From the Institute of Biochemistry, Department of
Biochemistry and Biotechnology, Martin-Luther-University
Halle-Wittenberg, 06120 Halle/Saale, Kurt-Mothes-Stra
e 3, Germany
and the ¶ Division of Molecular Structural Biology, Department of
Medical Biochemistry and Biophysics, Karolinska Institute,
Tomtebodavägen 6, 171 77 Stockholm, Sweden
Received for publication, August 30, 2000, and in revised form, January 31, 2001
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ABSTRACT |
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The cleavage of the donor substrate
D-xylulose 5-phosphate by wild-type and H263A mutant
yeast transketolase was studied using enzyme kinetics and circular
dichroism spectroscopy. The enzymes are able to catalyze the cleavage
of donor substrates, the first half-reaction, even in the absence of
any acceptor substrate yielding D-glyceraldehyde
3-phosphate as measured in the coupled optical test according to
Kochetov (Kochetov, G. A. (1982) Methods Enzymol. 90, 209-223) and compared with the H263A variant. Overall, the H263A
mutant enzyme is less active than the wild-type. However, an increase
in the rate constant of the release of the enzyme-bound glycolyl moiety
was observed and related to a stabilization of the "active
glycolaldehyde" ( The pentose phosphate pathway is a major metabolic pathway in all
cells. It consists of a dehydrogenase-decarboxylating system that
converts D-glucose 6-phosphate to D-ribulose
5-phosphate, generating NADPH+H+ for use in reductive
biosynthesis, an isomerizing system that interconverts
D-ribulose 5-phosphate to D-xylulose
5-phosphate and D-ribose 5-phosphate, and a sugar
rearrangement system that converts D-ribose 5-phosphate and
D-xylulose 5-phosphate to the glycolytic intermediates
D-fructose 6-phosphate and D-glyceraldehyde 3-phosphate. Transketolase (EC 2.2.1.1) plays an important part in the
rearrangement system, since it creates, together with transaldolase, a
reversible link between the pentose phosphate pathway and glycolysis.
The catalytic activity of transketolase is dependent on thiamin
diphosphate (ThDP)1 and
divalent cations such as Mg2+ or Ca2+. The
enzyme from Saccharomyces cerevisiae is composed of two identical subunits with a molecular mass of 74 kDa per monomer (1). The
thiamin diphosphate molecule binds in a cleft at the interface between
the two subunits and is completely inaccessible from the outer
solution, except for the C2 atom of the thiazolium ring to which the
donor substrate binds covalently (2). Transketolase catalyzes the
cleavage of a carbon-carbon bond adjacent to a carbonyl group in
ketosugars and transfers a two-carbon unit to aldosugars. The reaction
cycle of transketolase can be separated into two parts. The first half
of the reaction consists of the cleavage of the donor substrate and
formation of the first product, an aldose, and a covalently bound
intermediate, the The first crystal structure of a ThDP-dependent enzyme to
be solved was that of holotransketolase from S. cerevisiae
(2). This structure has been refined to 2.0-Å resolution (5). Beside transketolase, the crystal structures of other
ThDP-dependent enzymes, such as pyruvate oxidase from
Lactobacillus plantarum (6, 7), pyruvate decarboxylase from
brewers' yeast (8, 9), pyruvate decarboxylase from Zymomonas
mobilis (10), pyruvamide-activated pyruvate decarboxylase from
S. cerevisiae (11), benzoylformate decarboxylase from
Pseudomonas putida (12), and pyruvate ferredoxin oxidoreductase from Desulfovibrio africanus (13), have been solved and revealed the ThDP molecule to be bound in the
V-conformation. The specificity of the coenzyme binding was extended to
complexes of transketolase with ThDP analogues (14).
