Observation of thiamin-bound intermediates and microscopic rate constants for their interconversion on 1-deoxy- d -xylulose 5-phosphate synthase: 600-Fold rate acceleration of pyruvate decarboxylation by d -glyceraldehyde-3-phosphate.

Article abstract of DOI:10.1021/ja307315u

The thiamin diphosphate (ThDP)-dependent enzyme 1-deoxy-d-xylulose 5-phosphate (DXP) synthase carries out the condensation of pyruvate as a 2-hydroxyethyl donor with d-glyceraldehyde-3-phosphate (d-GAP) as acceptor forming DXP. Toward understanding catalysis of this potential anti-infective drug target, we examined the pathway of the enzyme using steady state and presteady state kinetic methods. It was found that DXP synthase stabilizes the ThDP-bound predecarboxylation intermediate formed between ThDP and pyruvate (C2α-lactylThDP or LThDP) in the absence of d-GAP, while addition of d-GAP enhanced the rate of decarboxylation by at least 600-fold. We postulate that decarboxylation requires formation of a ternary complex with both LThDP and d-GAP bound, and the central enzyme-bound enamine reacts with d-GAP to form DXP. This appears to be the first study of a ThDP enzyme where the individual rate constants could be evaluated by time-resolved circular dichroism spectroscopy, and the results could have relevance to other ThDP enzymes in which decarboxylation is coupled to a ligation reaction. The acceleration of the rate of decarboxylation of enzyme-bound LThDP in the presence of d-GAP suggests a new approach to inhibitor design.

Full text of DOI:10.1021/ja307315u

Article
pubs.acs.org/JACS
Observation of Thiamin-Bound Intermediates and Microscopic Rate
Constants for Their Interconversion on 1‑Deoxy‑D‑xylulose
5‑Phosphate Synthase: 600-Fold Rate Acceleration of Pyruvate
Decarboxylation by D‑Glyceraldehyde-3-phosphate.
Hetalben Patel,† Natalia S. Nemeria,† Leighanne A. Brammer,‡ Caren L. Freel Meyers,*,‡
and Frank Jordan*,†
†Department of Chemistry, Rutgers University, Newark, New Jersey 07102, United States
‡Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland
21205, United States
S
* Supporting Information
ABSTRACT: The thiamin diphosphate (ThDP)-dependent enzyme 1-deoxy-D-
xylulose 5-phosphate (DXP) synthase carries out the condensation of pyruvate as a 2-
hydroxyethyl donor with D-glyceraldehyde-3-phosphate (D-GAP) as acceptor forming
DXP. Toward understanding catalysis of this potential anti-infective drug target, we
examined the pathway of the enzyme using steady state and presteady state kinetic
methods. It was found that DXP synthase stabilizes the ThDP-bound
predecarboxylation intermediate formed between ThDP and pyruvate (C2α-
lactylThDP or LThDP) in the absence of D-GAP, while addition of D-GAP enhanced the rate of decarboxylation by at least
600-fold. We postulate that decarboxylation requires formation of a ternary complex with both LThDP and D-GAP bound, and
the central enzyme-bound enamine reacts with D-GAP to form DXP. This appears to be the first study of a ThDP enzyme where
the individual rate constants could be evaluated by time-resolved circular dichroism spectroscopy, and the results could have
relevance to other ThDP enzymes in which decarboxylation is coupled to a ligation reaction. The acceleration of the rate of
decarboxylation of enzyme-bound LThDP in the presence of D-GAP suggests a new approach to inhibitor design.
