Donorsubstrateregulationof transketolase
Olga A. Esakova
1
, Ludmilla E. Meshalkina
1
, Ralph Golbik
2
, Gerhard Hu¨bner
2
and German A. Kochetov
1
1
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia;
2
Martin-Luther-University
Halle-Wittenberg, Halle/Saale, Germany
The i nfluence of substrates on the interaction of apotrans-
ketolase with thiamin diphosphate was investigated in the
presence of magnesium ions. It was shown that the donor
substrates, but not the acceptor substrates, enhance the
affinity of the coenzyme either to only one active center of
transketolase or to both active centers, but to different
degrees in each, r esulting in a negative coop erativity for
coenzyme binding. I n the absence ofdonor substrate, neg-
ative cooperativity is not observed. The donorsubstrate did
not affect t he interaction o f t he apoenzyme w ith t he inactive
coenzyme analogue, N3¢-pyridyl-thiamin diphosphate.
The influence of the donorsubstrate on the coenzyme–
apotransketolase interaction was predicted as a result
of formation of the transketolase reaction intermediate
2-(a,b-dihydroxyethyl)-thiamin diphosphate, which exhib-
ited a higher affinity to the enzyme than thiamin diphos-
phate
1
. The enhancement of thiamin diphosphate’s affinity
to apotransketolase in the presence ofdonorsubstrate is
probably one of the mechanisms underlying the sub-
strate-affected transketolaseregulation at low coenzyme
concentrations.
Keywords:2-(a,b-dihydroxyethyl)-thiamin diphosphate;
regulation of enzyme activity; s pectrophotometric t itration;
thiamin diphosphate; transketolase.
Transketolase (TK, E C 2 .2.1.1), containing divalent c ations
and t hiamin diphosphate (ThDP) as cofactors, catalyzes
one of the key reactions of the pentosephosphate pathway
in carbohydrate transformation, namely the cleavage of a
carbon–carbon bond adjacent to a carbonyl group in
ketoses ( donor substrates) with subsequent transfer of a
two-carbon unit to a ldoses (acceptor substrates) [1]. The TK
enzyme is a homodimer with two active centers located at
the interface between the contacting surfaces of the mono-
mers. The active centers are characterized by the same
enzymatic a ctivity, regardless o f the d ivalent cation u sed as a
cofactor. A negative cooperativity in ThDP binding is
observed in the presence of calcium ions [2–5]. However,
contrasting data have been published regarding the affinity
of the coenzyme to t he apoenzyme of TK (apoTK) in the
presence of magnesium ions.
2
Some authors report a
negative cooperativity, albeit slightly pronounced [5], while
others call into question the nonequivalency of the e nzyme’s
active centers on ThDP binding [6,7].
ThDP–apoTK binding requires at least a two-step
mechanism [8].
TK þ Th DP
!
TKÁÁÁThDP
!
k
þ1
k
À1
TK
Ã
-ThDP
ðScheme 1Þ
The first step, fast and easily reversible, yields an inter-
mediate: a catalytically inactive , primary TKÆÆÆThDP com-
plex. The second step is slow and accompanied by
conformational changes necessary for the formation of the
catalytically active holoenzyme, TK*-ThDP. The initially
identical TK active centers become nonequivalent in the
course of ThDP binding. It has been inferred [9] that the
nonequivalency of the TK active centers in coenzyme
binding is determined by the increase of the backward
conformational transfer rate constant (k
)1
in Scheme 1) for
the one active center with respect to the other. The X-ray
data have shown that the structures of apoTK and
holoenzyme oftransketolase (holoTK) differ in the position
of two loops in the two subunits (residues 187–198 and
383–394) – t hey are relatively flexible in the apoenzyme and
structured in the holoenzyme. These t wo loops are charac-
terized by high mobility, and in holoTK they directly
contact the coenzyme [10–12]. It cannot be ruled out that
the interdependent counter-phase movement of these loops
determines the alternative destabilization of the secondary
complexes (TK*-ThDP in Scheme 1) of the TK active
centers with the coenzyme [9].
