Glyoxylate carboligase: A unique thiamin diphosphate-dependent enzyme that can cycle between the 4′-aminopyrimidinium and 1′,4′- iminopyrimidine tautomeric forms in the absence of the conserved glutamate

Article abstract of DOI:10.1021/bi300893v

Glyoxylate carboligase (GCL) is a thiamin diphosphate (ThDP)-dependent enzyme, which catalyzes the decarboxylation of glyoxylate and ligation to a second molecule of glyoxylate to form tartronate semialdehyde (TSA). This enzyme is unique among ThDP enzyme

Full text of DOI:10.1021/bi300893v

Article
pubs.acs.org/biochemistry
Glyoxylate Carboligase: A Unique Thiamin Diphosphate-Dependent
Enzyme That Can Cycle between the 4′-Aminopyrimidinium and
1′,4′-Iminopyrimidine Tautomeric Forms in the Absence of the
Conserved Glutamate
Natalia Nemeria,‡ Elad Binshtein,† Hetalben Patel,‡ Anand Balakrishnan,‡ Ilan Vered,† Boaz Shaanan,†
Ze’ev Barak,† David Chipman,*,† and Frank Jordan*,‡
†Department of Life Sciences, Ben-Gurion University, P.O. Box 653, Beer-Sheva 84105, Israel
‡Department of Chemistry, Rutgers University, 73 Warren Street, Newark, New Jersey 07102, United States
S
* Supporting Information
ABSTRACT: Glyoxylate carboligase (GCL) is a thiamin
diphosphate (ThDP)-dependent enzyme, which catalyzes the
decarboxylation of glyoxylate and ligation to a second
molecule of glyoxylate to form tartronate semialdehyde
(TSA). This enzyme is unique among ThDP enzymes in
that it lacks a conserved glutamate near the N1′ atom of ThDP
(replaced by Val51) or any other potential acid−base side
chains near ThDP. The V51D substitution shifts the pH
optimum to 6.0−6.2 (pKa of 6.2) for TSA formation from pH
7.0−7.7 in wild-type GCL. This pKa is similar to the pKa of 6.1 for the 1′,4′-iminopyrimidine (IP)−4′-aminopyrimidinium
(APH+) protonic equilibrium, suggesting that the same groups control both ThDP protonation and TSA formation. The key
covalent ThDP-bound intermediates were identified on V51D GCL by a combination of steady-state and stopped-flow circular
dichroism methods, yielding rate constants for their formation and decomposition. It was demonstrated that active center
variants with substitution at I393 could synthesize (S)-acetolactate from pyruvate solely, and acetylglycolate derived from
pyruvate as the acetyl donor and glyoxylate as the acceptor, implying that this substitutent favored pyruvate as the donor in
carboligase reactions. Consistent with these observations, the I393A GLC variants could stabilize the predecarboxylation
intermediate analogues derived from acetylphosphinate, propionylphosphinate, and methyl acetylphosphonate in their IP
tautomeric forms notwithstanding the absence of the conserved glutamate. The role of the residue at the position occupied
typically by the conserved Glu controls the pH dependence of kinetic parameters, while the entire reaction sequence could be
catalyzed by ThDP itself, once the APH+ form is accessible.
he enzyme glyoxylate carboligase (GCL) catalyzes the
thiamin diphosphate (ThDP)-assisted ligation of two
valine (residue V51 in the wild-type Escherichia coli enzyme)
appears in the position expected for this Glu residue. It had
been assumed that the conserved glutamate is essential for
activation of the coenzyme in ThDP-dependent enzymes by
facilitating the tautomerization of the weakly basic 4′-
aminopyrimidine (AP) to the more basic 1′,4′-iminopyrimidine
(IP) tautomer (Scheme 1A). Surprisingly, steady-state kinetic
analyses revealed that the V51D and V51E variants are less
active than GCL despite having higher rates of activation of the
coenzyme (deprotonation at C2 of the thiazolium moiety). The
turnover rates are 2 orders of magnitude lower for the V51D
variant and 7-fold lower for the V51E variant.5 However, as the
coenzyme analogue N3′-pyridyl-ThDP (in which N′1 is
replaced by CH) leads to an inactive GCL holoenzyme, the
1′,4′-iminopyrimidine tautomer of ThDP must have a role in
T
molecules of glyoxylate to form (R)-tartronate semialdehyde
(TSA) and CO2.1−7 GCL is a member of the homologous
family of ThDP-dependent enzymes catalyzing reactions that
begin with the decarboxylation of a 2-oxoacid substrate.2 It is a
homotetramer and requires FAD in addition to ThDP and
Mg2+ for catalysis.1−3 Despite its structural similarity to
enzymes whose physiological substrate is pyruvate (pyruvate
decarboxylases, pyruvate oxidases, and especially acetohydrox-
yacid synthases), the sole substrate of GCL is the 2-aldoacid
glyoxylate and the enzyme is unreactive with 2-ketoacids
(Scheme 1A). It is important to note that GCL lacks both the
conserved glutamate residue that is found in all other known
ThDP enzymes, where its carboxylate group is within hydrogen
bonding distance of N1′ of the 4′-aminopyrimidine ring of
ThDP5,6 and any other potential acid−base side chain near
ThDP. This unique property among ThDP enzymes makes
GCL particularly useful for mechanistic studies where
interpretation of pH-dependent behavior is sought. In GCL,
Received: July 3, 2012
Revised: August 17, 2012
Published: September 12, 2012
© 2012 American Chemical Society
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a
Scheme 1
a
(A) Mechanism of the GCL reaction, including tautomerization/ionization states of thiamin. Possible reactions of a decarboxylase carboligase with
glyoxylate. (B) Possible reactions of a decarboxylase carboligase with pyruvate as the sole substrate, in which acetolactate is produced. With I393A
GCL, in the presence of pyruvate and glyoxylate the mixed product acetylglycolic acid is the expected product.
Scheme 2. Formation of Covalent Adducts of ThDP with Pyruvate and with Pyruvate Analogues MAP and AcPhi
catalysis despite the absence of the conserved Glu.5 Circular
dichroism (CD) evidence suggested that the V51D variant can
in fact stabilize the IP tautomer of ThDP.5 To understand the
mechanism of GCL in more detail, it is important to identify
the intermediate species on the reaction pathway or analogues
for such species (Scheme 1A). Covalent adducts of ThDP with
the pyruvate analogues (Scheme 2) methyl acetylphosphonate
(MAP) and acetylphosphinate (AcPhi) are stable analogues of
the first predecarboxylation intermediate formed between
pyruvate and ThDP, C2α-lactylThDP (LThDP). These
adducts, as well as LThDP itself, have provided many insights
into mechanisms of ThDP-dependent enzymes that act on
pyruvate.8−12 Importantly, it has been demonstrated for several
ThDP enzymes that incremental addition of MAP and AcPhi
produced a positive CD band near 300−310 nm, a band
assigned to the 1′,4′-iminophosphonolactylThDP (PLThDP)
and 1′,4′-iminophosphinolactylThDP, respectively.12−14 Un-
fortunately, no synthesis of phosphonyl or phosphinyl
analogues of glyoxylate (e.g., formylphosphinate) has yet
been published. To gain further understanding of substrate
binding and specificity in GCL, we used the I393A GCL and
V51D/I393A GCL, predicted by modeling based on the crystal
structure of GCL,5 to allow GCL to accept pyruvate or its
analogues as donor substrates. Modeling indicates that the side
chain of Ile393 creates steric hindrance for substrates larger
than glyoxylate, like the molecular determinants for substrate
specificity identified on acetohydroxyacid synthase II from E.
coli.36
In this study, we employ kinetic studies using steady-state
and stopped-flow circular dichroism (CD) spectroscopy to
further investigate the mechanistic ability of GCL to function
despite the absence of the “conserved” glutamate, as well as the
determinants of its specificity for glyoxylate as the substrate. By
using V51D GLC, we demonstrated that the pH optimum of
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TSA formation and the pKa for the IP−APH+ equilibrium for
this variant are both significantly shifted to the acid side by the
same magnitude compared with those of GLC. Using the
unfavorable pH of 7.6 and a low temperature in the CD
experiments allowed us to identify the key ThDP-bound
intermediates on V51D GCL. By using MAP, AcPhi, and
propionylphosphinate (PrPhi), we now present CD evidence
that GCL variants with Ile393 substituted with Ala can stabilize
the predecarboxylation intermediates derived from pyruvate
and 2-oxobutanoate analogues even in the absence of the V51D
substitution.
Reaction of I393A GCL with Pyruvate Monitored by
Reduction of 2,6-Dichlorophenolindophenol. The 2,6-di-
chlorophenolindophenol (DCPIP) reaction was conducted at
5 °C by monitoring its reduction at 600 nm in a 1 mL reaction
mixture containing 0.5 mM ThDP, 5 mM MgCl2, 80 μM
DCPIP, and 20 μg of protein in 50 mM KH2PO4 (pH 7.0). The
reaction was initiated by addition of pyruvate (0.025−5 mM)
after equilibration of I393A in the reaction mixture for 5 min
because of the trace amounts of DTT present in the protein.