According to Scheme 1 several proton
transfer steps occur in the catalytic cycle, which are likely mediated
by the 4'-amino group of the pyrimidine moiety of the enzyme-bound ThDP
as the major source of charge stabilization (2, 15, 16). On the basis
of the x-ray structure of transketolase, catalytically important residues in the reaction cycle have been suggested (2, 4, 5) and probed
by site-directed mutagenesis (4, 15, 17-19). For instance, four
histidine residues, located in the active site of transketolase from
S. cerevisiae, have been replaced by alanine using
site-directed mutagenesis and the enzymatic activity of the variants
investigated (18). The overall crystal structure of the H263A variant
is very similar to the wild-type holotransketolase structure, the amino
acid replacement did not introduce significant structural changes in
the mutant enzyme or in the apparent Km values for
ThDP, the donor substrate D-xylulose 5-phosphate, and the
acceptor substrate D-ribose 5-phosphate, respectively. The mutation is most deleterious in terms of kcat
(0.23 s-carbanion) by histidine 263. Chemically synthesized DL-(
,
-dihydroxyethyl)thiamin diphosphate
is bound to wild-type transketolase with an apparent
KD of 4.3 ± 0.8 µM (racemate)
calculated from titration experiments using circular dichroism
spectroscopy. Both enantiomers are cleaved by the enzyme at different
rates. In contrast to the enzyme-generated
-carbanion of
(
,
-dihydroxyethyl)thiamin diphosphate formed by decarboxylation
of hydroxylactylthiamin diphosphate after incubation of transketolase
with
-hydroxypyruvate, the synthesized
DL-(
,
-dihydroxyethyl)thiamin diphosphate did not work
as donor substrate when erythrose 4-phosphate is used as acceptor
substrate in the coupled enzymatic test according to Sprenger
(Sprenger, G. A., Schörken, U., Sprenger, G., and Sahm, H. (1995) Eur. J. Biochem. 230, 525-532).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-carbanion of
,
-dihydroxyethyl-ThDP
(DHEThDP). In the second half of the reaction the
-carbanion reacts
with the acceptor (aldose) substrate in a nucleophilic manner. The
two-carbon unit from the first reaction is transferred to the acceptor
substrate, and a ketose with its carbon skeleton extended by two carbon
atoms is formed. The enzyme has a high specificity for donor ketoses
with D-threo (C3-L,
C4-D) configuration and for
-hydroxylated acceptor
aldoses with C2-D configuration (3, 4).
1, wild-type 46.3 s
1; Ref. 18). The side chain of
His263 forms a hydrogen bond to a phosphate oxygen
of ThDP and thus, the second nitrogen atom of the imidazole ring, which
is not protonated at the pH optimum of the enzyme (pH 7.6), points
straight toward the C3 hydroxyl group of the donor substrate. In this
way, it is perfectly positioned to abstract a proton from the donor
substrate (Scheme 1) (20).
View larger version (24K):
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Scheme 1.
To shift the equilibrium established by transketolase,
-hydroxypyruvic acid (HPA) is used as the ketol donor (3). This strategy couples the formation of the glycolyl-ThDP complex
(
-carbanion of DHEThDP) with the decarboxylation of HPA and renders
the complete reaction practically irreversible. Scheme
2 shows the catalytic cycle of the
transketolase-mediated condensation of HPA and
-hydroxyaldehyde. Because of its strong stereospecificity, transketolase offers an
enormous synthetic potential (21-23).
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In this work we studied the cleavage of the donor substrate
D-xylulose 5-phosphate in the absence of any acceptor
substrate by wild-type and H263A mutant holotransketolase from S. cerevisiae by kinetics and circular dichroism spectroscopy.
Furthermore, we have chemically synthesized racemic DHEThDP
(DL-DHEThDP) and investigated its complex formation with
wild-type apotransketolase and the cleavage of the respective complexes
in the presence and absence of the acceptor erythrose 4-phosphate by
kinetics. These results are compared with the formation of enzyme-bound
-carbanion of DHEThDP (glycolyl-ThDP, "active glycolaldehyde";
Ref. 24) according to Scheme 2 using HPA as substrate donor and the
cleavage in the presence and absence of acceptor substrates.