As already known, 2-(a,b-dihydroxyethyl)-thiamin
diphosphate (DHEThDP) is a n intermediate of t he TK
reaction for donorsubstrate transformation. According to
the data obtained by X-ray crystallography [13], there are
additional bonds of DHEThDP to amino acid residues in
Correspondence to G. A. Kochetov, A. N. Belozersky Institute of
Physico-Chemical Biology, Moscow State U niversity, 119992,
Moscow, GSP-2, Russia. Fax: +7 95 939 31 81,
Tel.: +7 95 939 14 56, E-mail: kochetov@genebee.msu.su
Abbreviations: apoTK, transketolase apoenzyme; DHEThDP,
2-(a,b-dihydroxyethyl)-thiamin diphosphate; holoTK, transketolase
holoenzyme; HPA, hydroxypyruvic acid; ThDP, thiamin diphosphate;
TK, transketolase from Saccharomyces cerevisiae; X5P, xylulose
5-phosphate.
Enzyme: transketolase (EC 2.2.1.1).
(Received 29 June 2004, revised 10 August 2004,
accepted 3 September 2004)
Eur. J. Biochem. 271, 4189–4194 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04357.x
the active center of the enzyme compared to ThDP. The
enzymatically generated intermediate displays a higher
affinity to apoTK than ThDP [14]. Based on these data,
it is possible to assume that in the presence of the donor
substrate, ThDP will possess higher affinity to apoTK. The
present study is devoted to elucidation of a possible
regulatory role ofdonor substrates in holoTK formation.
Similarly to other ThDP
3
-dependent enzymes, TK is
capable of using, as cofactors, various bivalent cations.
Certain kinetic properties of TK are known to d epend on
bivalent cations, w hich are used as cofactors [5]. Comparing
the same kinetic characteristics obtained for ThDP-depend-
ent enzymes with diverse bivalent cations as cofactors i s a
matter o f undebatable interest. Only Mg
2+
has b een used as
a cofactor in the studies of all ThDP-dependent enzymes
(including, until recently, TK). This is the reason why Mg
2+
was also used as a cofactor in this work.
Materials and methods
Materials
ThDP and glycyl-glycine were purchased from Serva
Electrophoresis
4,54,5
(Heidelberg, Germany); hydroxypyruvic
acid (HPA), xylulose 5-phosphate (X5P), MgCl
2
, racemic
glyceraldehyde and ribose 5-phosphate were from Sigma
Chemical Co; N3¢-pyridyl-ThDP was synthesized as des-
cribed previously [15]. Other chemicals were of the highest
quality commercially available.
Purification of TK
Recombinant bakers yeast TK, with a specific activity of
22 UÆmg
)1
, was isolated by a m ethod described previously
[16]. The enzyme was obtained as apoTK and was
determined to be homogeneous by SDS/PAGE. The TK
concentration was determined spectrophotometrically,
using an A
1%
1cm
of 14.5 at 280 nm [17].
Measurement of TK activity
The activity of TK was determined spectrophotometrically
at 25 °C by measuring the rate of NAD
+
reduction in a
coupled system with glyceraldehyde-3-phosphate dehydro-
genase [1].
Determination of ThDP concentration
ThDP concentration was determined spectrophotometri-
cally at 272.5 nm using a molar extinction coefficient of
7800 [18].
Absorption spectra
Absorption spectra were recorded at 25 °Cusingan
AMINCO DW 2000 spectrophotometer
6
(SLM Instru-
ments, Rochester, NY, USA) (optical path length of
1 cm). M edium components were 1 mgÆmL
)1
TK, 50 m
M
glycyl-glycine buffer (pH 7.6), 2.5 m
M
MgCl
2
,0.5m
M
ThDP or 0.04 m
M
N3¢-pyridyl-ThDP, and 2.5 m
M
HPA,
if indicated. The difference spectra of holoTK in the
presence or absen ce of HPA were obtained b y subtraction
of the individual spectra of apoTK, ThDP or N3¢-pyridyl-
ThDP, and HPA, correspondingly.