The linear part of the progress curves was used to calculate the
initial velocity (slope per minute).
CD Spectroscopy. CD experiments were conducted on a
Chirascan CD spectrometer from Applied Photophysics in a 1
cm path length cell in the near-UV (250−450 nm) wavelength
region. For each experiment, conditions are presented in the
figure legend.
pH Dependence of CD Bands. To study the pH
dependence of the CD bands pertinent to the IP or AP
forms, the pH of the protein solution was adjusted using a
sympHony pH electrode (VWR, Batavia, IL) and spectra were
recorded after each adjustment.22 The pKa was determined
from the fit of the log CD302−304 versus pH according to eq 1
for a single ionizing group or according to eq 2 for two ionizing
groups using SigmaPlot version 10.0
EXPERIMENTAL PROCEDURES
■
Materials. Sodium glyoxylate, pyruvate, FAD, ThDP,
ampicillin, tetracycline, 2,3-dimethoxy-5-methyl-1,4-benzoqui-
none (Coenzyme Q0), DTT, and NADH were obtained from
Sigma (St. Louis, MO). Ammonium sulfate and SDS were
obtained from BDH Chemicals Ltd. (Poole, U.K.). Yeast
extract, peptone, and agar were from DIFCO (Detroit, MI).
Restriction enzymes were from New England BioLabs Inc.
(Beverly, MA), Novagen (Darmstadt, Germany), and Promega
(Madison, WI). All reagents were of analytical grade.
Synthesis of AcPhi. was conducted according to the
procedure reported by Baillie et al.15 PrPhi was prepared as
described previously.16 Methyl acetylphosphonate (MAP) was
synthesized using trimethylphosphite and acetylchloride as
reagents according to the method of Kluger et al.17 Purity and
correct synthesis of all compounds were confirmed by NMR
spectroscopy and mass spectrometry.
log(CD) = log(CDmax) − log(1 + 10pK −x
)
1
(1)
log(CD) = log(CDmax) − log(1 + 10pK −x + 10x−pK
)
1
2
(2)
where x is pH.
Expression, Purification, and Site-Directed Muta-
genesis of GCL. E. coli gene gcl encoding GCL was cloned
as previously described.18 GCL and its variants were expressed
from the pQE60-GCL plasmid and purified by affinity
chromatography using a HiTrap Chelating HP Ni column (5
The pH dependence of the TSA activity catalyzed by V51D
GCL and GCL as measured by direct CD detection of TSA was
determined in 50 mM KH2PO4, adjusted to the desired pH
(pH 5.9−7.5), containing 0.06 mM KCl, 0.1 mM ThDP, 5 mM
MgCl2, and 50 μM FAD. Glyoxylate was added to a final
concentration of 10 mM, and the reaction was initiated by
addition of GCL (25 μg) or V51D GCL (30−50 μg). The
CD290 per second slope was calculated at different pH values,
and a plot of log CD290 per second versus pH was prepared.
Data were fit using eq 1 or 2.
Titration of GCL with AcPhi, PrPhi, and MAP. CD
spectra of GCL and its variants in the absence and presence of
pyruvate analogues and PrPhi were recorded in the near-UV
region at 290−550 nm and 25 °C. Conditions of each
experiment are described in the figure legends. A plot of
ellipticity at 302 nm versus substrate analogue concentration
was constructed by subtracting the ellipticity of the enzyme at
the same wavelength, but recorded in the absence of a substrate
analogue, as reported previously.23 The Kd values were
calculated using the Hill equation (eq 3):
̈
mL, Pharmacia) and the Pharmacia AKTA prime FPLC
system.5 The single substitutions were introduced into
pQE60-GCL using the overlap extension site-directed muta-
genesis method with the FailSafe kit (EPICENTRE Bio-
technologies). The primers used were designed to introduce a
modification into the restriction site as an aid in screening for
clones with mutated plasmids. The mutations were verified by
sequencing of the coding region of each plasmid used for
expression of variant proteins.
Enzyme Assays. TSA Activity Measurements. The rate of
steady-state formation of TSA by GCL was measured in a
coupled enzyme assay that follows the disappearance of NADH
with tartronate semialdehyde reductase (TSAR).19 TSAR was
cloned, expressed, and purified as previously described.5 The
TSA activity was also measured by direct detection of TSA
formation using a Chirascan CD spectrometer at 290 nm
(Applied Photophysics, Leatherhead, U.K.).18 The reaction
medium in a volume of 2.4 mL contained 50 mM KH2PO4 (pH
7.7), 0.06 M KCl, 0.1 mM ThDP, 5 mM MgCl2, and 50 μM
FAD. The glyoxylate was added to a final concentration of 10
mM, and the reaction was started by addition of GCL (25 μg)
or V51D GCL (30−50 μg) at 37 °C and monitored for 300 s.
The linear part of the progress curves was used to calculate the
initial velocity (slope per second) using the Pro-Data Viewer
program supplied by the manufacturer.
nH
CD302 = (CDmax302[ligand]nH )/(S0.5 + [ligand]nH )
(3)
where nH is a Hill coefficient. When nH = 1.0, S0.5 = Kd.
CD Titration of I393A GCL with Pyruvate. I393A GCL
(2.5 mg/mL, active center concentration of 38.6 μM) in 0.10 M
KH2PO4 (pH 7.6) containing 5 mM MgCl2 and 0.50 mM
ThDP was titrated with pyruvate (0.02−4.0 mM) at 4 °C. After
15 h at 4 °C, the protein was removed from the reaction
mixture with an Ultracel-30K Centrifugal Filter Unit (Milli-
pore) and CD spectra were recorded at 250−400 nm for (S)-
acetolactate detection.
Acetolactate Determination. The modified Westerfeld
creatine-naphthol colorimetric method was used for assaying
acetolactate (AL) formation20,21 with absorbance measure-
ments taken with a Beckman DU640 spectrophotometer.
Stopped-Flow CD Spectroscopy. Kinetic traces were
recorded on a Pi*-180 stopped-flow CD spectrometer (Applied
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Photophysics) using a 10 mm path length cell at the specified
wavelengths. The temperature was maintained at 6 or 15 °C as
stated in the figure legends.
substitution I393A or double substitutions V51D and I393A,
GCL is converted to an acetolactate synthase that can use
pyruvate as a substrate with a catalytic efficiency (kcat/Km) ∼20
times higher than that of wild-type GCL (Table 1). Formation
of acetolactate was detected by a colorimetric measurement20,21
and by direct CD detection of (S)-acetolactate (see below).18
The I393V, L478A, and I479V variants did not exhibit marked
increases in the level of acetolactate synthesis compared to that
of the GCL.
The Val51 residue in GCL is critical for TSA synthesis.
Although the V51D variant catalyzes proton exchange at C2 of
the thiazolium ring of ThDP ∼6-fold more rapidly than GCL,
the TSA−ThDP complex accumulates in this enzyme and its
turnover rate is lower by 2 orders of magnitude.5 The X-ray
structure of GCL suggested that aspartate can be easily
accommodated in place of the V51 position in the active center
of GCL with its carboxylate group positioned within hydrogen
bonding distance of N1′ of the 4′-aminopyrimidine ring. On
the other hand, we suggested that the carboxylate in the V51E
variant can make such a hydrogen bond only if the structure is
distorted.5 Hence, the V51D substitution appeared to be more
fruitful for our investigation.
The doubly substituted V51D/I393A GCL was created to
address whether the combination of a carboxylate group, which
can accelerate the activation of ThDP, with relief of the steric
hindrance to the larger substrate pyruvate by replacement of
Ile393 with Ala might allow the variant to effectively stabilize
the 1′,4′-iminopyrimidine tautomeric intermediates derived
from pyruvate and to effectively catalyze the synthesis of
acetolactate. As shown in Table 1, the catalytic efficiency for
formation of acetolactate by V51D/I393A GCL is similar to
that of the singly substituted I393A GCL despite the presence
of the Asp residue at position 51. The formation of TSA by this
doubly substituted variant cannot be detected in a coupled
enzyme assay. The observation that the presence or absence of
an aspartate residue at position 51 has little effect on the activity
of a V51D/I393A variant in the formation of acetolactate
(compared to the result with the single I393A substitution)
implies that the effect of Asp51 on TSA formation is specifically
related to the chemistry of the natural substrate glyoxylate.
Identification of ThDP-Bound Intermediates and
Their Tautomeric Forms on V51D GCL by CD. It was
suggested that ThDP enzymes with a variety of active center
environments can stabilize the IP form of ThDP, and that the
presence of a glutamate residue within hydrogen bonding
distance of the N1′ atom of the 4′-aminopyrimidine ring is
important for catalyzing the tautomeric equilibration and
stabilizing the IP form.12,13,24 It was further suggested that all
C2α-tetrahedral ThDP adducts on the pathway for an
enzymatic reaction will likewise exist as their 1′,4′-iminopyr-
imidine tautomers at pH values above the pKa of the APH+
ionization state. With these findings in mind, CD has now been
applied to GCL and its variants.