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MATERIALS AND METHODS |
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Chemicals and Enzymes-- Thiamin diphosphate and formaldehyde were purchased from Merck Eurolab GmbH (Darmstadt, Germany) and D-ribose 5-phosphate, D-xylulose 5-phosphate, D-erythrose 4-phosphate, D-glyceraldehyde 3-phosphate from Sigma Aldrich Chemie GmbH (Deisenhofen, Germany). The auxiliary enzymes glycerol-3-phosphate dehydrogenase, triose phosphate isomerase, phosphoglucose isomerase, and glucose-6-phosphate dehydrogenase were obtained from Roche Diagnostics GmbH (Mannheim, Germany). All other chemicals used were commercially available.
Protein Expression and Purification-- H402 (tkl1::HIS3 derivative of W303-1A) yeast cells carrying wild-type plasmid pTKL1 (1) or the H263A mutant (18) were cultured in a leucine-deficient medium, containing galactose as a carbon source to obtain a high copy number of the plasmid (25). Cells were collected by centrifugation and disrupted in a beat beater. The protein purification was performed according to the established protocol of Wikner et al. (15) with some modifications. Sephacryl S-200 HR was used instead of Sephacryl S-300 HR and Source 15Q instead of Mono Q (Amersham Pharmacia Biotech, Europe GmbH). About 90 mg of pure transketolase having a specific catalytic activity of 33-40 units/mg for the wild-type and 0.2 unit/mg for the H263A variant could be obtained from 5 liters of cell culture.
The protein concentration of pure transketolase was determined
spectrophotometrically at 280 nm using a molar absorption coefficient of 157,600 M1·cm
1
according to the method of Gill and von Hippel (26).
Activity Measurements--
The specific activities of wild-type
transketolase and of the H263A variant were measured
spectrophotometrically using two different assays. In the assay
according to Kochetov (27) the transketolase reaction becomes
practically irreversible, and the cleavage of the donor substrate is
detected. One unit is defined as the formation of 1 µmol of
D-glyceraldehyde 3-phosphate (corresponding to 1 µmol of
glycerol 3-phosphate)/min. The assay mixture contained 1 mM
D-xylulose 5-phosphate, 1.8 mM
D-ribose 5-phosphate, 0.3 mM
NADH+H+, 0.1 mM ThDP, 2.5 mM
CaCl2, 5 units of triose phosphate isomerase, 5 units of
glycerol-3-phosphate dehydrogenase, and 1-100 milliunits of
transketolase in 50 mM glycyl glycine (pH 7.6). The
temperature was set to 25 °C. To test the catalytic activity of
transketolase toward -hydroxypyruvate and chemically synthesized
racemic DHEThDP (DL-DHEThDP), the assay according to
Sprenger et al. (28) was used. In this assay, 1 unit is
defined as the formation of 1 µmol of D-fructose
6-phosphate (corresponding to 1 µmol of D-glucono 5-lactone)/min. The assay mixture contained 0.6 mM
D-erythrose 4-phosphate, 4 mM
-hydroxypyruvate, 0.37 mM NAD+, 0.1 mM ThDP, 2.5 mM CaCl2, 4 units of
each phosphoglucose isomerase and glucose-6-phosphate dehydrogenase.
The temperature was set to 25 °C. If DL-DHEThDP (1 mM) was used as donor of the glycolyl moiety, the cofactor
ThDP and
-hydroxypyruvate had been omitted in the assay mixture. The
commercially available D-erythrose 4-phosphate contained
some D-glucose 6-phosphate. Therefore, the side reaction of
the D-erythrose 4-phosphate with the auxiliary enzymes was run until the NADH+H+ formation finished. The entire
enzymatic reaction was started by the addition of transketolase. In
both assay systems either the oxidation of NADH+H+
or the reduction of NAD+ was followed by the absorbance
change at a wavelength of 340 nm on a Kontron Uvicon-941 absorption spectrophotometer.