Spectrophotometric titration
The binding of ThDP to apoTK, and the formation of a
catalytically active holoenzyme, is accompanied by the
appearance of a new absorption band (in the 290–340 n m
range), the intensity of which is strictly correlated with the
amount of coenzyme bound in the a ctive centers of TK [3,4].
This approach was used to determine the affinity of the
coenzyme to TK [4,9] and to study the interaction of ThDP
with the a poenzyme. In this way, the kinetics of the distinct
stages during this process were measured [9]. In the present
study, this method was used to investigate holoTK recon-
stitution in the presence of substrates. The donor substrate
does not provide a n e ssential contribution t o the absorption
spectrum o f holoTK a t 320 nm (curves 1 and 2 in Fig. 1).
Therefore, titration of a poTK with T hDP w as carried out at
320 n m. The acceptor substrate, however, has no effect on
the absorption spectrum of the holoenzyme. The registra-
tion was conducted in a two-wavelength mode (k ¼
320 nm, k ¼ 400 nm), using an AMINCO DW 2000
spectrophotometer.
ApoTK
7
(3 mL; 0.7 mgÆmL
)1
)in50m
M
glycyl-glycine
buffer, pH 7.6, containing 2.5 m
M
MgCl
2
,wasaddedtoa
quartz cuvette. After recording the initial absorption a t
320 nm, the first 10 lL(2.3l
M
) of ThDP was added
8
and any absorption change was registered. The next 10–
40 lL (4.5–452.6 l
M
)ofThDP
9
were added after recording
the absorption change. No further change in a bsorption
after a ddition of the final s ample of T hDP was used as a sign
of full reconstitution of holoTK.
10
The final absorption level
characterizes the a mount of holoTK with ThDP bound in
two active sites. A typical exp eriment is presen ted in Fig. 2.
The influence of t he substrate on reconstitution of holoTK
Fig. 1.
17
Difference absorption spectra oftransketolase from Saccharo-
myces cerevisiae (TK) (1 mgÆmL
)1
)in50m
M
glycyl-glycine buffer,
pH 7.6, containing 2.5 m
M
MgCl
2
,at25°C. (1) Holoenzyme of
transketolase (holoTK) i n the absence ofsubstrate after subtraction of
the spectra of the transketolase apoenz yme (apoTK) and thiamin
diphosphate (ThDP); ( 2) holoTK in the presenc e of 2.5 m
M
hydroxypyruvic acid (HPA) after subtraction of the spectra of apoT K,
thiamin diphosphate (ThDP) and HPA; (3) complex of TK with
N3¢-pyridyl-ThD P after subtraction of the spectra of apoTK and
N3¢-pyridyl-ThDP.
4190 O. A. Esakova et al.(Eur. J. Biochem. 271) Ó FEBS 2004
was studied when apoenzyme w as incubated i n t he presence
of 2.5 m
M
HPA and 2.5 m
M
MgCl
2
prior to the addition of
ThDP.
Reconstitution of the TK–N3¢-pyridyl-ThDP (an inactive
analogue of ThDP) c omplex, was monitored b y a c hange in
the optical density at 345 nm (absorption maximum of the
band induced as a result o f formation of the c omplex
11
;curve
3 in Fig. 1). Spectrophotometric titration was carried out
similarly to the experiments with ThDP, using a two-
wavelength mode (k ¼ 345 nm, k ¼ 420 nm) , on an
AMINCO DW 2000 spectrophotometer in 50 m
M
glycyl-
glycine buffer, pH 7.6, containing 2.5 m
M
MgCl
2
. Binding
of N3¢-pyridyl-ThDP (K
i
¼ 1.3 n
M
[19]) is i mpaired in the
presence of 20 m
M
inorganic sodium diphosphate, resulting
in a decrease of the apparent binding constant. In experi-
ments where substrate influence on the reconstitution of
complex TK with N3¢-pyridyl-ThDP was studied, the
enzyme was incubated in the p resence of 2.5 m
M
HPA
and 2.5 m
M
MgCl
2
, prior to the addition of analo gue.