Pre-Steady-State Formation of Covalent 1′,4′-Imino-
pyrimidylThDP-Bound Intermediates on V51D GCL. A
solution of V51D GCL (3.6 mg/mL, active center concen-
tration of 55.6 μM) in 0.1 M KH2PO4 (pH 7.6) containing 0.5
mM ThDP, 2.5 mM MgCl2, 1.0 mM DTT, 10 μM FAD, and
1% glycerol in one syringe was mixed rapidly with an equal
volume of glyoxylate (2.0 mM) in the same buffer placed in the
second syringe at 6 °C. The data points were collected at 302
nm over a period of 50 s. Data from four or five repetitive shots
were averaged and were fit to a double-exponential (eq 4),
single-exponential (eq 5), or triple-exponential (eq 6) model
using SigmaPlot version 10.0:
CD302−304(t) = CD1e−k t + CD2e−k t + c
1
2
(4)
(5)
CD302−304(t) = CD1e−k t + c
1
CD302−304(t) = CD1e−k t + CD2e−k t − CD3e−k t + c
1
2
3
(6)
where k1, k2, and k3 are the apparent rate constants and c is
CDmax
in the exponential rise to maximum model or
302
CDmin in the exponential decay model.
302
RESULTS
■
Rationale for the Selection of Active Center Residues
V51 and I393 for Substitution. According to the X-ray
structure of GCL [Figure 1, Protein Data Bank (PDB) entry
Figure 1. Structure of the active center of GCL demonstrating the
hydrophobic environment of the thiazolium ring of ThDP. This figure
was created using PyMOL based on the published structure of GCL
(PDB entry 2PAN).5
2PAN], residues I393, L478, and I479 are located close to the
thiazolium ring of ThDP and apparently form a pocket
proximate to C2 that can accommodate a glyoxylate moiety but
cannot easily accommodate a pyruvate molecule, with an extra
methyl group.5 To understand the structure−function relation-
ship, these residues were substituted with alanine or the amino
acids conserved at the homologous positions in the AHAS
(Table 1). The single I393A, I393V, L478A, and I479V
substitutions led to a lower catalytic efficiency (kcat/Km) of
GCL in TSA formation [approximately 10−36-fold lower
compared with that of GCL, as detected, in a coupled enzyme
assay with TSA reductase (TSAR)19] (Table 1). As a result of
The 1′,4′-Iminopyrimidine Tautomeric Form of ThDP Is
Stabilized on V51D GCL. It was demonstrated earlier that
V51D GCL displayed a positive CD band at 302 nm in the
resting state, suggested to correspond to the IP form of ThDP
bound to this variant, not displayed on the V51E variant.5 The
following experimental evidence is presented here to confirm
this suggestion. (1) The CD band at 302 nm was partially
reduced on replacement of ThDP by thiamin 2-thiothiazolone
diphosphate (ThTTDP), an analogue of ThDP with the C2−H
bond in the thiazolium ring substituted with a C2S bond
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Table 1. Kinetic Properties of GCL and Its Active Center Variants
a
b
tartronate semialdehyde formation
specific activity (μmol min−1 mg−1) (%)
acetolactate formation
specific activity (nmol min−1 mg−1
enzyme
kcat/Km (s−1 mM−1
)
)
kcat/Km (s−1 M−1
)
wild type
I393A
17.5 2.5 (100%)
0.68 0.02 (3.9%)
21.0
2.06
0.65
1.79
0.58
8.2 0.9
82.0 3.4
3.0 0.5
3.0 0.14
4.0 0.2
−
0.05
0.86
0.11
0.37
0.30
−
c
I393V
0.93 (5.3%)
c
L478A
0.06 (0.34%)
I479V
0.84 0.05 (4.8%)
d
d
V51D
0.2 0.02 (1.2%)
1.3
e
e
V51D/I393A
nd
nd
90.2 3.0
0.94
a
b
The rate of TSA formation was measured in a coupled enzyme assay with TSAR.19 The modified colorimetric method by Westerfeld was used for
measurement of acetolactate activity.20,21 For these two variants, the activity was over a range of substrate levels and was measured once, so that the
errors cannot be estimated. Data from ref 5 were used. No activity could be detected.
c
d
e
(not shown). (2) The amplitude of the CD band at 302 nm
was reduced upon addition of AcPhi, an analogue of pyruvate;
however, no signature for formation of 1′4′- iminophosphino-
lactylThDP was in evidence. (3) The positive CD band at 302
nm is replaced by a negative one at 290 nm upon addition of
glyoxylate, because of formation of the (R)-TSA product. (4)
The intensity of the positive CD band at 302 nm depends on
pH, in a manner similar to that reported previously by the
Jordan group for other ThDP-dependent enzymes.22
Effect of the V51D Substitution on Equilibria of
Tautomeric and Ionization States of ThDP. The AP form of
ThDP on benzaldehyde lyase and benzoylformate decarbox-
ylase, and both the AP and IP forms on pyruvate oxidase from
Lactobacillus plantarum, as well as on the human pyruvate
dehydrogenase E1 component all exhibited pH-dependent
behavior.22,25 Here, we conduct a pH titration to determine the
pKa for the IP−APH+ equilibrium (there is no evidence of the
presence of a detectable concentration of the AP form on GCL
so far) on V51D GCL. When the pH is reduced from 7.95 to
5.72, the amplitude of the positive CD band at 302−304 nm
observed on V51D GCL was reduced (Figure 2), indicating
that the IP form of ThDP is dominant at pH 7.95, while the
APH+ form is likely dominant at pH 5.72, with no CD band
detected for this form so far. The pH titration data fit to a single
proton titrating with a pKa of 6.1 for the IP−APH+ equilibrium
(Figure 2B). On several ThDP enzymes studied so far, the pKa
for the IP−APH+ equilibrium is near the pH optimum of
enzyme activity,22 which is pH 7.7 for GCL. The data thus
Figure 2. Effect of pH on CD spectra of V51D GCL. (A) Near-UV
suggest that the V51D substitution significantly shifted the pH
optimum of enzyme activity to the acid side.
CD spectra of V51D GCL at different pH values. The V51D GCL
from stock was diluted to a concentration of 2.6 mg/mL (active center
concentration of 40 μM) in 0.1 M KH2PO4 containing 0.5 mM ThDP
and 2.5 mM MgCl2. CD spectra were recorded after the pH had been
adjusted to the desired value (pH 5.72−7.95). (B) Dependence of log
CD304 on pH. Data were fit to a single ionizing group (eq 1).
To test this hypothesis, the rate of formation of (R)-TSA by
GCL and V51D GCL was estimated directly by using CD290, as
reported previously.18 Both GCL and V51D GCL displayed a
pH dependence for TSA formation. A plot of the log of the
reaction slope detected from the progress curves (Figure 3A)
[the molar ellipticity for (R)-TSA is not available in the
literature] versus pH revealed a maximal activity at pH 7.0−7.7
for the GCL (Figure 3B), with the bell-shaped plot being
described by two acid−base groups with an apparent pK1 of 6.1
and a pK2 of >8.0. For V51D GCL, the pH optimum of activity
was shifted to pH 6.0−6.2 (Figure 4). The apparent pKa of 6.2
calculated from activity measurements correlates well with the
pKa of 6.1 determined for the IP−APH+ equilibrium from the
previous CD experiment, suggesting that the same groups
control both events, probably the D51COO−−APH+ dyad
(kinetically equivalent to, and indistinguishable from, the
D51COOH−IP dyad), perhaps assisting with release of
product from the TSA−ThDP covalent complex (see below).
Stabilization of the 1′,4′-Iminopyrimidine Form of ThDP-
Bound Intermediates Derived from Glyoxylate on V51D GCL.
Because V51D GCL is a slow variant [0.20 μmol min−1 (mg of
protein)−1] compared to GCL [17.5 μmol min−1 (mg of
protein)−1], the ThDP-bound intermediates could be detected
by steady-state CD at a low temperature (5 °C). Upon titration
of V51D GCL by glyoxylate using concentrations in excess of
the concentration of active centers (1, 5, and 8 mM as
compared with the active center concentration of 9.5 μM), the
magnitude of the positive CD band at 302 nm assigned to the
IP form of ThDP on V51D GCL was reduced and was finally
replaced by a negative band when the temperature was
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Figure 4. Effect of pH on the TSA activity of V51D GCL. (A)
Progress curves of TSA formation at different pH values using CD at
290 nm. Conditions of the experiment are presented in Experimental
Procedures. The linear part of the progress curves was used to
calculate the initial velocity (CD290 per second) using the Pro-Data
Viewer program. (B) pH dependence of log(CD290 per second). Data
were fit to eq 1 for a single ionizing group.