The entire cleavage of the donor substrate without the acceptor was followed by the absorbance change at 340 nm and 25 °C in a stopped-flow machine (SX.18MV, Applied Photophysics, Leatherhead, Great Britain) working in the single mixing mode. The two syringes had the same volume, resulting in a 1:2 dilution (1+1 mixing) of the solutions. One syringe contained the reaction mixture mentioned for the assay according to Kochetov (27), but without the acceptor substrate D-ribose 5-phosphate. The concentration of D-xylulose 5-phosphate was 2 mM. The other syringe was filled with the same reaction mixture containing different amounts of transketolase and no substrate. Furthermore, the activity of the auxiliary enzymes was increased to 1550 units/ml by desalting on HiTrap G25 columns (5 × 5 ml) and subsequent concentration using Palfiltron concentration tubes (cutoff: 5000 Da). The programs SigmaPlot® (Jandel Scientific Software) and Dynafit (29) were used for nonlinear regression analyses of the kinetics.
Direct Measurement of Donor Substrate Binding to Transketolase-- According to Kochetov et al. (30) the binding of the donor substrate D-fructose 6-phosphate was directly followed by the absorbance change at 300 nm and 25 °C in a stopped-flow machine with the same arrangement mentioned above. One syringe contained the wild-type enzyme (12 µM), 100 µM ThDP, 2.5 mM CaCl2 in 50 mM glycyl glycine (pH 7.6). The other syringe contained the donor substrate D-fructose 6-phosphate at various concentrations (0.05-1 mM) and 100 µM ThDP, 2.5 mM CaCl2 in 50 mM glycyl glycine (pH 7.6). The kinetics were analyzed using the program Dynafit (29).
Direct Measurement of the Transketolase Catalyzed Reaction of
-Hydroxypyruvate and D-Glyceraldehyde 3-Phosphate with
Transketolase--
The binding of the donor substrate HPA was directly
followed by the absorbance change at 300 nm and 25 °C in a
stopped-flow machine but with the double mixing mode. One syringe
contained the wild-type enzyme (8.25 µM), 30 µM ThDP, 2.5 mM CaCl2 in 50 mM glycyl glycine (pH 7.6). The other syringe contained the
donor substrate HPA (8.25 µM) and 30 µM
ThDP, 2.5 mM CaCl2 in 50 mM glycyl
glycine (pH 7.6). After a reaction time of 8 s,
D-glyceraldehyde 3-phosphate (8.25 µM) was added.
Circular Dichroism Measurements-- Near-UV circular dichroism spectra of transketolase were recorded on a Jasco J710 circular dichroism spectrophotometer using cuvettes with an optical path length of 1 cm at a temperature of 25 °C. All samples were measured in 50 mM glycyl glycine (pH 7.6) containing 2.5 mM CaCl2.
To determine the KD value of chemically synthesized racemic DHEThDP (DL-DHEThDP) to apotransketolase, the titration was followed by the change in ellipticity at an analytical wavelength of 320 nm until binding kinetics were finished (about 3 min).
H/D-exchange Measurements on Wild-type and H263A Mutant Enzyme-- The kinetics of the H/D-exchange of the C2-H of ThDP in the transketolase proteins were measured by rapid quenched flow and 1H NMR as described previously (16).
Chemical Synthesis of DL-DHEThDP-- DL-DHEThDP was synthesized according to Krampitz and Votaw (31). To separate DL-DHEThDP from nonconverted ThDP, the reaction mixture was supplied to an anion exchange chromatography on QAE-Sephadex A25 (Amersham Pharmacia Biochemicals, Uppsala, Sweden). The components were eluted by an acetic acid gradient (0-50 mM). DL-DHEThDP eluted in front of ThDP. Afterward, the samples were lyophilized and analyzed by mass spectroscopy (VG BIO Q Tripel-Quadrupol mass spectrometer) and 1H NMR (Bruker ARX Avance 500). Synthesis and chromatography yielded about 40-50 mg of pure DL-DHEThDP. The stability of chemically synthesized DL-DHEThDP was followed by 1H NMR in 100 mM sodium phosphate (pH 7.6) at 10 °C. After 2 weeks only 7% DL-DHEThDP converted to ThDP under these conditions.