covalent intermediates (Scheme 1, right): stopped-flow circular
dichroism (CD) and chemical quench followed by NMR
detection. The CD method can monitor events on the enzyme
itself, the NMR method monitors ThDP-bound intermediates
after release from the enzyme (fortuitously, all the major ones
are stable in acid), and is also useful to provide positive
identification of the species seen in the CD spectrum. The CD
studies take advantage of (a) identification of an electronic
absorption for the 1′,4′-iminopyrimidine (IP form, Scheme 1
left) of ThDP at 300−310 nm8 and (b) demonstration on 10
ThDP enzymes so far that at pH values at or above the pKa of
the 4′-aminopyrimidinium form (APH+) of ThDP, the IP
tautomer predominates in ThDP-bound covalent intermediates
where the C2α-carbon carries four substituents.9
INTRODUCTION
■
The enzyme 1-deoxy-D-xylulose 5-phosphate (DXP) synthase
generates the first crucial intermediate in the biosynthesis of
both thiamin and pyridoxal as well as in isoprenoid biosynthesis
essential in human pathogens.1−3 DXP synthase uses thiamin
diphosphate (ThDP) as coenzyme and pyruvate and D-GAP as
substrates. The reaction catalyzed by DXP synthase combines
aspects of decarboxylase and carboligase enzymes. The
chemical mechanism of the first step is likely to parallel typical
ThDP-dependent pyruvate and other 2-oxoacid decarboxylase
enzymes, the pyruvate forming a predecarboxylation covalent
intermediate with ThDP (C2α-lactylThDP or LThDP)
followed by decarboxylation to an enamine/C2α-carbanion/
C2α-hydroxyethylidene-ThDP, which undergoes carboligation
with the aldehyde functional group of D-GAP to form DXP
(Scheme 1 right).
EXPERIMENTAL PROCEDURES
■
Materials. ThDP, pyruvate, yeast alcohol dehydrogenase, nicoti-
namide adenine dinucleotide, 2,6-dichlorophenolindophenol
(DCPIP), D,L-GAP, and 1-deoxy-D-xylulose 5-phosphate (DXP)
were from Sigma-Aldrich (St. Louis, MO). HEPES and dithiothreitol
were from USB (Cleveland, OH). DXP synthase was purified as
reported previously.7
Previous mechanistic studies of DXP synthase were
performed under steady-state conditions.4−7 We report the
first study of DXP synthase using tools capable of observing
and determining the time-resolved behavior of ThDP-bound
Received: July 25, 2012
Published: October 16, 2012
© 2012 American Chemical Society
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Scheme 1. Presteady State Detection of Forward Rate Constant for Individual Steps in DXP Synthase Catalytic Cycle at 6 °C
a
with Both Pyruvate and GAP Present in LThDP Formation
a
k1 is formation of LThDP; k2 is decarboxylation to enamine; k3 is carboligation with D-GAP to provide DXP∼ThDP; k4 is release of DXP product.
CD studies assume that LThDP and DXP∼ThDP when bound to DXP synthase are both in the IP tautomeric form while NMR can differentiate
them by the method of Tittmann et al.14 The decarboxylation rate constant 4.2 × 101 s−1 pertains to k2.
Activity Measurement of DXP Formation Using Coupled
Enzyme (IspC). DXP synthase activity was measured spectrophoto-
metrically using pyruvate and D,L-GAP, and IspC (DXP reductoiso-
merase) as a coupling enzyme as reported previously.7 Under these
conditions D-GAP from a solution of D,L-GAP reacts exclusively to
form DXP.10 Here we have used commercially available racemic D,L-
GAP by knowing that L-GAP would not affect DXP synthase
activity.1,5,6 The concentration of D-GAP was calculated as one-half
of the D,L-GAP concentration.
CD Spectroscopy. CD spectra were recorded on a Applied
Photophysics Chirascan CD spectrometer (Leatherhead, U.K.) in 2.4
mL volume with 1 cm path length cell.
Formation of LThDP from Pyruvate by DXP Synthase. DXP
synthase (55.9 μM active centers) was titrated with pyruvate (0.05−1
mM) in 100 mM HEPES (pH 8.0) containing 100 mM NaCl, 0.2 mM
ThDP, and 1 mM MgCl2 at 4 °C. After apparent saturation with 1 mM
pyruvate, 250 μM D-GAP was added. The Kd,app was calculated by
fitting the data to a Hill function (equation S1, see Supporting
Information).
reaction was monitored at 313 nm for 7 s. Data were fit to a single-
exponential model as in eq 1.
Single Turnover Experiments of DXP Synthase with Pyruvate in
the Absence of D-GAP. DXP synthase (81.4 μM active centers) in one
syringe was mixed with equal volume of pyruvate (50 μM) in the
second syringe, both in buffer A at 6 °C. The reaction was monitored
at 313 nm for 50 s, and data were fit to a triple exponential model as in
eq 2. The same experiment was carried out at 37 °C, and data were fit
to double exponential model as in eq 3.
