganded wild-type TIM
(22). We therefore do not expect that the K12G mutation will
result in a large change in protein structure, but it should leave a
small water-filled cleft at the protein surface.
Figure 5 shows the dependence of the apparent second-
order rate constant (kcat/Km)app for K12G TIM-catalyzed
isomerization of GAP (determined as the slopes of plots of
vi/[E] vs [GAP]) on the concentration of 2-phosphoglycolate
(PGA) at pH 7.5, 25 °C, and an ionic strength of 0.10 (NaCl).
The Ki value of 1.1 mM (Table 3) was determined from the
We report here a second-order rate constant kcat/Km of
12 M-1 s-1 for isomerization of GAP catalyzed by K12G mutant
yeast TIM at pH 7.5, 25 °C, and an ionic strength of 0.10
(Table 3). In a previous study of the K12M mutant, the apparent
kcat/Km value of 30 M-1 s-1 was attributed to the presence of
∼0.0005% contaminating wild-type TIM in the mutant enzyme

5384 Biochemistry, Vol. 49, No. 25, 2010
Go et al.
Table 3: Kinetic Parameters for Isomerization of GAP by Wild-Type and K12G Mutant Yeast Triosephosphate Isomerasea
yeast TIM
kcatb (s-1
)
Kmb (mM)
kcat/Km (M-1 s-1
)
Ki for PGA (mM)
wild type
7300 ( 400
0.6 ( 0.2
1.1 ( 0.2
50 ( 20
6.6 ꢀ 106c
12 ( 0.4e
0.019 ( 0.004d
1.1 ( 0.2f
K12G
effectg
1.2 ꢀ 104
50
5.5 ꢀ 105
60
ΔΔG or ΔΔGq (kcal/mol)
5.6
2.3
7.8
2.4
aAt pH 7.5 (30 mM TEA), 25 °C, and an ionic strength of 0.10 (NaCl). Quoted errors are standard errors obtained from least-squares analysis. bDetermined
from the fit of initial velocity data to the Michaelis-Menten equation. cCalculated as the ratio of the values of kcat and Km. dDetermined by global nonlinear
least-squares analysis of initial velocity data in the presence of 0, 21, and 130 μM PGA. eDetermined as the slope of the plot of vi/[E] vs [GAP] at e3 mM GAP
(Figure 4, inset). fDetermined from the nonlinear least-squares fit of the data in Figure 5 to eq 7. gEffect of the K12G mutation on the kinetic parameter.
determined in this work, which are in good agreement with the
previously published literature values (36).
In an earlier study of K12M TIM, this mutant was thought to
have a very weak affinity for phosphodianion ligands. The Ki
value of 1.1 mM determined here for inhibition of K12G TIM by
PGA at pH 7.5 (Figure 5 and Table 3) shows that the K12G
mutant forms a moderately stable complex with this inhibitor
trianion. However, it is more difficult to evaluate the stability of
the complex of K12G TIM with GAP. The slight curvature in the
Michaelis-Menten plot of vi/[E] versus [GAP] (Figure 4) is con-
sistent with a Km of 50 ( 20 mM, but these data also show a
reasonable fit to a linear equation. We note that (1) if the K12G
mutation results in a similar 60-fold increase in the dissociation
constants for both PGA and GAP, then the value of Km for GAP
would be ca. 60 mM which is close to the Km of ≈50 mM obtained
F
IGURE 5: Dependence of the apparent second-order rate constant
from the data in Figure 4 and (2) the effect of the K12G mutation
on the affinity of the enzyme for GAP should not be any larger
than the effect on its affinity for PGA, because the additional
negative charge at PGA will tend to strengthen, not weaken, the
interaction of the ligand with the cationic side chain of Lys-12 of
the wild-type enzyme. Therefore, we conclude that K12G TIM
has a weak affinity for GAP with a Km of 50-60 mM.