Preparation of "Acceptor-free" D-Xylulose
5-Phosphate--
The progress curve of transketolase action using
commercially available D-xylulose 5-phosphate in the
absence of an acceptor substrate shows a background reaction of
D-glyceraldehyde 3-phosphate formation with 6% of that
rate measured in the presence of the acceptor substrate in performing
the test according to Kochetov (27). Furthermore, an initial burst
phase could be detected, the amplitude of which was dependent on the
initial concentration of D-xylulose 5-phosphate. The jump
could be repeated by additional D-xylulose 5-phosphate, but
not by additional transketolase and must, therefore, be caused by
acceptor compound(s) present in the commercially available donor
substrate (Fig. 1). To remove these
impurities in all measurements, the D-xylulose 5-phosphate was first preincubated with 20 µg/ml wild-type or 50 µg/ml H263A mutant holotransketolase for a time period of 5 or 20 min. This procedure yielded acceptor-free donor substrate that was used in
the kinetic experiments.
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Determination of DL-DHEThDP Turnover by Transketolase-- Transketolase (75 µM) was incubated with an equimolar concentration of racemic DHEThDP in 0.02 M sodium phosphate (pH 7.6) at 25 °C. 300-µl samples were quenched with 200 µl of 12.5% (w/v) trichloroacetic acid, 1 M DCl. After separation of the denatured protein by centrifugation the supernatant containing DHEThDP and ThDP was analyzed by 1H NMR spectroscopy. The formation of ThDP due to the enzymatic cleavage of DHEThDP by transketolase was monitored by the decrease of the relative integral of the C6'-H signal (singlet) of DHEThDP at 7.30 ppm and the increase of the relative integral of the C6'-H signal (singlet) of ThDP at 8.01 ppm.
Chromatographic Determination of
DL-DHEThDP Turnover by
Transketolase--
The apoenzyme of wild-type transketolase was
incubated with equimolar amounts of chemically synthesized
DL-DHEThDP in 50 mM glycyl glycine (pH 7.6),
containing 2.5 mM CaCl2 for 10 and 30 min at
25 °C. The reaction was stopped by the addition of 10 volumes of
boiling methanol. The precipitated protein was removed by
centrifugation at 15,000 × g for 10 min at 4 °C.
The supernatant was evaporated to dryness and afterward dissolved in
water. The pH of the solution was adjusted to 6.7 and supplied to an
anion exchange chromatography on QAE-Sephadex A25 (Amersham Pharmacia
Biochemicals, Uppsala, Sweden) at 6-8 °C. The fractions containing
either DHEThDP or ThDP were lyophilized and analyzed by mass
spectroscopy (VG BIO Q Tripel-Quadrupol mass spectrometer) and
1H NMR spectroscopy (Bruker ARX Avance 500).
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RESULTS |
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Cleavage of the Donor Substrate D-Xylulose 5-Phosphate
in the Absence of an Acceptor Substrate by Wild-type and H263A Mutant
Transketolase--
The cleavage of the acceptor-free donor substrate
by wild-type transketolase was measured according to Kochetov (27)
using a stopped-flow machine. The progress curves are shown in Fig. 2A. The initial burst phase
observed corresponds to a single turnover of the donor substrate per
active site and is directly proportional to the enzyme concentration
used (Fig. 2B). A turnover number of 0.65 per active site
has been measured at a specific transketolase activity of 26 units/mg.
An extrapolated turnover number of 1 resulted for an enzyme having a
specific activity of 40 units/mg. The following slower steady state
phase is indicative for the cleavage of the enzyme-bound glycolyl
moiety (-carbanion/enamine state of enzyme-bound DHEThDP) to ThDP
and glycolaldehyde and/or a replacement of the DHEThDP by ThDP and its
further reaction with D-xylulose 5-phosphate. The product
D-glyceraldehyde 3-phosphate is converted by the auxiliary
enzymes in the test system according to Kochetov (27), making
the reaction practically irreversible. As a control, the rate of the
complete transketolase reaction could be regained after addition of the
acceptor substrate D-ribose 5-phosphate in the course of
the steady state phase. On the other hand, glycolaldehyde, occurring
from the cleavage of the enzyme-bound
-carbanion of DHEThDP may also
react as an acceptor substrate in a side reaction. The product of this
(futile) cycle reaction is D-erythrulose, which is not
detected by the assay system used and not considered in this context
because of the small concentration of glycolaldehyde and its high
Km value.