Presteady State Decarboxylation of 1′,4′-IminoLThDP in the
Presence of D-GAP. DXP synthase (68.1 μM active centers) was
premixed with 500 μM pyruvate at 6 °C to form LThDP in one
syringe, then rapidly mixed with an equal volume of 100 μM D-GAP in
the second syringe, both in buffer A at 6 °C. A similar experiment was
performed using D-GAP (0.05−0.25 mM), and data were fit to eq 1.
Presteady State Formation of DXP. DXP synthase (1.04 μM active
centers) in buffer A placed in one syringe was mixed with an equal
volume of 4 mM pyruvate and 2 mM D-GAP in the same buffer placed
in the second syringe. Reaction was monitored by CD over a period of
100 s at 6 °C at 297 nm, and data were fitted to a double-exponential
model as in eq 4.
Stopped-Flow CD Spectroscopy. Kinetic traces were recorded
on a Pi*-180 stopped-flow CD spectrometer (Applied Photophysics,
U.K.) using 10 mm path length. ThDP-bound intermediates (LThDP
specifically) were detected at 313 nm and DXP at 297 nm, as dictated
by the high sensitivity of the lamp at those wavelengths. Data from 10
repetitive shots were averaged and fit to the appropriate equation using
Sigma Plot v.10.0.
NMR Spectroscopy. NMR spectra were acquired on a Varian
INOVA 600 MHz instrument. The water signal was suppressed by
presaturation.
Detection of LThDP by 1H NMR Spectroscopy. The reaction
mixture containing DXP synthase (459.3 μM active centers) in 20 mM
Tris (pH 8.0), 100 mM NaCl, 0.1 mM ThDP, and 0.5 mM MgCl2 was
mixed with 1 mM pyruvate in the same buffer. The reaction mixture
was incubated at 5 °C for 5 s (and an identical sample for 20 s) and
quenched with 12.5% TCA in 1 M DCl/D2O. The mixture was
CD (t) = CD1e−k t + c
1
(1)
(2)
(3)
(4)
λ
CD (t) = CD1e−k t + CD2e−k t − CD3e−k t + c
1
2
3
λ
CD (t) = CD1e−k t − CD3e−k t + c
1
3
λ
1
CD (t) = CD1e−k t + CD2e−k t + c
1
2
centrifuged at 15 700 g for 20 min, and the H NMR spectrum of the
λ
supernatant was recorded using 16 384 scans with a recycle delay of
2.0 s.
where k1, k2, and k3 are the apparent rate constants, c is CDmaxλ in the
exponential rise to maximum model or CDmin in the exponential
1
λ
Detection of DXP Product by H NMR Spectroscopy. A reaction
decay model.
mixture containing DXP synthase (22.22 μM active centers) in 20 mM
KH2PO4 (pH 7.5) containing 100 mM NaCl, 0.1 mM ThDP, 0.5 mM
MgCl2, and 1 mM DTT was mixed with 15 mM D-GAP and 10 mM
pyruvate in the same buffer. After overnight incubation at 4 °C, the 1H
NMR spectrum of the supernatant was recorded.
Presteady State Formation of 1′,4′-IminoLThDP. DXP synthase
(68.1 μM active centers) in one syringe was mixed with equal volume
of 2 mM pyruvate in the second syringe under presteady state
conditions in buffer A (50 mM Tris (pH 8.0), 100 mM NaCl, 0.5 mM
ThDP, 2.0 mM MgCl2, 1 mM DTT, 1% glycerol) at 6 °C. The
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the presteady state experiment, DXP synthase (68.1 μM active
centers) in one syringe was rapidly mixed with saturating
concentration of pyruvate (2 mM) in the second syringe, giving
rise to an increase at 313 nm and reaching a steady state in 2 s
with rate constant of 1.1 0.03 s−1 (Figure 1, right).