Determination of
K
D
for ThDP in the presence and
absence of HPA
Based on the spectrophotometric titration data, the disso-
ciation constants of ThDP binding to each of the enzyme’s
active centers were determined. At a s aturating c oncentra-
tion of ThDP, t he maximum a lteration i n the absorbance at
320 n m corresponds to 100% formation of holoTK. The
K
D
for ThDP was calculated using the program
SCIENTIST
.
Calculation
12
based on the model for two active centers,
according to Dixon’s method [20]
½holoTK¼
0:5 ½TK½ThDP
free
½ThDP
free
þK
1
D
þ
0:5 ½TK½ThDP
free
½ThDP
free
þK
2
D
:
The concentration of free ThDP was determined according
to the following equation:
½ThDP
free
¼½ThDP
total
À½ThD P
bound
where [ThDP
bound
] is equivalent to the concentration of the
active centers occupied by ThDP.
Determination of
K
d
for ThDP in the presence and
absence of X5P
The apoTK (1–2 lgÆmL
)1
) was preincubated at 25 °Cin
50 m
M
glycyl-glycine buffer, pH 7.6, containing 2.5 m
M
MgCl
2
and 0.1% BSA (for TK stabilization) at different
concentrations of ThDP (0.5–120 l
M
) in the presence or
absence of 0.5 m
M
X5P. The reconstitution reaction was
allowed to proce ed for 90–150 min, which was the time
usually required for completion of the process. After-
wards, the activity of the holoenzyme was measured by
adding all the components necessary for determining
TK activity (1m
M
sodium arsenate, 0.37 m
M
NAD
+
,
3 U glyceraldehyde 3-phosphate dehydrogenase, 3.2 m
M
dithiothreitol, 1 m
M
ribose 5-phosphate). The changes in
optical density at 340 nm were measured as described
above.
Based o n the data, the K
d
for T hDP in the presence
and absence of X5P was determined using the program
SCIENTIST
.Calculation
13
as discussed above.
m ¼
0:5 Â V
max
½ThDP
½ThDPþK
1
d
þ
0:5 Â V
max
½ThDP
½ThDPþK
2
d
:
Results
Influence of the donorsubstrate on the reconstitution
of apoTK with ThDP
The influence o f the donorsubstrate on the binding of
ThDP to apoTK i n the presence of Mg
2+
, a s investigated
by the s pectrophotometric titration method, i s shown in
Fig. 3. The affinity exhibited by the two active centers of
apoTK to ThDP in the absence ofsubstrate (curve 1) is
Fig. 2.
18
Reconstitution of holotransketolase
from apotransketolase (0.7 mgÆmL
)1
)and
thiamin diphosphate (ThDP) ( 0–0.453 m
M
).
Data of spectrophotometric titration in
50 m
M
glycyl-glycine buffer, pH 7.6, in the
presence of 2.5 m
M
MgCl
2
,at25°C.
Ó FEBS 2004 Regulationoftransketolase (Eur. J. Biochem. 271) 4191
the same in both cases, revealing K
1
D
¼ K
2
D
¼ 5.2 l
M
(Table 1). The addition of HPA (an artificial donor
substrate for TK, cleaved in an irreversible manner) caused
a s ignificant increase in the affinities of the two active centers
to ThDP (Fig. 3, curve 2 a nd Table 1); moreover, these
affinities were exhibited to different degrees: for one active
center, the dissociation constant (K
2
D
) decreased to 1.6 l
M
,
while the affinity of the other increased to such an extent
that K
1
D
could not be determined under the experimental
conditions used. T he affinity of the first active center of TK
for ThDP could not be estimated by the method employed
herein because the affinity was too high: all the ThDP a dded
to the sample was stoich iometrically bound to the fi rst active
center. Thus, in the presence of HPA, the a ffinity of ap oTK
to ThDP increased a nd a negative cooperative effect on
coenzyme binding was induced that is not observed in the
absence ofsubstrate (Table 1).