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Residue His263 in transketolase plays an important role in
the donor substrate binding (18). The ability of the H263A mutant transketolase to cleave the acceptor-free donor substrate
D-xylulose 5-phosphate was studied in the same approach as
described for the wild-type enzyme; the progress curves are presented
in Fig. 2C. The experimental data were fitted according to
the reaction sequence in Scheme 3; the
resulting rate constants are summarized in Table
I and illustrated in Fig. 2, A
and C.
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It was also possible to follow the binding of the donor substrate directly by absorbance, but because of the impure D-xylulose 5-phosphate, rate constants were only calculated for the binding of D-fructose 6-phosphate to the wild-type of holotransketolase (Table I).
H/D-exchange Measurements on Wild-type and H263A Mutant
Enzyme--
The rate constant of the H/D-exchange of the C2-H atom of
the enzyme-bound cofactor in H263A mutant transketolase was estimated to 50 ± 5 s1 and seems, therefore, to
be unaltered with respect to the wild-type enzyme (61 ± 5 s
1; Ref. 16) within the experimental error.
Near-UV Circular Dichroism Spectroscopy-- The binding of the cofactor ThDP to apotransketolase, as well as the binding of the artificial donor substrate HPA and the acceptor substrate glycolaldehyde to holotransketolase, can be monitored by near-UV circular dichroism (32, 33). This approach has already been used for the spectroscopic characterization of the H263A mutant enzyme (18).
On incubation of wild-type holotransketolase with only the donor
substrates D-xylulose 5-phosphate or D-fructose
6-phosphate, a near-UV circular dichroism spectrum was measured showing
a positive ellipticity between 300 and 310 nm (Fig.
3). Nearly the same spectrum could be
obtained on incubation of holotransketolase with the artificial donor
substrate HPA and the acceptor substrate D-glyceraldehyde 3-phosphate (Fig. 4). This is in
agreement with the results in kinetics showing a cleavage of the donor
substrate in the absence of the acceptor substrate. Furthermore, the
products occurring from the first part of the transketolase reaction
(cleavage) are able to be reaction partners in the second part
(condensation) in a futile cycle or equilibrium. After the reaction of
holotransketolase with HPA, the decarboxylation of the enzyme-bound
hydroxylactyl-ThDP yields enzyme-bound -carbanion of DHEThDP, which
forms D-xylulose 5-phosphate as a result of the
condensation reaction with D-glyceraldehyde 3-phosphate.
However, in the absence of the auxiliary enzymes used in the assays
mentioned above for the kinetics, D-xylulose 5-phosphate is
able to initiate the next reaction cycle (futile cycle).
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Reconstitution and Enzymatic Activity of Wild-type Apotransketolase
with DL-DHEThDP--
Chemically synthesized
racemic DHEThDP (DL-DHEThDP) shows no circular dichroism
spectrum in the wavelength range between 240 and 380 nm. On incubation
of DL-DHEThDP with wild-type apotransketolase, a near-UV
circular dichroism spectrum showing a negative ellipticity with an
extremum at 320 nm was measured, which is identical to that of
holotransketolase. The ellipticity was used as an analytical tool in a
titration experiment of DL-DHEThDP to apotransketolase (Fig. 5), yielding a
KD value of 4.3 ± 0.8 µM for the racemate (for comparison, KD value of ThDP was
determined to 0.6 µM). The near-UV circular dichroism
spectra, however, do not prove the cleavage of the synthesized
DL-DHEThDP by transketolase, because they are identical to
those of ThDP reconstituted enzyme.
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Therefore, the determination of DHEThDP and ThDP from incubation
mixtures of wild-type apotransketolase and DL-DHEThDP was performed by NMR via the changes of the C6'-H signal (singlet) of
DHEThDP at 7.30 ppm, and of ThDP at 8.01 ppm, respectively, as
described under "Materials and Methods." As illustrated in Fig.
6, both enantiomers were accepted by the
enzyme, but converted with different rates (k = 0.0012 s1 and k' = 0.0002 s
1). The preparative separation of the
intermediate DHEThDP and the cofactor ThDP after 10- and 30-min
incubation is shown in the insets of Fig. 6. A circular
dichroism signal of the DHEThDP fraction could be detected in the
wavelength range between 260 and 280 nm (not shown) and the degree of
the signal, which depends on the incubation time, confirms the
different reaction rates calculated from the NMR measurements.