RESULTS
■
The Tautomeric and Ionization States of ThDP on
DXP Synthase. The near-UV CD spectrum of DXP synthase
displays a negative band at 320 nm (Figure S1A of the
Supporting Information), suggesting the preponderance of the
AP tautomer of ThDP bound to the enzyme.9 Next, the CD
spectrum was recorded at different pH values to convert the AP
form of ThDP to the APH+ form (Scheme 1, left). Reducing
the pH from 7.98 to 7.07 gradually diminished the amplitude of
the CD band at 320 nm (Figure S1A of the Supporting
Information), indicating that the AP form of ThDP is dominant
at pH 7.98, and the APH+ form is dominant at pH 7.07 [while
the APH+ form has no CD signature identified so far, its
existence on three ThDP enzymes was recently demonstrated
by solid-state NMR].11 A plot of the CD320 against pH could
be fitted to a single proton titrating with a pKa of 7.5 (Figure
S1A of the Supporting Information, inset) for the protonic
equilibrium ([AP] + [IP])/[APH+]. This pKa elevation
compared to the value of 4.85 in water12 has been attributed
to interaction via the short hydrogen bond between the N1′-
atom of the 4′-aminopyrimidine and the highly conserved
glutamate side chain.
Next, we determined the pH dependence of the rate of
formation of DXP by DXP synthase to establish whether the
pKa of the APH+ bears any relationship to the pH dependence
of the activity. The rate of formation of DXP by DXP synthase
determined at pH values of 6.65−8.08 (Figure S1B of the
Supporting Information) revealed a pKapp of 7.6 (Figure S1B of
the Supporting Information inset), which correlates well with
the pKa of 7.5 for the protonic equilibrium ([AP]+[IP])/
[APH+] (Figure S1A of the Supporting Information, inset).
That is the pKa of the enzyme-bound APH+ form is very near
the pH of optimal activity of the enzyme7 signaling the need for
all forms APH+, AP, and IP in the mechanism, in accord with
results on five other ThDP enzymes, thus creating a general
trend.13
The species represented by the positive CD band at 313 nm
(signifying the presence of the IP tautomeric form of ThDP)
could be assigned as either the predecarboxylation intermediate
(LThDP) or the postdecarboxylation ThDP-bound DXP
intermediate (Scheme 1, right), since both carry tetrahedral
substitution at C2α. To resolve the ambiguity, an NMR method
was used,14 a method that recognized that the chemical shift of
the C6′-H resonance is different for LThDP (7.26 ppm)14 and
C2α- hydroxyethylThDP (HEThDP, 7.33 ppm)14 and indeed
for carboligated product-ThDP adducts as well. Accordingly, an
NMR spectrum of a mixture of DXP synthase with pyruvate
quenched in 5 or 20 s revealed a chemical shift of 7.26 ppm,
confirming that the CD at 313 nm corresponds to LThDP
(Figure S3 of the Supporting Information).14 Integration of the
C2-H and C6′-H resonances showed that approximately the
same percent of ThDP was converted to LThDP in 5 s (∼50%)
and in 20 s (∼55%), thus confirming the remarkable stability of
LThDP on DXP synthase.
To interpret data once both pyruvate and D-GAP are present,
we need to first establish rates of LThDP formation and
decarboxylation in the absence of D-GAP. We accomplished
this by a single turnover stopped-flow CD experiment with
DXP synthase concentration in excess of pyruvate, carried out
at 6 and 37 °C under the same conditions, the latter to enable
comparison with alternative fates of the enamine intermediate.
DXP synthase (81.4 μM active centers) in one syringe was
mixed with an equal volume of pyruvate (50 μM) in the second
syringe. The rates of LThDP formation and decarboxylation are
k1 = 1.4 0.05 s−1 and k2 = (7 0.01) × 10−2 s−1 at 6 °C
(Figure 2A) and k1 = 2.8 0.15 s−1 and k2 = (4.9 0.01) ×
10−1 s−1 at 37 °C (Figure 2B, Table 1).
Direct Observation of the Predecarboxylation LThDP
Intermediate on DXP Synthase. We next turned to CD
studies of DXP synthase with the donor substrate pyruvate. On
addition of saturating pyruvate at 4 °C, there developed a
positive CD band at 313 nm (Figure 1, left), gradually replacing
Figure 2. Formation and decarboxylation of LThDP on DXP synthase
in the absence of D-GAP under single turnover conditions (A) at 6 °C
and (B) at 37 °C.