(kcat/Km)app for the isomerization of GAP catalyzed by K12G mutant
yeast TIM on the concentration of 2-phosphoglycolate at pH 7.5 (30 mM
TEA), 25 °C, and an ionic strength of 0.10 (NaCl).
K12G TIM-Catalyzed Reaction of GAP in D2O. We repor-
ted previously that the reaction of GAP catalyzed by wild-type
rabbit or chicken muscle TIM in D2O, monitored for 2-5 h (32),
results in the formation of the three products of the enzyme-
catalyzed reaction along with substantial formation of methyl-
glyoxal from the competing nonenzymatic elimination reaction
(Scheme 2) (14). We observe here that the K12G mutation in
yeast TIM results in a large increase in the velocity of formation
of methylglyoxal over that predicted for the competing none-
nzymatic elimination reaction (Table 1). This shows that K12G
TIM also catalyzes the elimination reaction of GAP to give
methylglyoxal (Scheme 4). Table 1 reports the normalized frac-
tional yields fE of the products of the enzymatic reaction of GAP
catalyzed by 85 and 12 μM K12G TIM in D2O at pD 7.9. These
F
IGURE 6: pH dependence of the observed second-order rate con-
yields were calculated using eq 9, where (fMG E
yield of methylglyoxal from the enzymatic reaction that was cal-
culated from the total fractional yield of methylglyoxal (fMG tot
as described in Results.
) is the fractional
stant (kcat/Km)obs for the isomerization of GAP catalyzed by wild-
type and K12G TIM. ([) Data of Plaut and Knowles (35) for wild-
type TIM from chicken muscle at 30 °C. The solid line shows the fit of
the data to eq 8 with a (kcat/Km)* of 0. (b) Data from this work for
K12G mutant yeast TIM at 25 °C. The solid and dashed lines
compare the nonlinear least-squares fits of these data to eq 8 using
(kcat/Km)* values of 0 and 1.1 M-1 s-1, respectively, for turnover of
GAP monoanion (Scheme 3).
)
ð fPÞo
fE
¼
ð9Þ
ð fDHAPÞo þ ð fd-DHAPÞo þ ð fd-GAPÞo þ ð fMGÞE
preparation (21). By comparison, the lowered affinity of K12G
TIM for GAP and PGA determined here (Table 3) shows that the
observed activity of the K12G mutant cannot be due to a
contaminating wild-type TIM activity. Table 3 also reports the
kinetic parameters for turnover of GAP by wild-type yeast TIM
ð fPÞo
ðfEÞPT
¼
ð10Þ
ð fDHAPÞo þ ð fd-DHAPÞo þ ð fd-GAPÞo
A rate constant ratio kPT/kelim of ≈1 ꢀ 106 was estimated
for partitioning of the enzyme-bound enediol(ate) phosphate

Article
Biochemistry, Vol. 49, No. 25, 2010 5385
Scheme 4
the very large 6ꢀ105-fold effect of the K12G mutation on kcat/Km
for isomerization of GAP (Table 3).
We conclude that Lys-12 plays an important role in stabilizing
both the transition state for deprotonation of GAP and of the
bound enediol(ate) intermediate toward elimination of phosphate
dianion. However, it plays a much less important role in con-
trolling the partitioning of the enediol(ate) intermediate in D2O
among DHAP, d-GAP, and d-DHAP.
K12G TIM-Catalyzed Reaction of [1-13C]GA in D2O.
The large difference between the kcat/Km for the isomerization of
GAP (1.0 ꢀ 108 M-1 s-1)3 and that for the deprotonation of
glycolaldehyde (∼0.10 M-1 s-1)4 by wild-type chicken muscle
TIM shows that interactions between the substrate phosphodia-
nion and TIM provide an ∼12 kcal/mol stabilization (intrinsic
phosphate binding energy) of the transition state for proton
transfer from GAP (12, 16). Truncation of GAP to give the neut-
ral substrate glycolaldehyde eliminates transition state stabiliza-
tion resulting from interactions with the phosphodianion group.