In order to study the influence of the native donor
substrate, X5P (which is c leaved by the enzyme in r eversible
manner), on the affinity of the coenzyme to apoTK, the
enzymatic activity of apoTK was measured after preincu-
bation with different concentrations of ThDP in the
presence or absence of X5P
14
. The results of the experiment
are presented in Fig. 4 and Table 1. Both active centers of
apoTK showed the same affinity to ThDP in the absence of
X5P, displaying a K
d
of 4.6 l
M
(curve 1, Fig. 4). In the
presence of X5P (curve 2, Fig. 4) the values K
1
d
¼ 0.22 l
M
and K
2
d
¼ 4.4 l
M
for ThDP were d etermined (Table 1).
Thus, the addition of X5P caused a significant increase in
ThDP affinity to one of the two TK active centers.
Consequently, as in the case of HPA, X5P not only
increases the affinity of TK to ThDP, but also causes
nonequivalency of the enzyme’s active centers in coenzyme
binding. I n c ontrast, the acceptor s ubstrates (glyceraldehyde
and ribose 5-phosphate) exert no influence on the affinity
of the enzyme’s active centers to ThDP (Table 1).
The interaction of N3¢-pyridyl-ThDP with apoTK
N3¢-p yridyl-ThDP is an inactive a nalogue of ThDP, in
which the N1¢ atom is replaced with CH [19,21]. The
presence of the induced band in the difference absorption
spectrum on the TK-N3¢-pyridyl-ThDP complex (curve 3,
Fig. 1) enabled us to measure the b inding of this analogue
to the apoenzyme using the spectrophotometric titration
method. Figure 5 shows the formation of an inactive
complex o f TK with N3¢-pyridyl-ThDP in the presence or
Table 1. Dissociation constants of thiamin diphosphate (ThDP) of the
two active sites oftransketolase from Saccharomyces cerevisiae in the
presence of Mg
2+
, as determined by using spectrophotometric titration
(K
D
) and by assaying the holoenzyme activity (K
d
). Thedatawerecal-
culated
17
using the program
SCIENTIST
. K
D
and K
d
were determined
basedonthedatapresentedinFigs3and4,respectively.
Substrate
K
1
D
(l
M
)
K
2
D
(l
M
)
K
1
d
(l
M
)
K
2
d
(l
M
)
No substrate 5.2 5.2 4.6 4.6
2.5 m
M
HPA
a
1.6
b
––
0.5 m
M
X5P – – 0.22
b
4.4
b
10 m
M
glyceraldehyde 5.4 5.4 – –
0.7 m
M
ribose 5-phosphate – – 4.8 4.8
a
In the experiments using hydroxypyruvic acid (HPA), the affinity
of ThDP to TK is so high that the method for determination of K
D
for the first active site is not qualified.
b
In this case, the value of the
dissociation constant is apparent, i.e. the value was determined in
the presence ofdonor substrate.
Fig. 4.
20
Activity of the transketolase ho loenzyme (holoTK), reconstituted
at different concentrations of thiamin diphosphate (ThDP) in 50 m
M
glycyl-glycine buffe r, pH 7.6, in the presence of 2.5 m
M
MgCl
2
at 25 °C.
The activity of holoTK was determined as described in the Materials
and methods: (1) reconstitution of ho loTK with out s ubstrate; a nd ( 2)
reconstitution of holoTK in the presence of 0.5 m
M
xylulose 5-phos-
phate (X5P). The concentrations of TK used are 2 and 1 lgÆmL
)1
and
the T hDP concentrations u sed ranged from 0 t o 20 l
M
and from 20 to
120 l
M
, respectively. T he d ata were fi tted to th e T K conc entratio n of
1 lgÆmL
)1
. The points are obtained experimentally; the lines are cal-
culated for a set of parameters presented in Table 1.
