Donor substrate regulation of 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 of donor substrate, neg-
ative cooperativity is not observed. The donor substrate 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 donor substrate 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-

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 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 of donor 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

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

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 of transketolase 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 of substrate 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

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

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

0:5 Â V
max
½ThDP
½ThDPþK
1
d
þ
0:5 Â V
max
½ThDP
½ThDPþK
2
d
:
Results
Influence 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 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 of substrate (curve 1) is
Fig. 2.
18
Reconstitution of holotransketolase
from apotransketolase (0.7 mgÆmL
)1
)and

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 of substrate (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 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

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 of donor 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

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 donor substrate 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 donor substrate 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 of donor substrates may be
explained by the formation o f t he TK reaction intermediate,

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 donor substrate 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 of donor substrate 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

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 Regulation of transketolase (Eur. J. Biochem. 271) 4193
data were received in t he absence of the donor substrate 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 donor substrate 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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