Figure 3. Effect of pH on the TSA activity of GCL. (A) Progress
curves of TSA formation were recorded at different pH values using
CD at 290 nm. Conditions of the experiment are presented in
Experimental Procedures. The linear part of the progress curves was
used to calculate the initial velocity (CD290 per second) using the Pro-
Data Viewer program. (B) Dependence of log(CD290 per second) on
pH. The log(CD290 per second) was plotted vs pH, and data were fit
to eq 2 for two ionizing groups.
increased to 20 °C (Figure 5). This is largely due to the
formation of (R)-TSA, which has a strong negative CD band at
290 nm.18 Formation of this product was confirmed by the CD
spectrum of the reaction mixture after removal of protein: the
negative CD band detected at 290 nm is what was expected for
(R)-TSA (not shown). A similar decrease in the amplitude of
the positive CD band at 302 nm was observed when the
experiment was conducted at pH 7.6 or 6.5 (Figure 5 presents
data at pH 6.5), giving clear evidence of (R)-TSA (product)
formation.
Time-Resolved CD Experiments with ThDP-Bound Inter-
mediates on V51D GCL. Next, in a series of experiments, the
reaction of V51D GCL with glyoxylate was monitored by
stopped-flow CD at 302 nm and pH 7.6. In the first experiment
conducted at 6 °C, the V51D GCL (active center concentration
of 56 μM) in one syringe was mixed with 2 mM glyoxylate in
the second syringe, resulting in a time-dependent accumulation
of a species with a positive ellipticity at 302 nm (Figure 6A).
The reaction reached steady state within 5 s with a k1 of 6.2 s−1
and a k1′ of 0.58 s−1 (the data points were analyzed as a double
exponential resulting in two rate constants, k1 and k1′, for the
fast and slow phases of the reaction, respectively), suggesting
formation of a covalent glycolyl-ThDP predecarboxylation
Figure 5. CD spectra of V51D GCL titrated by glyoxylate. The V51D
GCL (1.23 mg/mL, active center concentration of 19.0 μM) in 0.1 M
KH2PO4 (pH 6.5) containing 0.5 mM ThDP, 2.5 mM MgCl2,1.0 mM
DTT, 10 μM FAD, and 1% glycerol was titrated by glyoxylate (0.010−
8 mM) at 5 °C. CD spectra were recorded in the near-UV region after
each addition of glyoxylate. Observable changes in the intensity of the
CD band at 302 nm were detected with 1, 5, and 8 mM glyoxylate, but
not with 0.01−0.5 mM glyoxylate. When the temperature was
increased to 20 °C, formation of (R)-TSA was evident.
intermediate (Schemes 1A and 3 for rate constant assignment
and Table 2 for values).
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Figure 6. continued
glyoxylate in the second syringe at 15 °C. (C) Time-dependent
decarboxylation of 1′,4′-iminoglycolylThDP with data from Figure 6B
expanded. Data were fit to a single exponential (eq 5). (D) Formation
of the TSA−ThDP complex and release of TSA by V51D GCL at pH
7.6. The V51D GCL (1.7 mg/mL, active center concentration of 26
μM) was preincubated with 0.5 mM glyoxylate for 15 min in one
syringe and then mixed at 6 °C with 16 mM glyoxylate in the second
syringe. The data were fit to a triple exponential (eq 6).
The second experiment was conducted under similar
conditions but at 15 °C. Initially, the intensity of the CD
signal at 302 nm decreased for at least the first 0.2 s (Figure 6B
for early times and Figure 6C for details), followed by an
increase in CD302 with an exponential rise to maximum. We
assign the final positive CD signal developed in this experiment
to the TSA−ThDP covalent adduct, identified by a quench-
NMR method.5 We assign the rate constant in Figure 6C
(expansion of Figure 6B, the initial decrease) to decarbox-
ylation of the glycolyl-ThDP predecarboxylation intermediate
(k2 = 35 s−1 in Figure 6C; see Schemes 1A and 3 and Table 2)
and k3 = 2.9 s−1 and k3′ = 0.56 s−1 to the formation of the
TSA−ThDP complex on GCL (Figure 6B). The formation of
the glycolyl-ThDP predecarboxylation complex was too fast to
be detected at 15 °C, but it most likely was detected at a low
concentration of glyoxylate at 6 °C in Figure 6A.
In the third experiment, the V51D GCL was preincubated
with 0.5 mM glyoxylate in one syringe to preform the covalent
glycolyl-ThDP predecarboxylation intermediate and then mixed
with 16 mM glyoxylate in the second syringe at 6 °C. An initial
fast rise was observed [providing the rate constants for
formation of the (R)-TSA−ThDP complex with a k3 of 3.2
s−1 and a k3′ of 0.34 s−1] followed by a much slower decrease
associated with a rate constant k4 of 0.16 s−1 for the release of
(R)-TSA (product) from the enzyme (Figure 6D).
This sequence of experiments could be realized only because
pH 7.6 is unfavorable for the V51D GCL variant. At the same
time, it allowed us to detect all key ThDP-bound intermediates
on the reaction path and to calculate rate constants for
formation of glycolyl-ThDP, its decarboxylation to the
enamine, carboligation of the enamine with the second
molecule of glyoxylate to provide the TSA−ThDP complex,
and finally the release of product from GCL. Under these
experimental conditions, product release is clearly the rate-
limiting step.
Finally, when the experiment was conducted at pH 6.5 (more
favorable pH for V51D GCL) and 6 °C, only the TSA−ThDP
product accumulated, and its release was also detected (Figure
7); the positive ellipticity at 302 nm was developed within 5 s
with rate constants k3 of 3.4 s−1 and k3′ of 0.55 s−1, followed by
a slow decrease in the ellipticity with a rate constant k4 of 0.04
s−1, again confirming TSA release as the rate-limiting step in
agreement with NMR data.5
CD Titration of V51D GCL with Analogues of Pyruvate.
Upon addition of AcPhi (0.001−5 mM) or MAP (0.001−2.6
mM) to V51D GCL (active center concentrations of 15.2 and
38 μM, respectively), the amplitude of the CD band at 302 nm
assigned to the IP form of ThDP was reduced by approximately
57% in both cases but no 1′,4′-iminopyrimidine tautomeric
forms of the predecarboxylation adduct resulting from addition
of AcPhi and MAP were detected (not shown). The S0.5,AcPhi
calculated for half of the reduction of the CD band at 302 nm
Figure 6. Time course of the reaction of V51D GCL with glyoxylate
monitored by stopped-flow CD. (A) Formation of 1′,4′-iminoglyco-
lylThDP at pH 7.6 and 6 °C. V51D GCL (3.6 mg/mL, active center
concentration of 55.6 μM) in 0.1 M KH2PO4 (pH 7.6) containing 0.5
mM ThDP, 2.5 mM MgCl2, 1.0 mM DTT, and 10 μM FAD in one
syringe was mixed with an equal volume of 2.0 mM glyoxylate in the
same buffer in the second syringe. Data were fit to a double-
exponential equation (eq 4). (B) Decarboxylation of 1′,4′-
iminoglycolyl-ThDP and formation of the TSA−ThDP complex at
pH 7.6 and 15 °C. V51D GCL (2.3 mg/mL, active center
concentration of 35.5 μM) in one syringe was mixed with 2.0 mM
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a
Scheme 3. Minimal Mechanism and Microscopic Rate Constants for V51D GCL
a
k1 represents the formation of glycolylThDP, the predecarboxylation intermediate, positive CD band at 302 nm. k2 represents the decarboxylation
to enamine. k3 represents carboligation with glyoxylate to provide the TSA−ThDP complex, the postdecarboxylation intermediate, positive CD band
near 300 nm. The values represent the average from three independent experiments. k4 represents the release of TSA product from ThDP·GCL
(identified by NMR5). k1, k3, and k4 were determined at 6 °C, and k2 was determined at 15 °C.
Table 2. Kinetic Analysis of the Mechanism of V51D GCL by Stopped-Flow CD under Different Experimental Conditions
conditions (T, pH,
glycolyl-ThDP formation, k1
(s−1
glycolyl-ThDP decarboxylation, k2
TSA−ThDP complex, k3
(s−1
TSA release, k4
(s−1
a
expt
1
[glyoxylate])
)
(s−1
)
)
)
b
c
c
c
6 °C, pH 7.6, 1 mM
15 °C, pH 7.6, 1 mM
6 °C, pH 7.6, 8 mM
6.2 0.6
nd
nd
nd
0.58 0.03
c
b
c
2
3
4
nd
35
nd
nd
4
2.9 0.6
nd
0.56 0.09
c
c
c
b
nd
3.2 1.1
0.16 0.12
0.04 0.01
0.34 0.13
d
c
b
6 °C, pH 6.5, 1 mM
nd
3.4 0.4
0.55 0.04
a
b
Experiments are numbered according to the text in Results. The experimental data are presented in Figure 6A−D. The experimental curves were
c
d
treated as a double exponential, leading to two rate constants (fast and slow). Not detected. Experiment conducted at pH 6.5, favorable for V51D
GCL as demonstrated in this work.