Therefore, the observation of a large effect of the K12G mutation
on the rate constant for deprotonation of glycolaldehyde would
provide direct evidence of stabilizing interactions between the
excised cationic side chain of Lys-12 and the carbon acid sub-
strate piece. This two-part substrate approach was used to probe
the interactions of amino acid side chains with the phosphodia-
nion and nucleoside portions of the substrate in the decarboxy-
lation of orotidine 50-monophosphate catalyzed by orotidine
50-monophosphate decarboxylase (41).
intermediate of the wild-type TIM-catalyzed reaction between
protonation at carbon and elimination of inorganic phos-
phate (37). This is much larger than the kPT/kelim of 6.5 for
partitioning of the same enediol(ate) phosphate between proton
transfer at carbon and elimination of phosphate within a loose
complex with a small tertiary ammonium cation in solution (14).
These observations show that interactions with wild-type TIM
strongly stabilize the bound enediol(ate) phosphate intermediate
toward elimination of inorganic phosphate (37, 38). The absence of
the alkylammonium side chain of Lys-12 of K12G mutant yeast
TIM results in a dramatic change in the ratio of the yields of the
products of proton transfer and elimination (Scheme 4), from a
kPT/kelim of ≈1ꢀ106 (37) to a value of 4.5 (0.81/0.18) (Table 1). This
dramatic change in the partitioning of the enediol(ate) phosphate
intermediate shows that interaction of the cationic side chain of
Lys-12 with the bound enediol(ate) phosphate strongly protects
this species from elimination of inorganic phosphate. Good yields
of methylglyoxal are also observed from the reactions of GAP and
DHAP catalyzed by a loop deletion mutant of TIM (38), because
interactions between the enediol(ate) phosphate and the flexible
loop (loop 6) of TIM play a vital role in preventing breakdown
of the intermediate with the loss of inorganic phosphate.
Table 1 also reports the normalized fractional yields (fE)PT of
the three products of the transfer of a proton to the enzyme-
bound enediol(ate) intermediate of the reactions of GAP cata-
lyzed by wild-type TIM from rabbit or chicken muscle (32) and
K12G mutant yeast TIM (this work, calculated using eq 10).
These data show that the K12G mutation causes the yield of
DHAP formed by intramolecular transfer of hydrogen from sub-
strate GAP to decrease from 49% for the wild-type enzyme to
33% for the K12G mutant. There is good evidence that the
We reported previously that the reaction of [1-13C]GA cata-
lyzed by wild-type chicken muscle TIM gives an ∼50% combined
yield of isomerization product [2-13C]GA, the product of iso-
merization with deuterium exchange ([2-13C,2-2H]GA), and the
product of deuterium exchange ([1-13C,2-2H]GA) resulting from
the “specific” reactions of [1-13C]GA at the enzyme active site (28)
(Scheme 5). By contrast, the only clearly detectable product of the
reaction of [1-13C]GA catalyzed by K12G mutant yeast TIM is
the dideuteriated isotopomer [1-13C,2,2-di-2H]GA which is for-
med in ∼56% yield (Table 2). This isotopomer was also observed
as a minor product (∼20% yield) of the wild-type chicken muscle
TIM-catalyzed reaction, where it was proposed to form by a
“nonspecific” protein-catalyzed reaction that occurs outside the
enzyme active site (28).
Scheme 5
H-labeled carboxylic acid side chain of Glu-165 of the TIM enediol-
3
(ate) complex is sequestered from bulk solvent D2O and that this
residue undergoes exchange with a small pool of similarly
sequestered hydrons followed by the transfer of a proton to the
enediol(ate) to form d-GAP and d-DHAP (Scheme 4) (39, 40).