Table 1. Rate Constants for LThDP Formation and
Decarboxylation on DXP Synthase under Presteady State
Conditions
Figure 1. Formation of 1′,4′-iminoLThDP from pyruvate by DXP
synthase. Left: CD titration of DXP synthase with pyruvate (1) 50 μM,
(2) 100 μM, (3) 150 μM, (4) 1 mM. Inset: addition of 250 μM D-GAP
generated DXP. Right; rate of 1′,4′-iminoLThDP formation at 6 °C.
a
reaction with DXP synthase
k1 (s−1
)
k2 (s−1
)
pyruvate, 6 °C
pyruvate (single turnover,
6 °C)
pyruvate (single turnover,
37 °C)
pyruvate + D-GAP, 6 °C
1.1 0.03
1.4 0.05
(7 0.01) × 10−2
(4.9 0.01) × 10−1
(4.2 1.0) × 101
the negative CD band at 320 nm assigned to the AP form of
ThDP (Scheme 1). The CD band at 313 nm revealed apparent
saturation with pyruvate (apparent Kd,pyruvate ∼90 μM, Figure S2
of the Supporting Information) and was tentatively assigned to
the 1′,4′-iminopyrimidinylLThDP. A stopped-flow CD experi-
ment was designed to determine the rate of interconversion of
the ThDP-bound covalent intermediates on DXP synthase. In
2.8 0.15
(6.8 0.01) × 10−1
a
See detailed conditions in Experimental section; see Scheme 1 right
for assignment of rate constants to individual steps.
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Direct Observation of the Rate of LThDP Decarbox-
ylation in the Presence of D-GAP. Next, we wished to
determine the effect of the acceptor substrate D-GAP on the
rate of decarboxylation of LThDP. For these experiments,
commercially available D,L-GAP was used as the source of D-
GAP; L-GAP does not act as substrate for the enzyme (Figure
S6 of the Supporting Information).1,5,6,10 Addition of 250 μM
D-GAP to DXP synthase with preformed LThDP (displayed a
positive CD band at 313 nm in Figure 1, inset) resulted in
formation of a new positive band at 288 nm, replacing the 313-
nm band (Figure 1 left, inset). The CD spectrum of the
supernatant after protein was removed indicated the persistence
of the band at 288 nm, suggesting that it corresponds to the
DXP product. Confirmation of the product was obtained with
an NMR sample, prepared using DXP synthase (22.2 μM active
centers) with pyruvate (10 mM) and D-GAP (15 mM). The 1H
NMR spectrum identified the DXP in the supernatant by
comparison with the spectrum of an “authentic” commercially
available DXP sample and that reported.1
Having observed accumulation of LThDP on the enzyme
(Figure 1, left), we reasoned that direct monitoring of the CD
at 313 nm (corresponding to LThDP) would enable us to
directly measure the rate of decarboxylation upon the addition
of D-GAP. As seen in Figure 1 left inset, the decarboxylation of
LThDP is accelerated in the presence of the acceptor substrate
D-GAP. To obtain quantitative support for this hypothesis,
LThDP was preformed in one syringe by mixing DXP synthase
(68.1 μM active centers) and pyruvate (500 μM) at 6 °C and
then was rapidly mixed with D-GAP (100 μM) placed in the
second syringe on the stopped-flow CD instrument. Time-
dependent decarboxylation of LThDP (CD at 313 nm, Figure
3A) was observed with a rate constant of (3.0 0.9) × 101 s−1
pyruvate (4 mM) and D-GAP (2 mM) in the second syringe at
6 °C, providing a rate constant k4 = 1.2 0.03 s−1 (Figure S4 of
the Supporting Information, bottom). The lower concentration
of DXP synthase assured that the CD observations pertain to
formation of free, rather than enzyme-bound DXP.