Table 3. Kinetic Parameters for Equilibrium Binding of
Pyruvate and 2-Oxobutanoate Analogues to GCL Variants
a
GCL
substitution
Kd (S0.5) for AcPhi
Kd (S0.5) for PrPhi
Kd for MAP
(mM)
(mM)
(mM)
I393A
0.32 0.02
0.46 0.03
1.4 0.09
(nH = 2.23)
(nH = 1.7)
b
b
V51D
0.18 0.11
−
0.11 0.09
0.11 0.1
V51D/I393A 0.37 0.09
0.28 0.04
(nH = 1.1)
(nH = 1.29)
a
Kinetic parameters were determined in steady-state CD experiments.
For V51D GCL, no 1′,4′-iminopyrimidine tautomers from addition
b
of AcPhi and MAP were detected. Instead, the intensity of the CD302
band was reduced by AcPhi and MAP with the values of S0.5 listed
here.
experiment with MAP revealed an S0.5,MAP of 0.11
0.09
mM for the CD band at 302 nm, indicating similar behavior
with MAP (Table 3). Hence, the evidence for covalent complex
formation with these substrate analogues is tentative.
Figure 7. Reaction of V51D GCL with glyoxylate monitored by
stopped-flow CD at pH 6.5. The V51D GCL (6.45 mg/mL, active
center concentration of 99.6 μM) in 0.10 M KH2PO4 (pH 6.5)
containing 0.5 mM ThDP, 2.5 mM MgCl2, 1.0 mM DTT, and 10 μM
FAD in one syringe was mixed with an equal volume of 2 mM
glyoxylate in the second syringe. The reaction was monitored over 45 s
at 6 °C. Data were fit to a triple exponential (eq 6).
Interestingly, with the doubly substituted V51D/I393A GCL,
we did observe changes in the CD spectrum upon binding of
these pyruvate analogues (Table 3). We must emphasize,
however, that while we have assigned CD spectroscopic
signatures for the IP and AP forms of ThDP on the enzymes,
we do not have such a characteristic signature for the APH+
form. It is therefore possible that addition of the pyruvate
analogues to the V51D variant produces this “CD silent” form
was 0.18 0.11 mM, indicating that AcPhi is likely bound in
the active centers of V51D GCL (Table 3). A similar
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(now identified on three ThDP enzymes by solid-state NMR
methods26,27). Alternatively, an adduct whose thiazolium ring is
sufficiently distorted to eliminate or radically shift the CD band
may be formed, which is a less likely scenario.
Stabilization of the 1′,4′-Iminopyrimidine Form of
ThDP-Bound Intermediates Derived from Glyoxylate on
GCL. Upon titration of GCL with glyoxylate in a steady-state
CD experiment, formation of positive CD bands at 302 and 337
nm was observed. The maximal intensity of the CD band at 302
nm was reached at concentrations of glyoxylate equivalent or
slightly higher than the concentration of active centers.
Increasing the concentration of glyoxylate produced (R)-TSA
(Figure S1 of the Supporting Information, top).
In a single-turnover experiment (where [GCL subunits] >
[glyoxylate]) using stopped-flow CD at 302 nm, the reaction
reached steady state within approximately 5 s with rate
constants k1 of 1.13 s−1 and k1′ of 0.28 s−1 (Figure S1 of the
Supporting Information, bottom), at least 5 times slower than
with V51D GCL (Figure 6A). The CD band at 302 nm can be
assigned to the first covalent predecarboxylation intermediate,
1′,4′-iminopyrimidinylglycolylThDP. Support for this assign-
ment comes from a comparison of the rate constants
determined for the individual steps in GCL by NMR methods.5
It was demonstrated that formation of the first covalent
predecarboxylation intermediate from glyoxylate was the rate-
limiting step for GCL in TSA (product) formation.5 The CD
band at 337 nm has not yet been assigned. It developed
simultaneously with that at 302 nm upon addition of low
concentrations of glyoxylate, but it did not respond to
increasing concentrations of glyoxylate: the CD band at 302
nm was replaced by a negative CD band at 290 nm due to TSA
release (Figure S1 of the Supporting Information, top).
Identification of ThDP-Bound Intermediates on GCL
Variants with the I393A Substitution. Stabilization of
1′,4′-IminopyrimidinylThDP Predecarboxylation Intermedi-
ates from Pyruvate on I393A GCL and V51D/I393A GCL. It is
evident from kinetic studies (Table 1) that with the I393A
substitution the GCL becomes an acetolactate synthase,
accepting pyruvate as a substrate. In this study, by using
pyruvate and 2-oxobutanoic acid analogues, we were able to
demonstrate that I393A GCL can stabilize the 1′,4′-
iminopyrimidine ThDP predecarboxylation intermediates
even in the absence of aspartate at position 51. V51D/I393A
GCL behaves like I393A GCL.
Figure 8. CD titration of I393A GCL by pyruvate at 6 °C. (A) CD
spectra of I393A GCL (2.0 mg/mL, active center concentration of
30.8 μM) in 0.1 M KH2PO4 (pH 7.6) containing 0.5 mM ThDP, 2.5
mM MgCl2, 1.0 mM DTT, and 10 μM FAD in the absence and
presence of 0.020−0.70 mM pyruvate. Each spectrum was recorded
after preincubation of I393A GCL with pyruvate for 20 min at 6 °C.
(B) Dependence of the ellipticity at 302 nm on pyruvate
concentration. The inset shows the plot of the CD302 data points vs
pyruvate concentration at concentrations of <0.80 mM. Data were fit
to a Hill equation (eq 3).
concentrations of <500 μM with an S0.5,pyruvate of 1.72 μM (not
shown).
The production of (S)-acetolactate requires decarboxylation
of pyruvate in the active centers of I393A GCL and V51D/
I393A GCL, resulting in the formation of the enamine
intermediate, and then subsequent ligation to a second pyruvate
(Scheme 1B, bottom). The intermediacy of the enamine was
confirmed by the reaction of I393A GCL with pyruvate in the
presence of DCPIP under conditions similar to those in the CD
experiment. The plot of the initial velocity (slope per minute)
of DCPIP reduction versus pyruvate concentration was again
biphasic, displaying saturation at low pyruvate concentrations
(Kd,pyruvate = 39 μM) (not shown).
Next, we demonstrated that glyoxylate could also be an
acceptor of the enamine intermediate derived from pyruvate on
I393A GCL. The positive CD band formed at 302−304 nm on
I393A GCL upon addition of stoichiometric amounts of
pyruvate was replaced by a negative CD band in the presence of
glyoxylate indicating formation of acetylglycolic acid derived
from pyruvate as the acetyl donor and glyoxylate as the
acceptor (Scheme 1B, top) (Figure 9A). However, the
acetylglycolate was not optically stable and could no longer
A steady-state CD titration of I393A GCL by pyruvate at 4
°C revealed two positive CD bands, one at 302 nm and the
second at 335 nm (Figure 8A). The CD band at 335 nm was
not assigned but was similar to the CD band at 330 nm
observed for V51D GCL (so far unassigned, but also seen at
337 nm on GCL). It was suggested5 that the weak CD band at
330 nm possibly originated from FAD.
The CD band at 302 nm approached its maximal intensity at
concentrations of pyruvate equivalent to the concentration of
active centers (Figure 8A). A plot of ellipticity at 302 nm versus
pyruvate concentration clearly shows a biphasic curve with
saturation at concentrations of pyruvate of <1 mM (Kd,pyruvate
=
8.2 μM) (Figure 8B). At pyruvate concentrations of >1 mM,
the plot was not saturated; rather, the release of (S)-
acetolactate was detected and was confirmed by a CD spectrum
of the reaction mixture after the protein had been removed
(Figure S2 of the Supporting Information). Similar data were
obtained for V51D/I393A GCL; a plot of ellipticity at 302 nm
versus pyruvate concentration displayed saturation at pyruvate
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syringe was mixed with glyoxylate in the second syringe in a
stopped-flow CD experiment, positive ellipticity developed at
302 nm within 5 s with a k3 of 2.69 s−1 and a k3′ of 0.63 s−1,
followed by a decrease in ellipticity with a k4 of 0.02 s−1
suggesting formation of a acetylglycolate−ThDP complex on
the enzyme (it being responsible for the CD band at 302 nm)
and its release (Figure 9B and Scheme 1B, top). When the
concentration of glyoxylate was increased to 4 mM, only the
release of acetylglycolate could be detected (k4 = 0.01 s−1)
(Figure 9C and Scheme 1B, top). These experiments suggest
that the acetylglycolate−ThDP complex can be detected by
stopped-flow CD at 302 nm and its release is the rate-limiting
step. In a stopped-flow CD experiment with V51D/I393A
GCL, the enzyme in one syringe was mixed with pyruvate in
the second syringe but in the absence of glyoxylate (Figure S3
of the Supporting Information). Again, the positive ellipticity at
302 nm was developed within 5 s with a k3 of 2.22 s−1 and a k3′
of 0.50 s−1, which can be assigned to the formation of the
acetolactate−ThDP complex. The origin of the CD band at 302
nm will be discussed below.