Therefore, the decrease in the yield of the product of intramo-
lecular transfer of hydrogen is nominally consistent with the con-
clusion that the K12G mutation increases the accessibility of the
active site to the bulk solvent. The K12G mutation also results in
an increase in the ratio of the yields of d-GAP and d-DHAP, from
0.7 for wild-type TIM to 1.3 for the K12G mutant. However,
these small effects are difficult to rationalize in comparison with
3Values for kcat of 2300 s-1 and Km of 0.45 mM for isomerization of
GAP by chicken muscle TIM at pH 7.5 and 25 °C were reported in our
earlier work (28). GAP exists as 95% hydrate and 5% free carbonyl (the
reactive form) in D2O at pD 7.9, 25 °C, and an ionic strength of 0.10
(NaCl) (32).

5386 Biochemistry, Vol. 49, No. 25, 2010
Go et al.
The second-order rate constant (kcat/Km) for the nonspecific
K12G TIM-catalyzed reaction of [1-13C]GA to give [1-13C,2,2-
di-2H]GA that occurs outside the active site can be calculated as
(0.11 M-1 s-1)(0.56)=0.062 M-1 s-1, where 0.11 M-1 s-1 is the
observed second-order rate constant for the K12G TIM-cata-
lyzed disappearance of [1-13C]GA and 0.56 is the fractional yield
of [1-13C,2,2-di-2H]GA (Table 2). This rate constant is at least
90-fold larger than the (kcat/Km)iso of e7ꢀ10-4 M-1 s-1 for the
isomerization of [1-13C]GA at the active site to give [2-13C]GA
(Table 2). The corresponding second-order rate constant for the
isomerization of [1-13C]GA catalyzed by wild-type chicken
muscle TIM can be calculated from data in our previous work
as (kcat/Km)iso = 9.5ꢀ 10-3 M-1 s-1 (28).5 Therefore, the K12G
mutation results in an at least 14-fold decrease in (kcat/Km)iso for
the isomerization of [1-13C]GA. This provides direct evidence
that the cationic side chain of Lys-12 stabilizes the transition state
for the transfer of a proton from glycolaldehyde by interaction
with the carbon acid substrate piece. It is reasonable to conclude
that this side chain also acts similarly to stabilize the transition
sate for isomerization of GAP by interaction with both the
nonreacting phosphodianion and the reacting carbon acid frag-
ments of the whole substrate.
turnover of GAP monoanion results in a small improvement in
the fit of the experimental data to eq 8 (see Results). This suggests
that K12G TIM exhibits an only ∼10-fold selectivity for catalysis
of the isomerization of GAP dianion [(kcat/Km) = 12 M-1 s-1
(Table 3)] over GAP monoanion [(kcat/Km)* = 1.1 M-1 s-1].
The downward break centered at pH 9.2 for wild-type TIM
cannot be due to deprotonation of the cationic side chain of Lys-
12 because the same break is observed in the profile for K12G
TIM (Figure 6).6 This demonstrates that pKa > 9.2 for the
cationic side chain of Lys-12, which is consistent with the salt
bridge with the carboxylate side chain of Glu-97 observed for
TIM PGA and TIM PGH complexes (10, 17, 43). It also sug-
3
3
gests that there is a large distance between the side chains of Lys-12
and this second critical residue.
Specificity in Transition State Binding. The K12G muta-
tion in yeast TIM results in a 5.5ꢀ105-fold decrease in kcat/Km for
the isomerization of GAP (Table 3), corresponding to a 7.8 kcal/
mol stabilization of the transition state by the cationic side chain
of Lys-12. This effect can be partitioned into an ∼50-fold effect
on Km (ΔΔG = 2.3 kcal/mol) and a much larger ∼12000-fold
effect on kcat for the isomerization of the bound substrate [ΔΔGq=
5.6 kcal/mol (Table 3)]. The effect of the mutation on Km can be
directly attributed to the loss of ground state electrostatic
interactions between the substrate phosphodianion group and
the cationic side chain of Lys-12. More importantly, the large
effect on kcat shows that there is a significant strengthening of the
interactions between the ligand and the alkylammonium side
chain of Lys-12 on moving from the ground state Michaelis
complex to the transition state for carbon deprotonation. The
most striking structural change on moving from the Michaelis
complex to the enediol(ate)-like transition state for deprotona-
tion of GAP is the change in formal charge at the substrate
carbonyl oxygen, from 0 to -1, and the change in the total charge
at bound ligand, from -2 to -3. This increase in negative charge
is expected to result in an increase in the strength of the stabilizing
electrostatic interactions between the alkylammonium side chain
of Lys-12 and the altered substrate in the transition state (44).