Putative Acceptors for Reactions of the Enamine. As
shown in Scheme 2, one could envision alternative competing
trapping of the enamine resulting from LThDP decarbox-
ylation, and the rates of these reactions needed to be
established under the reaction conditions. A CD method was
developed to measure the steady-state kinetic parameters for
DXP synthase providing data consistent with the previous
coupled assay.7 (1) With D-GAP as acceptor, the rate of DXP
formation (4.21 s−1) at 37 °C was calculated from the slope of
the progress curves recorded by CD with time at 290 nm. To
calculate the rate constant, the molar ellipticity of DXP (13 200
deg cm2 dmol−1) was determined (enzyme assays shown in the
Supporting Information). (2) With a proton electrophile the
enamine would be converted to HEThDP followed by release
of acetaldehyde, whose rate of formation with the YADH/
NADH coupled assay was 0.01 s−1. (3) With pyruvate as
acceptor, carboligation of the enamine leads to (R)-acetolactate
(negative CD band at 300 nm) in accord with the findings by
the Johns Hopkins group.10 A CD titration of DXP synthase
with pyruvate (1−90 mM) revealed a negative band centered at
300 nm, and the CD amplitude at 300 nm increased with
increasing pyruvate concentration (Figure S5 of the Supporting
Information). Plotting the CD ellipticity at 300 nm against
[pyruvate] gave S0.5 for pyruvate of 51.0 1.7 mM (Figure S5
of the Supporting Information inset), and a kcat = 0.21 s−1 was
calculated. (4) Finally, the DCPIP reduction assay was carried
out as evidence of decarboxylation, giving a rate constant of
0.15 s−1.
The rates of product formation from these putative acceptors
at 37 °C (Scheme 2) indicate that formation of DXP is the
preferred outcome by a significant factor but that DXP synthase
could indeed also produce significant (R)-acetolactate under
some conditions.
DISCUSSION
■
Perhaps the most significant observation made from steady
state CD was of a “stable” ThDP-bound predecarboxylation
intermediate with tetrahedral C2α substitution at 4 °C on DXP
synthase in the absence of D-GAP; confirmed by NMR to be
LThDP. Stopped-flow CD experiments enabled us to measure
the rates of key steps on the putative reaction pathway of DXP
synthase at 6 °C: the rate constant for formation of LThDP
(1.1 s−1, k1 in Scheme 1), the rate constant of decarboxylation
(4.2 × 101 s−1, k2 in Scheme 1 in the presence of D-GAP and 7
× 10−2 s−1 in its absence), the rate constant for carboligation k3
remains unknown but is almost certainly not rate limiting, and
the rate constant for DXP release (1.2 s−1, k4 in Scheme 1). On
the basis of this information, LThDP formation appears to be
rate limiting. Most notable is the sizable rate acceleration on the
decarboxylation step caused by the acceptor substrate D-GAP
(Scheme 1). We note that in accord with our findings, Eubanks
and Poulter4 first proposed a unique requirement for ternary
complex formation in DXP synthase catalysis on the basis of
CO2 trapping studies that showed significant reduction in rate
of CO2 release in the absence of D-GAP. That study suggested
that the presence of D-GAP is required for CO2 release;
however, until now it was unknown whether binding of D-GAP
facilitated formation of LThDP when both substrates are
Figure 3. (A) Time course of LThDP decarboxylation and resynthesis
by DXP synthase in the presence of D-GAP at 6 °C. (B) Expansion of
early behavior in A fitting data to eq 1. (C) Dependence of the rate of
LThDP decarboxylation on final concentration of D-GAP.
(Figure 3B), a rate constant that increases to (4.2 1) × 101
s−1 for DXP synthase saturated with D-GAP (Figure 3C). An
interpretation of the total behavior at 313 nm (Figure 3A) is
that decarboxylation proceeds until all of the D-GAP is
consumed (drop in the curve; the concentration of D-GAP
reacting is 50 μM after mixing), after which time the remaining
pyruvate forms more LThDP.
To determine the rate of DXP release by CD, DXP synthase
(1.04 μM active centers) in one syringe was mixed with
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Scheme 2. Putative Acceptors for the Enamine Reactions under Steady State Conditions at 37 °C
bound, or promoted decarboxylation of the LThDP inter-
mediate. The CD approach used here permits the interrogation
of these individual steps. Here, we show that the LThDP
intermediate readily forms upon binding of pyruvate to DXP
synthase in the absence of D-GAP, and persists at low
temperature until addition of D-GAP.
hence the rate constant for decarboxylation of enzyme-bound
LThDP must be at least this large, more in the range of the rate
constant here observed with D-GAP present.