The I393A Substitution Allows GCL To Stabilize the
1′,4′-Iminopyrimidyl Tautomer of Stable Predecarbox-
ylation ThDP-Bound Intermediates from Pyruvate
Analogues. Upon addition of AcPhi to I393A GCL (Figure
10) and V51D/I393A GCL (not shown), the positive CD band
again developed at 302 nm and reached a plateau, indicating
formation of the 1′,4′-iminophosphinolactyl-ThDP intermedi-
ate and providing half-saturation values (S0.5,AcPhi) of 0.32 mM
(I393A GCL) and 0.37 mM (V51D/I393A GCL) (Table 3).
Figure 9. Formation of an acetylglycolate product by I393A GCL as
detected by CD. (A) CD spectra of I393A GCL recorded in the
presence of 1 mM pyruvate and upon addition of 1 mM glyoxylate at 6
°C. The I393A GCL was diluted to a concentration of active centers of
30.8 μM in 0.1 M KH2PO4 (pH 7.6) containing 0.5 mM ThDP, 2.5
mM MgCl2, 1.0 mM DTT, 10 μM FAD, and 1% glycerol. (B) Time-
dependent formation of an acetylglycolate−ThDP complex and
product release. The I393A GCL (4.69 mg/mL, active center
concentration of 72.4 μM) in buffer as in panel A was preincubated
with 1 mM pyruvate in one syringe and then mixed at 6 °C with 1.0
mM glyoxylate in the second syringe. Data were fit to a triple
exponential (eq 6). (C) Time-dependent release of an acetylglycolate
product. Conditions were the same as in panel B, but 4 mM glyoxylate
was present in the second syringe.
be detected by CD after the protein had been removed from
the reaction mixture. This is readily explained as the chiral
carbon can undergo racemization, being located between two
carbonyl functions, a ketone and a carboxylic acid. The
formation of acetylglycolate as the only carboligase product was
proven by the absence of CD bands corresponding to (S)-
acetolactate (positive band at 300 nm) or (R)-TSA (negative
band at 290 nm). Both products are optically stable and should
be detected if they are formed after the protein had been
removed. When I393A GCL incubated with pyruvate in one
Figure 10. CD spectra of I393A GCL titrated with AcPhi. (A) The
I393A GCL (2.0 mg/mL, active center concentration of 30.8 μM) in
0.10 M KH2PO4 (pH 7.6) containing 5 mM MgCl2 and 0.50 mM
ThDP was titrated with AcPhi (0.050−4.0 mM) at 25 °C. (B)
Dependence of the CD at 302 nm on the concentration of AcPhi. Data
were fit to a Hill equation (eq 3).
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The I393A GCL displayed positive cooperativity on AcPhi
binding (nH = 2.23). In contrast, for I393V GCL and for GCL,
there was no significant change in the CD spectra upon
addition of AcPhi, most likely because there is little or no
binding of AcPhi to the active centers (data not shown).
Similar experiments conducted with I393A and MAP
displayed the band at 302 nm, which we ascribe to the 1′,4′-
iminophosphonolactylThDP (data not shown), and allowed
calculation of a Kd,MAP of 1.4 mM (Table 3). MAP thus also
binds to I393A GCL, although it is a weaker substrate analogue
than AcPhi, as was found with other ThDP enzymes.24 For
V51D/I393A GCL, the Kd,MAP was 0.11 mM.
CD Evidence of the Formation of the 1′,4′-Iminophos-
phinopropionylThDP from PrPhi. In addition to AcPhi, the
I393A GCL and V51D/I393A GCL could also bind PrPhi,
[CH3CH2C(O)P(H)(=O)O−], an analogue of 2-oxobuta-
noate, and form the 1′,4′-iminophosphinopropionylThDP,
providing Kd,PrPhi values of 0.46 mM (I393A GCL) and 0.28
mM (V51D/I393A GCL), similar to the value obtained with
AcPhi (Table 3). The I393A GCL displayed positive
cooperativity on PrPhi binding, similar to that observed with
AcPhi. Apparently, the V51D substitution does indeed affect
the cooperativity of binding of a pyruvate analogue to the
I393A variant (Table 3) for reasons unclear at this time.
A major conclusion from the CD studies cited above is that
the predecarboxylation intermediate analogues derived from
AcPhi, PrPhi, and MAP can be stabilized on I393A and V51D/
I393A variants in their 1′,4′-iminopyrimidine tautomeric forms.
It is significant that the V51D/I393A variant behaves like the
I393A variant in this regard. The observation that the presence
or absence of an aspartate at position 51 has little effect on the
production of acetolactate by V51D/I393A GCL in comparison
with a singly substituted I393A GCL implies that its effect is
specifically on the formation of TSA.
increase in the pyruvate concentration to >1 mM shifts this
equilibrium to acetolactate release. Both the HEThDP and
acetolactylThDP would be expected to exist in their 1′,4′-
iminopyrimidyl tautomeric form on the bases of much
precedent for several ThDP enzymes,12 and either species
could be responsible for the CD band at 302 nm.
The V51D/I393A GCL behaves like the I393A GCL
according to a kinetic study of acetolactate formation, and
from CD studies of the stabilization of the predecarboxylation
intermediate analogues derived from AcPhi, MAP, and PrPhi.
The predecarboxylation intermediate analogues derived from
AcPhi, PrPhi, and MAP could be stabilized on I393A GCL and
V51D/I393A GCL in their 1′,4′-iminopyrimidine tautomeric
forms.
The observation that the presence or absence of an aspartate
at position 51 has little effect on the V51D/I393A GCL implies
that it is specifically effective on TSA formation from the
natural substrate glyoxylate.
With these observations on the I393-substituted GCLs, we
suggest that variants engineered at this position would be
expected to convert this enzyme to a useful tool in chiral
synthesis. For example, both pyruvate and glyoxalate are
excellent acceptors for the enamine derived from pyruvate for
acetolactate formation (k3 = 2.2 s−1) and for acetylglyoxalate
formation (k3 = 2.7 s−1) (Scheme 1B).
As an aside, we also emphasize that CD has clearly been
shown to provide an excellent direct kinetic assay for product
formation, much simpler than any coupled assay, or the classical
Westerfeld method for acetoin determination.
Role of the Conserved Glutamate. GCL has several
properties, which set it apart from the other members of the
pyruvate decarboxylase-pyruvate oxidase subfamily of ThDP-
dependent enzymes.28−31 The conserved glutamate residue
hydrogen bonded to N1′ of ThDP, found in all the other
ThDP-dependent enzymes, is replaced with a valine residue,
and the sole substrate of GCL is the 2-aldoacid glyoxylate
rather than a 2-ketoacid (Scheme 1A). In addition, not only is
the conserved glutamate missing, there are no potential acid−
base side chains in the proximity of the ThDP near the C2
thiazolium atom. The recently accepted paradigm regarding the
mechanism of ThDP-dependent enzymes12,24 suggests that the
conserved Glu residue is essential to the tautomerization of
ThDP, and that the 1′,4′-iminopyrimidine, but not the 4′-
aminopyrimidine, tautomer is sufficiently basic to deprotonate
the thiazolium C2−H group and thus lead to the active
carbanion/ylide form of the coenzyme. The studies reported
here provide convincing evidence that with GCL, (1) only the
unusual 1′,4′-iminopyrimidine tautomer, rather than the
canonical AP form, is detected and (2) the IP−APH+
proteolytic equilibrium functions in GCL, allowing the IP
form to play its central role in the absence and presence of a
group that is hydrogen bonded to N1′.
DISCUSSION
■
Following the initial report on the structure of GCL,5 we
explore two important issues about the enzyme remaining to be
elucidated: (1) control of substrate specificity and (2) role of
the residue opposite the N1′ atom of ThDP, typically occupied
by the highly conserved glutamate.
Control of Substrate Specificity at Position 393.
Experiments exploiting different techniques show that the
putative higher reactivity of the aldehyde group of glyoxylate is
not the important determinant of the substrate specificity of
GCL, but, rather, the structure of the active site pocket. In
particular, residue Ile393 restricts the ability of GCL to form
covalent complexes of ThDP with donor substrates larger than
glyoxylate.