The K12G mutation also results in an at least 14-fold decrease
in (kcat/Km)iso for the isomerization of bound glycolaldehyde
(vide infra), which shows that the cationic side chain of Lys-12
acts to stabilize the transition state for the transfer of a proton
from a neutral substrate piece that lacks a phosphodianion
group. This provides evidence of a significant stabilizing inter-
action between the cationic side chain of Lys-12 and the deve-
loping negative charge on the sugar substrate piece in an enediol-
(ate)-like transition state. It is in accord with computational
studies that pointed to the dominant role of Lys-12 in the stabi-
lization of the transition state for formation of an enediolate
intermediate by carbon deprotonation of the whole substrate
DHAP or GAP (44, 45).
Phosphite dianion strongly activates wild-type rabbit and
chicken muscle TIM toward the isomerization of glycolalde-
hyde (28, 30), and we have also found that alkylammonium cations
strongly activate K12G mutant yeast TIM toward the isomeriza-
tion of GAP (42). We therefore examined the K12G TIM-cata-
lyzed reaction of [1-13C]GA iþn D2O in the presence of 100 mM
þ
EtND3 or 50 mM EtND3 and 10 mM phosphite dianion
(Table 2). These reagents result in small increases in the observed
second-order rate constant (kcat/Km)obs for the disappearance of
[1-13C]GA (Table 2). However, these increases do not result from
catalysis of isomerization at the enzyme active site because there
is no detectable formation of the isomerization product [2-13C]GA
(Table 2). No attempt was made to characterize the additional
products of these slow reactions because the products do not
appear to be formed at the active site of TIM. We reach the
following conclusions. (1) Interactions between the phosphite
dianion activator and the cationic side chain of Lys-12 are essen-
tial for the observation of phosphite activation of the TIM-
catalyzed deprotonation of GA (30). (2) Interactions between the
phosphodianion group of GAP and exogenous ammonium
cations are essential for the observation of rescue of the activity
of K12G mutant TIM by these cations (42). However, K12G
mutant TIM shows no detectable activity for isomerization of
[1-13C]GA in the presence of EtND3 and phosphite dianion,
þ
presumably because these ions show a weak affinity for binding
to the mutant enzyme.
pH-Rate Profile for Isomerization of GAP by K12G
Mutant Yeast TIM. Figure 6 shows the pH-rate profiles for
kcat/Km for the isomerization of GAP catalyzed by wild-type chicken
muscle TIM (data from ref 35) and K12G mutant yeast TIM
(data from this work). The solid lines show the fits of the data
to eq 8 with a (kcat/Km)* of 0. The dashed line for K12G TIM
shows that the inclusion of the (kcat/Km)* of 1.1 M-1 s-1 for the
The role of Lys-12 in catalysis of isomerization has also been
inferred from an inspection of the X-ray crystal structure of yeast
TIM in a complex with DHAP (Figure 7) (7). The alkylammo-
nium side chain of Lys-12 lies roughly equidistant from the
phosphodianion and carbonyl groups of bound DHAP and is
expected to interact with both centers. This side chain likely does
not form a hydrogen bond with the phosphate dianion in the
4Calculated using a (kcat/Km)obs of 0.19 M-1 s-1 for the enzyme-
catalyzed disappearance of glycolaldehyde and the observation that
50% of the products of this reaction result from deprotonation of
glycolaldehyde within the active site (28).
6The similarity of the kinetic parameters for wild-type TIM from
chicken muscle (35) and yeast (Table 3 and ref 36) at pH 7.5 suggests that
the appearance of the pH-rate profiles for these enzymes should also be
similar.