Fig. 3.
19
Influence of the donorsubstrate on the forma tion of transketolase
holoenzyme (holoTK) from the transketolase apoenzyme (apoTK)
(0.7 mgÆmL
)1
) and thiamin diphosphate (ThDP) i n 50 m
M
glycyl-glycine
buffer, pH 7.6, in the presence of 2.5 m
M
MgCl
2
,at25°C(1)inthe
absence ofsubstrate and (2) in the presence of 2.5 m
M
hydroxypyruvic
acid (HPA). The formation o f holoenzyme was o bserved by the c hange
in absorbance at 320 nm, as described in the Materials and methods.
The points are obtained experimentally; the lines are calculated for a
set o f parameters p resented in Table 1. Insertion shows t he initial p art
of the curves.
4192 O. A. Esakova et al.(Eur. J. Biochem. 271) Ó FEBS 2004
absence of 2.5 m
M
HPA. As shown, this compound had
no influence on the affinity of N3¢-pyridyl-ThDP to TK,
indicating that the donorsubstrate affects the formation of
the c atalytically active holoenzyme, but not the formation of
the catalytically inactive complex of TK with N3¢-pyridyl-
ThDP.
Discussion
In the pre senc e of Mg
2+
, the two active centers of TK have
the same affinity for ThDP (Table 1). Donor substrates,
converted both reversibly (X5P) and i rreversibly ( HPA),
enhance the affinity of the coenzyme for apoTK. In the
presence of any donorsubstrate during holoTK reconsti-
tution, the a ffinity fo r t he cofactor ThDP increased t ogether
with the manifestation of a negative cooperativity between
the active sites in this process. Research on the influence of
aldoses (glyceraldehyde and ribose 5 -phosphate) o n the
reconstitution of holoTK h ave shown t hat the acceptor
substrate, in contrast to the donor sub strate, exerts no
influence on the affinity of the enzyme’s active centers for
ThDP (Table 1).
It is suggested that an enhancement of ThDP affinity for
apoTK in the presence ofdonor substrates may be
explained by the formation o f t he TK reaction intermediate,
DHEThDP, which exhibits a h igher affinity than ThDP to
TK [14]. This conclusion was supported by the experiment
with the inactive coenzyme analogue N3¢-pyridyl-ThDP,
which i s similar t o the native coenzyme except fo r the lack of
activity. Indeed, with respect to ThDP, the N3¢-pyridyl-
ThDP is a competitive inhibitor of TK [19] and of other
thiamin diphosphate-dependen t enzymes [22,23]. The inhi-
bition constant of N3¢-p yridyl-ThDP for TK is 1.3 n
M
[19].
Binding of N3¢-pyridyl-ThDP to the active sites of TK is
accompanied by the appearance of a new absorption band
in the same region of the CD spectrum, in which it appears
on the interaction of TK with the native coenzyme [19,21].
This fact p oints to the competent (correct) b inding of th is
analogue to TK and indicates the same microenvironment
of the analogue in the active site, a s in the case of ThDP.
The X-ray crystallography structure o f the TK-N3 ¢-pyridyl-
ThDP complex shows t hat after reconstitution, the ana-
logue displays the same V -conformation typical of T hDP in
the holoenzyme. In the active site of TK from Saccharo-
myces cerevisiae ,N3¢-pyridyl-ThDP interacts with con-
served amino acid residues, as does the native coenzyme,
except for a hydrogen bond emerging between the first
nitrogen atom of the aminopyrimidine ring of ThDP
(lacking in the analogue) and Glu418 [24]. This distinction
is, in fact, the reason f or the inactivity of th e analogue.
The donorsubstrate has no effect on the binding of this
analogue to TK (Fig. 5 ).