Remarkably, once the size of the residue at position 393 is
reduced to alanine, pyruvate is accepted as an alternate
substrate, and evidence of ThDP-bound intermediates derived
from pyruvate could be detected. The CD band at 302 nm
observed on I393A at low pyruvate concentrations (Figure 8A)
indicates that a covalent intermediate (or intermediates) is
formed on GCL at low pyruvate concentrations. We believe
this intermediate is one with tetrahedral substitution at C2α but
is not the enamine. Because (R)-acetolactate is produced at
higher pyruvate concentrations, the enamine must be present,
albeit at a concentration too low to detect. A possible source of
the band at 302 nm is the HEThDP, the conjugate acid of and
at protolytic equilibrium with the enamine, or the bound
carboligase product acetolactylThDP (Scheme 1A,B). An
The key ThDP-bound intermediates derived from glyoxylate
were identified on the reaction path of V51D GCL. A
comparison of the rate constants for 1′,4′-iminoglycolyl-
ThDP formation, its decarboxylation to the enamine, TSA−
ThDP complex formation, and finally release of TSA product
from GCL indicates that under the experimental conditions,
TSA product release is the rate-limiting step, in agreement with
the NMR approach.5 In the NMR approach,5 covalent ThDP-
bound intermediates are released from the enzymes, while in
this study, we observe enzyme-bound intermediates directly;
both methods lead to the same conclusions.
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The 1′,4′-iminoglycolyl-ThDP was detected on GCL when
the enzyme was titrated by equivalent amounts of glyoxylate.
The rate constant of 1′,4′-iminoglycolyl-ThDP formation was
at least 5 times slower than that for V51D GCL.
A major surprise of our results in toto is that all the steps with
the exception of product release appear to have rates
comparable to or even better than (when adjusted to the pH
optimum of the variant) those with Asp in place of Val at
position 51 (Table 2).
base candidates near the thiazolium ring allows us to conclude
that (a) ThDP itself in its various forms can indeed catalyze all
of the reactions of the GCL pathway and (b) the amino acid
opposite the N1′ atom of ThDP (typically the conserved
glutamate) is responsible for both the pKa of the APH+ form
and the pH dependence of the activity profiles.
Perhaps the most interesting puzzle remaining concerning
GCL is the selective advantage of the valine residue (Val51) in
place of the conserved glutamate. It was previously shown that
V51D GCL and V51E GCL are less active than GCL in the
synthesis of TSA from glyoxylate, despite having higher rates of
activation (deprotonation at C2) of the coenzyme.5 With the
results presented here, it would appear that it is in the product
release rate, rather than the rates of any intermediate steps,
where the valine has a dramatic effect. The structural basis of
these findings remains to be elucidated.
There is also strong evidence that adding the acceptor
substrate significantly increases the rate of decarboxylation, as
seen in the glyoxylate concentration dependence of the kinetics
of product formation. This may be yet another example of
active site communication where glyoxylate in the active site of
one subunit increases the rate of decarboxylation in the active
site of a second subunit in the “functional dimer” typical of
ThDP decarboxylases. Similar observations had already been
reported for benzoylformate decarboxylase32 and yeast pyruvate
decarboxylase,33 suggesting that the behavior reflects an
alternating active center mechanism in the functional dimer.
The presence of a water molecule in the proximity of the N1′
atom might provide an explanation for the ability of GCL to
catalyze the tautomeric equilibration required for the reaction.
It should be noted that the structures of ThDP-dependent
enzymes in the PDB show water molecules in the proximity of
the N1′ atom.5 Given the high concentration of water in all of
these crystals, the presence of such water in GCL would not be
surprising. The transient charge transferred to the 4′-amino-
pyrimidine moiety in the course of ThDP activation (Scheme
1A) and other steps in the mechanistic cycle of GCL can
presumably be shared with a water molecule H-bonded to N1′
and with other groups in its hydrogen bonding network.
Significant residual activity is seen in a variant of the
homologous enzyme AHAS II when the conserved glutamate
(Glu47) is replaced with alanine.34 When the conserved
glutamates on the E1 component of the E. coli pyruvate
dehydrogenase complex11 and the yeast pyruvate decarbox-
ylase35 are replaced with alanine, there is similar residual
activity detected. It is reasonable to assume that a water
molecule can fit into the gap created by such substitutions and
that this water might assist the tautomerization. It is relevant to
mention that the pKa in models for the 4′-aminopyrimidinium
ring of ThDP is ∼4.85, so that only a modest pKa perturbation
would be required in the active centers of ThDP enzymes to
match this.22 It was pointed out that the ThDP binding site in
GCL appears in fact to have a larger proportion of aliphatic,
nonpolarizable residues in the region of the thiazolium and 4′-
aminopyrimidine rings versus what is observed for other ThDP-
dependent enzymes, which should favor the stability of the
formally charge-neutral ylide on the thiazolium ring (in Scheme
1A).5 We suggest that the major need is some manner of
protonation of N1′ so that the APH+ form can be generated,
the source of the IP tautomer. The fact that the APH+ form was
indeed observed by solid-state NMR experiments on each of
the three enzymes examined (yeast pyruvate decarboxylase and
the E1 components of both the pyruvate and 2-oxoglutarate
dehydrogenase complexes from E. coli) supports this
hypothesis.26
ASSOCIATED CONTENT
■
S
* Supporting Information
Figures S1−S3. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*D.C.: e-mail, chipman@bgu.ac.il; phone, [+972]-8-647 9212.
F.J.: e-mail, frjordan@newark.rutgers.edu; phone, (973) 353-
5470; fax, (973) 353-1264.
Author Contributions
N.N. and E.B. contributed equally to this work.
Funding
This work was supported by U.S.-Israel Binational Science
Foundation Grant 2007-129 (to B.S., D.C., and F.J.) and
National Institutes of Health Grant GM050380 to F.J.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
GCL, wild-type glyoxylate carboligase; I393A GCL, V51D
GCL, and V51D/I393A GCL, GCL variants at the indicated
positions; TSAR, tartronate semialdehyde reductase; AHAS,
acetohydroxyacid synthase; ThDP, thiamin diphosphate; TSA,
tartronate semialdehyde; AL, acetolactate; MAP, methyl
acetylphosphonate; AcPhi, acetylphosphinate; PrPhi, propio-
nylphosphinate; LThDP, C2α-lactylthiamin diphosphate;
PLThDP, phosphonolactylthiamin diphosphate (the MAP−
ThDP adduct); DCPIP, 2,6-dichlorophenolindophenol; PEG,
polyethylene glycol; AP, 4′-aminopyrimidine tautomeric form
of ThDP; IP, 1′,4′-iminopyrimidine tautomeric form of ThDP;
APH+, N1′-protonated 4′-aminopyrimidinium form of ThDP;
CD, circular dichroism.
REFERENCES
■
(1) Chang, Y. Y., Wang, A. Y., and Cronan, J. E., Jr. (1993) Molecular
cloning, DNA sequencing, and biochemical analyses of Escherichia coli
glyoxylate carboligase. An enzyme of the acetohydroxy acid synthase-
pyruvate oxidase family. J. Biol. Chem. 268, 3911−3919.
(2) Gupta, N. K., and Vennesland, B. (1964) Glyoxylate Carboxylase
of Escherichia coli: A Flavoprotein. J. Biol. Chem. 239, 3787−3789.
(3) Gupta, N., and Vennesland, B. (1966) Glyoxylate carboligase of
Escherichia coli: Some properties of the enzyme. Arch. Biochem. Biophys.
113, 255−264.
That said, the V51D substitution once more supports the
suggestion that the pKa for the (IP+AP)−APH+ equilibrium
coincides with the pH optimum of activity, ensuring the
presence of all three forms for catalysis. This is the sixth ThDP
enzyme to date supporting this notion. Having no other acid−
7951
dx.doi.org/10.1021/bi300893v | Biochemistry 2012, 51, 7940−7952

Biochemistry
Article
(4) Cromatie, T. H., and Walsh, C. T. (1976) Escherichia coli
glyoxalate carboligase. Properties and reconstitution with 5-deazaFAD
and 1,5-dihydrodeazaFADH2. J. Biol. Chem. 251, 329−333.
(5) Kaplun, A., Binshtein, E., Vyazmensky, M., Steinmetz, A., Barak,
Z., Chipman, D. M., Tittmann, K., and Shaanan, B. (2008) Glyoxylate
carboligase lacks the canonical active site glutamate of thiamin-
dependent enzymes. Nat. Chem. Biol. 4, 113−118.
(6) Shaanan, B., and Chipman, D. M. (2009) Reaction mechanism of
thiamin diphosphate enzymes: New insights into the role of a
conserved glutamate residue. FEBS J. 276, 2447−2453.
(7) Chipman, D. M., Barak, Z., Shaanan, B., Vyazmensky, M.,
Binshtein, E., Belenky, I., Temam, V., Steinmetz, A., Golbik, R., and
Tittmann, K. (2009) Origin of the specificities of acetohydroxyacid
synthases and glyoxylate carboligase. J. Mol. Catal. B: Enzym. 61, 50−
55.
(8) O’Brien, T. A., Kluger, R., Pike, D. C., and Gennis, R. B. (1980)
Phosphonate analogues of pyruvate. Probes of substrate binding to
pyruvate oxidase and other thiamin pyrophosphate-dependent
decarboxylases. Biochim. Biophys. Acta 613, 10−17.