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The activity of the wild-type apotransketolase reconstituted on incubation with DL-DHEThDP was measured either by the complete assay according to Kochetov (27) or the modified assay according to Sprenger et al. (28). The data are summarized in Table II and are related to the activity of wild-type holotransketolase. In the presence of the natural substrates D-xylulose-5 phosphate and D-ribose-5 phosphate, the enzyme reconstituted with DL-DHEThDP established full catalytic activity as found for holotransketolase. On the other hand, no catalytic activity could be detected for the wild-type apotransketolase-DHEThDP complex using the acceptor substrate D-erythrose 4-phosphate, which is one of the best acceptor substrates known for transketolase having a Km value of about 41 µM. The concentration of DL-DHEThDP in these experiments was between 0.5 and 1 mM and far above the KD value determined.
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DISCUSSION |
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Cleavage of Donor Substrates by Wild-type and H263A Mutant
Transketolase--
According to Scheme 1 the kinetic mechanism of
action of transketolase comprising two reaction sequences follows a
ping-pong mechanism. A donor substrate (naturally a ketosugar) is
cleaved, and a glycolyl moiety covalently bound to ThDP in the
holoenzyme yields enzyme-bound -carbanion of DHEThDP (24). The
two-carbon unit is transferred to an acceptor substrate (naturally an
aldosugar) in the second reaction under reformation of the holoenzyme.
It was unknown whether transketolase is able to cleave a donor
substrate in the absence of the acceptor substrates as well. We could
prove that the enzyme is indeed able to cleave the donor substrate
D-xylulose 5-phosphate in a single turnover reaction. The
release of the product D-glyceraldehyde 3-phosphate could
be monitored by a coupled kinetic test system using triose phosphate
isomerase and glycerol-3-phosphate dehydrogenase as auxiliary enzymes.
The cleavage of D-xylulose 5-phosphate can be divided into
two phases, a burst phase, the amplitude of which corresponds to a
single turnover, and a slower second phase proceeding continuously. The latter phase could be related to the production of
D-glyceraldehyde 3-phosphate from D-xylulose
5-phosphate. This continuous production of D-glyceraldehyde
3-phosphate could result either from a replacement of the
enzyme-bound -carbanion of DHEThDP by ThDP present in excess in the
incubation mixture or from a cleavage of enzyme-bound
-carbanion of
DHEThDP to enzyme-bound ThDP and glycolaldehyde. Both of these
reactions would result in the formation of holotransketolase being
available for a next cycle with donor substrate. However, the reaction
of chemically synthesized DL-DHEThDP with the apoenzyme of
transketolase, which shows a cleavage of this intermediate (Fig. 6) at
a low rate, should result from the cleavage of enzyme-bound
-carbanion of DHEThDP.
The cleavage of the donor substrate D-xylulose 5-phosphate
was measured both with wild-type and H263A mutant transketolase. Investigation of the progress curves at various donor substrate concentrations yielded at least four kinetic constants, which are
related to the catalytic steps illustrated in Scheme 3. A comparison of
the corresponding rate constants between the two proteins offers
differences in the catalytic action studied. It was impossible to
calculate precisely equilibrium constants (K in Table I,
reflecting the binding and dissociation of the donor substrate
according to Scheme 3) from the progress curves, but the nearly
identical H/D-exchange rate constants show that the lower overall
catalytic activity of the H263A variant cannot be due to the
decelerated deprotonation reaction of the enzyme-bound ThDP and
precludes a possible involvement of His263 in the acid-base
catalysis of the initial proton abstraction at the C2 atom.