Taking into consideration the long history of studies on rates
of decarboxylation using nonenzymatic LThDP models, it
could be inferred that the dielectric constant of the putative
DXP synthase active site of the ternary complex may be low.
Lienhard and co-workers showed that transferring the 2-(1-
carboxy-1-hydroxyethyl)-3,4-dimethyl-thiazolium ion from
water to ethanol led to a 104−105 fold increase in the
decarboxylation rate.17 Kluger et al. reported a rate constant of
4 × 10−5 s−1 for decarboxylation of a C2α-lactylthiamin18 at pH
7.0 and 25 °C. More recently, a study by Zhang et al.
demonstrated “spontaneous” decarboxylation of C2α-lactylth-
iazolium salts with rate constants exceeding 50 s−1 in THF,
simply by decreasing the dielectric constant of the solvent.19
Evidence supporting the idea of an apolar active site of yeast
pyruvate decarboxylase (YPDC) was reported by Jordan et al.20
in a study utilizing thiochrome pyrophosphate, a fluorescent
ThDP analog whose emission spectra correlates to solvent
polarity. The effective active site dielectric constant for YPDC
was interpolated to that between 1-hexanol and 1-pentnol (i.e.,
between 11 and 13), despite the existence of a Glu, an Asp, and
two His residues (in addition to the conserved glutamate) at
the active center.
In view of these nonenzymatic models for LThDP
decarboxylation rates reported in the previous paragraph,
apparently, DXP synthase accelerates the rate of decarbox-
ylation [k2 = (7 0.01) × 10−2 s−1 at 6 °C] compared to the
value reported in aqueous buffer (4 × 10−5 s−1). Yet, the
lifetime of LThDP on DXP synthase in the absence of D-GAP is
still surprisingly long in comparison with other enzymes
carrying out the same function (see above). The 600-fold rate
acceleration on addition of D-GAP on DXP synthase translates
to an energy barrier lowering of 3.5 kcal/mol, achieving a rate
constant very similar to that achieved in some nonenzymatic
model systems.
The collective reports on DXP synthase mechanism are
conflicting. Eubanks and Poulter4 provided the earliest
compelling evidence for the requirement of a ternary complex
and proposed an ordered mechanism with pyruvate binding
first. More recently, reports by Matsue et al.6 and Brammer et
al.7 have proposed ping-pong or random sequential mecha-
nisms, respectively. On the basis of the evidence here that the
LThDP intermediate persists at low temperature until addition
of D-GAP, a classical ping-pong mechanism seems unlikely. The
present results also exclude an ordered mechanism requiring
GAP binding in the first step, since LThDP formation can
clearly take place upon binding of pyruvate to free enzyme. The
ordered and random sequential mechanisms previously
proposed4,10 both support the idea that ternary complex
formation is uniquely required in DXP synthase catalysis, in
accordance with our findings here. A random substrate binding
model cannot be directly tested here, as the presence of GAP
facilitates rapid decarboxylation and precludes build-up of
LThDP. While these results could support an ordered
mechanism where pyruvate binds first, this CD analysis does
not exclude the possibility that pyruvate can bind to an E-D-
GAP complex and undergo conversion to LThDP in the
presence of GAP, prior to GAP-promoted decarboxylation.
The 600-fold increase in rate of decarboxylation by DXP
synthase as a result of D-GAP binding appears to be unique and
supports the idea that ternary complex formation is required.
The results should be evaluated in view of relevant studies on
the decarboxylation of enzyme-bound LThDP. Tittmann and
Wille summarized data on the ability to observe LThDP on a
variety of ThDP enzymes15 and concluded the “LThDP to be
extremely short lived and marginally accumulated at steady
state, rendering a structural characterization of LThDP under
steady state turnover or single turnover conditions almost
impossible;” although the NMR method did detect some
LThDP in certain cases, such as in the F479W pyruvate oxidase
variant.26 These observations were also summarized in a review
by Kluger and Tittmann.16 In the experience of the Rutgers
group, turnover numbers for ThDP-dependent pyruvate
decarboxylating enzymes are in the range of (5−6) × 101 s−1,
The slow rate of decarboxylation of LThDP on the enzyme
in the absence of D-GAP requires explanation.