(9) Wille, G., Meyer, D., Steinmetz, A., Hinze, E., Golbik, R., and
Tittmann, K. (2006) The catalytic cycle of a thiamin diphosphate
enzyme examined by cryocrystallography. Nat. Chem. Biol. 2, 324−328.
(10) Tittmann, K., and Wille, G. (2009) X-ray crystallographic
snapshots of reaction intermediates in pyruvate oxidase and trans-
ketolase illustrate common themes in thiamin catalysis. J. Mol. Catal. B:
Enzym. 61, 93−99.
(11) Nemeria, N. S., Arjunan, P., Chandrasekhar, K., Mossad, M.,
Tittmann, K., Furey, W., and Jordan, F. (2010) Communication
between thiamin cofactors in the Escherichia coli pyruvate dehydrogen-
ase complex E1 component active centers: Evidence for a “direct
pathway” between the 4′-aminopyrimidine N1′ atoms. J. Biol. Chem.
285, 11197−11209.
(21) Engel, S., Vyazmensky, M., Geresh, S., Barak, Z., and Chipman,
D. M. (2003) Acetohydroxyacid synthase: A new enzyme for chiral
synthesis of R-phenylacetylcarbinol. Biotechnol. Bioeng. 83, 833−840.
(22) Nemeria, N., Korotchkina, L., McLeish, M. J., Kenyon, G. L.,
Patel, M. S., and Jordan, F. (2007) Elucidation of the chemistry of
enzyme-bound thiamin diphosphate prior to substrate binding:
defining internal equilibria among tautomeric and ionization states.
Biochemistry 46, 10739−10744.
(23) Nemeria, N. S., Korotchkina, L. G., Chakraborty, S., Patel, M. S.,
and Jordan, F. (2006) Acetylphosphinate is the most potent
mechanism-based substrate-like inhibitor of both the human and
Escherichia coli pyruvate dehydrogenase components of the pyruvate
dehydrogenase complex. Bioorg. Chem. 34, 362−379.
(24) Kluger, R., and Tittmann, K. (2008) Thiamin Diphosphate
Catalysis: Enzymic and Nonenzymic Covalent Intermediates. Chem.
Rev. 108, 1797−1833.
(25) Nemeria, N. S., Chakraborty, S., Balakrishnan, A., and Jordan, F.
(2009) Reaction mechanisms of thiamin diphosphate enzymes:
Defining states of ionization and tautomerization of the cofactor at
individual steps (minireview). FEBS J. 276, 2432−2446.
(26) Balakrishnan, A., Paramasivam, S., Chakraborty, S., Polenova, T.,
and Jordan, F. (2012) Solid-State Nuclear Magnetic Resonance
Studies Delineate the Role of the Protein in Activation of Both
Aromatic Rings of Thiamin. J. Am. Chem. Soc. 134, 665−672.
(27) Paramasivam, S., Balakrishnan, A., Dmitrenko, O., Gobert, A.,
Begley, T. P., Jordan, F., and Polenova, T. (2011) Solid-state NMR
and density function theory studies of ionization states of thiamin. J.
Phys. Chem. 115, 730−736.
(28) Muller, Y. A., and Schulz, G. E. (1993) Structure of the thiamin-
and flavin-dependent enzyme pyruvate oxidase. Science 259, 965−967.
(29) Lindqvist, Y., Schneider, G., Ermler, U., and Sundstrom, M.
(1992) Three-dimensional structure of transketolase, a thiamine
diphosphate dependent enzyme, at 2.5 Å resolution. EMBO J. 11,
2373−2379.
(30) Dyda, F., Furey, W., Swamibathan, S., Sax, M., Farrenkopf, B.,
and Jordan, F. (1993) Catalytic centers in the thiamin diphosphate
dependent enzyme pyruvate decarboxylase at 2.4 Å resolution.
Biochemistry 32, 6165−6170.
(31) Arjunan, P., Umland, T., Dyda, F., Swaminathan, S., Furey, W.,
Sax, M., Farrenkopf, B., Gao, Y., Zhang, D., and Jordan, F. (1996)
Crystal structure of the thiamin diphosphate-dependent enzyme
pyruvate decarboxylase from yeast Saccharomyces cerevisiae at 2.3 Å
resolution. J. Mol. Biol. 256, 590−600.
(32) Sergienko, E. A., Wang, J., Polovnikova, L., Hasson, M. S.,
McLeish, M. J., Kenyon, G. L., and Jordan, F. (2000) Spectroscopic
Detection of Transient Thiamin Diphosphate-Bound Intermediates on
Benzoylformate Decarboxylase. Biochemistry 39, 13862−13869.
(33) Sergienko, E. A., and Jordan, F. (2002) Yeast pyruvate
decarboxylase tetramers can dissociate into dimers along two
interfaces. Hybrids of low-activity D28A (or D28N) and E477Q
variants, with substitution of adjacent active center acidic groups from
different subunits, display restored activity. Biochemistry 41, 6164−
6169.
(34) Bar-Ilan, A., Balan, V., Tittmann, K., Golbik, R., Vyazmensky,
M., Hubner, G., Barak, Z., and Chipman, D. M. (2001) Binding and
activation of thiamin diphosphate in acetohydroxyacid synthase.
Biochemistry 40, 11946−11954.
(35) Balakrishnan, A., Gao, Y., Moorjani, P., Nemeria, N., Tittmann,
K., and Jordan, F. (2012) Bifunctionality of the thiamin diphosphate
cofactor: Assignment of tautomeric/ionization states of the 4′-
aminopyrimidine ring when various intermediates occupy the active
sites during the catalysis of yeast pyruvate decarboxylase. J. Am. Chem.
Soc. 134, 3873−3885.
(12) Nemeria, N. S., Chakraborty, S., Balakrishnan, A., and Jordan, F.
(2009) Reaction mechanisms of thiamin diphosphate enzymes:
Defining states of ionization and tautomerization of the cofactor at
individual steps. FEBS J. 276, 2432−2446.
(13) Nemeria, N., Chakraborty, S., Baykal, A., Korotchkina, L. G.,
Patel, M. S., and Jordan, F. (2007) The 1′,4′-iminopyrimidine
tautomer of thiamin diphosphate is poised for catalysis in asymmetric
active centers on enzymes. Proc. Natl. Acad. Sci. U.S.A. 104, 78−82.
(14) Baykal, A. T., Kakalis, L., and Jordan, F. (2006) Electronic and
nuclear magnetic resonance spectroscopic features of the 1′,4′-
iminopyrimidine tautomeric form of thiamin diphosphate, a novel
intermediate on enzymes requiring this coenzyme. Biochemistry 45,
7522−7528.
(15) Baillie, A. C., Wright, B. J., and Wright, K. (1982) U.S. Patent
4,339,443.
(16) Shim, D. J., Nemeria, N. S., Balakrishnan, A., Patel, H., Song, J.,
Wang, J., Jordan, F., and Farinas, E. T. (2011) Assignment of function
to histidines 260 and 298 by engineering the E1 component of the
Escherichia coli 2-oxoglutarate dehydrogenase complex; substitutions
that lead to acceptance of substrates lacking the 5-carboxyl group.
Biochemistry 50, 7705−7709.
(17) Kluger, R., and Pike, D. C. (1977) Active site generated
analogues of reactive intermediates in enzymic reactions. Potent
inhibition of pyruvate dehydrogenase by a phosphonate analogue of
pyruvate. J. Am. Chem. Soc. 99, 4504−4506.
(18) Vinogradov, M., Kaplun, A., Vyazmensky, M., Engel, S., Golbik,
R., Tittmann, K., Uhlemann, K., Meshalkina, L., Barak, Z., Hubner, G.,
̈
and Chipman, D. M. (2005) Monitoring the acetohydroxy acid
synthase reaction and related carboligations by circular dichroism
spectroscopy. Anal. Biochem. 342, 126−133.
(36) Steinmetz, A., Vyazmensky, M., Meyer, D., Barak, Z., Golbik, R.,
Chipman, D. M., and Tittmann, K. (2010) Valine 375 and
Phenylalanine 109 Confer Affinity and Specificity for Pyruvate as
Donor Substrate in Acetohydroxy Acid Synthase Isozyme II from
Escherichia coli. Biochemistry 49, 5188−5199.
(19) Chung, S. T., Tan, R. T., and Suzuki, I. (1971) Glyoxylate
carboligase of Pseudomonas oxalaticus. A possible structural role for
flavine-adenine dinucleotide. Biochemistry 10, 1205−1209.
(20) Epelbaum, S., Chipman, D. M., and Barak, Z. (1990)
Determination of products of acetohydroxy acid synthase by the
colorimetric method, revised. Anal. Biochem. 191, 96−99.
7952
dx.doi.org/10.1021/bi300893v | Biochemistry 2012, 51, 7940−7952