Conversion of HPA resulted in a significant increase in
the a ffin ities of the t wo active centers to T hDP; however, the
affinities of the two centers were different (Table 1). These
data are in agreement with the results of X-ray analysis,
which show the appearance of DHEThDP in both active
centers of TK [13]. On the other hand, the nonequivalency
of the enzyme’s active centers in the intermediate complex
suggests that different states of the active centers occur
during catalysis.
15
Consequently, the influence o f t he donorsubstrate on the
reconstitution of the holoenzyme is dependent on the ability
of the reconstituted complex to form DHEThDP or the
corresponding intermediate of any analogue. We were able
to predict the data obtained as the same effect has been
shown on the pyruvate dehydrogenase complex from
Escherichia coli [22,23]. Moreover, the efficient reconstitu-
tion of holoTK in the presence ofdonorsubstrate h as
previously been reported
16
[25]. However, an unexpected
result was the appearance of the negative cooperativity on
the binding of ThDP to ap oTK i n t he presence of the donor
substrates.
In the presence of X5P (a reversible donor substrate), the
affinity of ThDP increases in on e of the two active centers
(Table 1), i.e. the reaction intermediate DHEThDP, having
a high affinity to the enzyme, is formed at one active site
only. Thus, the cooperativity as a result of this h alf-of-the-
site reactivity becomes apparent. The data obtained do not
contradict previous results on the reversible converted
donor substrate protection of only one active site from the
chemical modification [26].
When the concentration of ThDP in the cell is low, only
a proportion of TK is represented by the holoenzyme
(the catalytically active form of the enzyme). T he donor
substrate increases the amount of holoTK by increasing, for
example, the affinity of ThDP for apoTK. As a result, the
total TK activity increases.
Hence, based on the data obtained, a mechanism may be
postulated for the efficient regulationof TK by the donor
substrate at a low concentration of coenzyme.
The p roposed m echanism e xplains v arious data reporting
the coenzyme’s affinity to apoTK in the presence of
magnesium i ons in the literature [5–7,25]. Some authors
report a negative cooperativity, albeit slightly pronounced
[5], while others call into question t he noneq uivalency of the
enzyme’s active centers on ThDP binding [6,7]. All of these
Fig. 5.
21
Influence of the donorsubstrate on the formation of an inactive
complex oftransketolase (TK) (0.7 mgÆmL
)1
)withN3¢-pyridyl-thiamin
diphosphate (ThDP) in 50 m
M
glycyl-glycine buffer, pH 7.6, in the
presence of 2.5 m
M
MgCl
2
at 25 °C. Curve 1 wasmeasuredinthe
absence o f substrate; curve 2 was measured in t he p resen ce of 2.5 m
M
hydroxypyruvic acid (HPA). Owing to the high affi nity of N3¢-pyridyl-
ThDP to the trans ketolase apoenzyme (apoT K) (K
i
¼ 1.3 n
M
)[20],
20 m
M
inorganic d iph osphate was ad ded as describe d in the Materials
and methods.
Ó FEBS 2004 Regulationoftransketolase (Eur. J. Biochem. 271) 4193
data were received in t he absence of the donorsubstrate and
were correlated with the results obtained. Strongly pro-
nounced negative cooperativity on the binding of ThDP to
apoTK [25] was shown in the presence of the donor
substrate and could be explained by the different influence
of the donorsubstrate on the affinity of the TK active
centers to ThDP.
Acknowledgements
This research was supported by a grant from the Russian Foundation
for Basic Research (03-04-49025).
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4194 O. A. Esakova et al.(Eur. J. Biochem. 271) Ó FEBS 2004
. of the donor substrate on the reconstitution
of apoTK with ThDP
The influence o f the donor substrate on the binding of
ThDP to apoTK i n the presence of. on
the binding of ThDP to ap oTK i n t he presence of the donor
substrates.
In the presence of X5P (a reversible donor substrate) , the
affinity of ThDP increases