5Calculated using a (kcat/Km)obs of 0.19 M-1 s-1 for the enzyme-
catalyzed disappearance of glycolaldehyde and the observation that the
isomerization product [2-13C]GA is formed in a yield of 5% (28).

Article
Biochemistry, Vol. 49, No. 25, 2010 5387
FIGURE 7: Structure of the active site of TIM, taken from the X-ray crystal structure of McDermott and co-workers (Protein Data Bank entry
1NEY) (7), showing the distances between the ammonium nitrogen of Lys-12 and the functional groups of bound substrate DHAP.
ground state Michaelis complex because the X-ray crystal struc-
ture reveals the presence of a water molecule between the cationic
side chain of Lys-12 and the phosphodianion group of DHAP,
which attenuates the electrostatic interaction (Figure 7) (7). This
interaction might be strengthened by a shift in the position of the
water to allow for greater proximity between the interacting
charges at the transition state for proton transfer. However, we
have no evidence to support this proposal, and we note that a
bridging water molecule in this position is also observed for com-
plexes between TIM and the intermediate analogues PGH (43)
and PGA (10).
The stretching frequency for the C-2 carbonyl group of DHAP
bound to the H95Q and H95N mutants of yeast TIM lies bet-
ween 1732 and 1742 cm-1 (46), which is similar to the carbonyl
stretching frequency of 1732 cm-1 for DHAP in water (47). This
suggests that there is no additional polarization of the carbonyl
π-bond of enzyme-bound DHAP due to a hydrogen bonding
interaction with Lys-12 (21). Although there is no crystal struc-
ture for the complex between TIM and GAP, the large effect
of the K12G mutation on kcat for the isomerization of GAP
(Table 3) provides strong evidence that the cationic side chain of
Lys-12 stabilizes negative charge that develops on O-1 at the
transition state for deprotonation of GAP.
Charged Enediolate or Neutral Enediol Intermediate?
Studies of the nonenzymatic deprotonation of GAP in water
show that direct Brønsted base-catalyzed deprotonation of the
substrate to form a negatively charged enediolate intermediate
is favored energetically over any competing Brønsted acid-
catalyzed pathway to form the enediol (14). This is because the
enolate oxygen of the enediolate intermediate is relatively weakly
basic [pKa ≈ 11 for the enol (14, 48)], so that there is no significant
advantage to concerted catalysis by relatively weak Brønsted
acids (49). Brønsted general acid catalysis of the deprotonation
of enzyme-bound GAP to form an enediol intermediate would
be favored if the transfer of a proton from the enzyme to the
enediolate were strongly favorable (50).
oxyanion which dictates the use of a neutral imidazole side chain
of His-95 (25), rather than the more acidic imidazolium cation, as
the catalytic electrophile at TIM. Full proton transfer to the
enediolate oxyanion would eliminate the large stabilizing electro-
static interaction with the cationic side chain of Lys-12. However,
partial proton transfer from the neutral imidazole of His-95 to
the oxyanion allows for effective transition state stabilization by
hydrogen bonding (46), while at the same time maintaining the
critical stabilizing electrostatic interaction with the cationic side
chain of Lys-12.
It is not obvious that enolate anions, whose formation from
neutral molecules is intrinsically more difficult in solvents with
low dielectric constants than in water, should in fact be formed more
easily at the nonpolar active sites of protein catalysts (51-55)
than in aqueous solution. However, zwitterions are strongly
stabilized by their transfer from aqueous solution to organic
solvents (56-58), and the formation of the effectively intramo-
lecular (59, 60) ion pairs between enzyme catalysts and immobi-
lized bound substrates will be favored entropically over the
bimolecular formation of ion pairs between small molecules in
solution. Others have noted that enzyme active sites provide a
highly organized environment for chemical reactions (51, 61, 62),
where appropriately placed amino acid side chains act to stabilize
bound ions with opposite charges.
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