A likely explanation to consider for the effect of D-GAP is
that decarboxylation is reversible21−23 and CO2 release from
the enzyme is potentiated by D-GAP binding. Several reports by
Kluger et al. have focused on model studies of decarboxylation
that have led to the proposal that decarboxylation is reversible
on the ThDP enzyme benzoylformate decarboxylase.16,21 For
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Journal of the American Chemical Society
Article
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benzoylformate decarboxylase and A28S benzaldehyde lyase, it
was suggested that serine residue from the active site may act as
nucleophile in CO2 trapping.24 The fact that we only observed
LThDP both on the enzyme and that once it is released from
the enzyme suggests that if the LThDP is indeed at equilibrium
with the enamine + CO2, the acid quench should produce
HEThDP. Given that HEThDP is not observed does not argue
against this scenario, but suggests that the equilibrium lies far to
the LThDP side.
A different explanation for the effect of D-GAP on the
decarboxylation rate is based upon our understanding of the
decarboxylation mechanism provided by Lienhard’s work25 and
the need for a zwitterion charge distribution (positively charged
thiazolium ring with carboxylate ionization state of the lactyl
group) for optimal decarboxylation rate. Thus, it is possible that
protonation of the carboxylate group by DXP synthase active
site residues could reduce the rate of decarboxylation.
Understanding the origins of the slow decarboxylation rate in
the absence of D-GAP and its acceleration in its presence could
create a new opportunity for the design of selective inhibitors
against this important enzyme.
Anselmetti, D.; Imperial, S.; Fernan
24, 4203−4217.
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CONCLUSION
■
In summary, a combination of steady state and time-resolved
(presteady state) CD spectroscopy were used to study the
individual rate constants in the DXP synthase reaction (Table 1
and Scheme 1). The results clearly demonstrate that formation
of LThDP is the rate-limiting step. Apparently, the acceptor
substrate D-GAP accelerates decarboxylation of LThDP
significantly, while in the absence of D-GAP, decarboxylation
is very slow. It is also clear that, among the putative acceptors
for the reaction with the enamine, D-GAP is the preferred
acceptor.
G.; Hubner, G. Biochemistry 2003, 42, 7885−7891.
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(15) Tittmann, K.; Wille, G. J. Mol. Catal. B 2009, 61, 93−99.
(16) Kluger, R.; Tittmann, K. Chem. Rev. 2008, 108, 1797−1833.
(17) Crosby, J.; Lienhard, G. E. J. Am. Chem. Soc. 1970, 92, 5707−
5716.
(18) Kluger, R.; Chin, J.; Smyth, T. J. Am. Chem. Soc. 1981, 103,
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Sherwood Lollar, B.; Kluger, R. J. Am. Chem. Soc. 2009, 131, 11638−
11639.
ASSOCIATED CONTENT
■
S
* Supporting Information
(22) Haussermann, A.; Rominger, F.; Straub, B. F. Chem.−Eur. J.
̈
Experimental conditions for DXP synthase enzyme assays and
pH dependence of AP form of ThDP on DXP synthase and
DXP formation. Figures representing pH dependence,
Michaelis−Menten plot to determine the Km for D-GAP,
HPLC analysis demonstrating utilization of D-GAP only from a
racemic mixture, and NMR spectra of DXP product. This
material is available free of charge via the Internet at http://
pubs.acs.org.
2012, DOI: 10.1002/chem..201202298.
(23) Gonzalez-James, O. M.; Singleton, D. A. J. Am. Chem. Soc. 2010,
132, 6896−6897.
(24) Brandt, G. S.; Kneen, M. M.; Petsko, G. A.; Ringe, D.; McLeish,
M. J. J. Am. Chem. Soc. 2010, 132, 438−439.
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2891−2900.
(26) Wille, G.; Meyer, D.; Steinmetz, A.; Hinze, E.; Golbik, R.;
Tittmann, K. Nat. Chem. Biol. 2006, 2, 324−328.
AUTHOR INFORMATION
■
Corresponding Author
cmeyers@jhmi.edu; frjordan@rutgers.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Supported at JHU by NIH-GM084998 and at Rutgers by NIH-
GM-050380. We gratefully acknowledge Jessica Smith and
Katie Heflin for assistance with the purification and activity
determination of wild-type DXP synthase used in this study.
REFERENCES
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