Consequently, the His263 is either directly involved in
subsequent elementary steps of the catalytic cycle or in the
stabilization of reaction intermediates. The wild-type enzyme, on one
hand, displays a higher rate constant of the donor substrate cleavage
(k3 in Table I) than the H263A variant. On the
other hand, the cleavage of the glycolyl moiety (k4) in the H263A mutant enzyme is surprisingly
faster than in the wild-type transketolase. These observations indicate
that His263 is involved in the reaction leading to the
cleavage of the donor substrate, probably by acting as a catalytic base
abstracting a proton from the C3-hydroxyl group of the donor substrate
(18) and by stabilizing the formed -carbanion intermediate through its positive charge. The loss of charge stabilization in the H263A variant may then explain why the continuous production of
D-glyceraldehyde 3-phosphate is much faster in this mutant
enzyme than in the wild-type. Similar rate constants
k3 and k4 were obtained
for the wild-type reaction by substituting the directly calculated rate
constants for the primary binding (k1 and
k2) with the separately determined values from
the measurement of the direct binding of the donor substrate
D-fructose 6-phosphate (Table I).
Near-UV Circular Dichroism Spectroscopy--
The negative extremum
at 320 nm appearing in the near-UV circular dichroism spectrum of
holotransketolase disappeared after addition of the donor substrates
D-xylulose 5-phosphate and D-fructose 6-phosphate and a new positive band with an extremum at about 300 nm
appears in both cases (Fig. 3). This is indicative for the same
asymmetry (state) of the reactants in the futile cycle reaction with
the product(s) originating from the first half of the reaction being
the reactant(s) of the condensation reaction (second half-reaction).
After incubation of holotransketolase with HPA, an inversion of the
circular dichroism spectrum occurs (Fig. 4), which is very likely
related to enzyme-bound hydroxylactyl-ThDP or the -carbanion of
DHEThDP. Simultaneous incubation of holotransketolase with HPA as donor
and D-glyceraldehyde 3-phosphate as acceptor substrate
results in the production of D-xylulose 5-phosphate, which
is itself a substrate of the first half-reaction as described above.
The circular dichroism spectrum of holotransketolase after incubation
with HPA and D-glyceraldehyde 3-phosphate in equimolar amounts showed the same spectrum as measured for D-xylulose
5-phosphate (Figs. 3 and 4). The corresponding progress curve and the
calculated rate constant of this reaction (3.5 s
1) are illustrated in the inset
of Fig. 4.
Interaction of Wild-type Apotransketolase with
DL-DHEThDP--
Chemically synthesized
DL-DHEThDP was incubated with the apoenzyme of
transketolase, revealing a KD value of 4 µM (racemate) similar to that of ThDP (0.6 µM). Both enantiomers of the analogue are cleaved by
transketolase at rather low rates as shown by 1H NMR
spectroscopy (Fig. 6). The enzyme incubated with DL-DHEThDP was unable to transfer the glycolyl moiety to the acceptor substrate D-erythrose 4-phosphate. This was in contrast to the
control experiment performed with HPA as glycolyl donor and
D-erythrose 4-phosphate as acceptor substrate, where the
formation of D-fructose 6-phosphate is detected in the
coupled enzymatic test system (Table II). The failure of DHEThDP to act
as the precursor of reaction intermediate in the transketolase reaction
at catalytically relevant rates may be due to the lack of a suitably
positioned enzymic group able to abstract the proton at the C carbon
of DHEThDP, a prerequisite for the binding of acceptor substrate. The
presence of such a group might in fact counteract catalytic deficiency,
because of possible protonation of the
-carbanion intermediate,
giving rise to an unwanted side reaction.
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ACKNOWLEDGEMENT |
---|
We thank Christer Wikner for providing the plasmid of the mutant H263A and Georg Wille for his help in investigating the enzyme kinetics using the program Dynafit.
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FOOTNOTES |
---|
* This work was supported by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, and the Swedish Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors supported by grants from the Graduiertenförderung of Sachsen-Anhalt.
To whom correspondence should be addressed. Tel.:
49-345-5524828; Fax: 49-345-5527011; E-mail:
huebner@biochemtech. uni-halle.de.
Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.M007936200
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ABBREVIATIONS |
---|
The abbreviations used are:
ThDP, thiamin
diphosphate;
DHEThDP, 2-(,
-dihydroxyethyl)thiamin diphosphate;
HPA,
-hydroxypyruvate.
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