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 290–34
Trang 1Donor substrate regulation of transketolase
Olga A Esakova1, Ludmilla E Meshalkina1, Ralph Golbik2, Gerhard Hu¨bner2and German A Kochetov1
1 A N Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia; 2 Martin-Luther-University Halle-Wittenberg, Halle/Saale, Germany
The influence 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, resulting in a negative cooperativity for
coenzyme binding In the absence of donor substrate,
neg-ative cooperativity is not observed The donor substrate did
not affect the interaction of the apoenzyme with the 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-ited a higher affinity to the enzyme than thiamin diphos-phate
1 The enhancement of thiamin diphosphate’s affinity
to apotransketolase in the presence of donor substrate is probably one of the mechanisms underlying the sub-strate-affected transketolase regulation at low coenzyme concentrations
Keywords: 2-(a,b-dihydroxyethyl)-thiamin diphosphate; regulation of enzyme activity; spectrophotometric titration; thiamin diphosphate; transketolase
Transketolase (TK, EC 2.2.1.1), containing divalent cations
and thiamin 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 aldoses (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 activity, regardless of the divalent cation used 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 the apoenzyme of TK (apoTK) in the
presence of magnesium ions
negative cooperativity, albeit slightly pronounced [5], while
others call into question the nonequivalency of the enzyme’s
active centers on ThDP binding [6,7]
ThDP–apoTK binding requires at least a two-step mechanism [8]
TKþ ThDP ! TKThDP !kþ1
k 1
TK-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)1in 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 of transketolase (holoTK) differ in the position
of two loops in the two subunits (residues 187–198 and 383–394) – they are relatively flexible in the apoenzyme and structured in the holoenzyme These two 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 an intermediate of the TK reaction for donor substrate 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 University, 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)
Trang 2the 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
capable of using, as cofactors, various bivalent cations
Certain kinetic properties of TK are known to depend on
bivalent cations, which are used as cofactors [5] Comparing
the same kinetic characteristics obtained for
ThDP-depend-ent enzymes with diverse bivalThDP-depend-ent cations as cofactors is a
matter of undebatable interest Only Mg2+has been used as
a cofactor in the studies of all ThDP-dependent enzymes
(including, until recently, TK) This is the reason why Mg2+
was also used as a cofactor in this work
Materials and methods
Materials
ThDP and glycyl-glycine were purchased from Serva
Electrophoresis
acid (HPA), xylulose 5-phosphate (X5P), MgCl2, 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 method 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 A1%1 cmof 14.5 at 280 nm [17]
Measurement of TK activity
The activity of TK was determined spectrophotometrically
at 25C 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 25C using an
AMINCO DW 2000 spectrophotometer
Instru-ments, Rochester, NY, USA) (optical path length of
1 cm) Medium components were 1 mgÆmL)1TK, 50 mM
glycyl-glycine buffer (pH 7.6), 2.5 mM MgCl2, 0.5 mM
ThDP or 0.04 mMN3¢-pyridyl-ThDP, and 2.5 mM HPA,
if indicated The difference spectra of holoTK in the
presence or absence of HPA were obtained by 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 nm range), the intensity of which is strictly correlated with the amount of coenzyme bound in the active 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 apoenzyme 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 an essential contribution to the absorption spectrum of holoTK at 320 nm (curves 1 and 2 in Fig 1) Therefore, titration of apoTK with ThDP was carried out at
320 nm The acceptor substrate, however, has no effect on the absorption spectrum of the holoenzyme The registra-tion was conducted in a two-wavelength mode (k¼
320 nm, k¼ 400 nm), using an AMINCO DW 2000 spectrophotometer
ApoTK
7 (3 mL; 0.7 mgÆmL)1) in 50 mM glycyl-glycine buffer, pH 7.6, containing 2.5 mMMgCl2, was added to a quartz cuvette After recording the initial absorption at
320 nm, the first 10 lL (2.3 lM) of ThDP was added
8
and any absorption change was registered The next 10–
40 lL (4.5–452.6 lM) of ThDP
the absorption change No further change in absorption after addition of the final sample of ThDP was used as a sign
of full reconstitution of holoTK
characterizes the amount of holoTK with ThDP bound in two active sites A typical experiment is presented in Fig 2 The influence of the substrate on reconstitution of holoTK
Fig 1.
17 Difference absorption spectra of transketolase from Saccharo-myces cerevisiae (TK) (1 mgÆmL)1) in 50 m M glycyl-glycine buffer,
pH 7.6, containing 2.5 m M MgCl 2 , at 25 °C (1) Holoenzyme of transketolase (holoTK) in the absence of substrate after subtraction of the spectra of the transketolase apoenzyme (apoTK) and thiamin diphosphate (ThDP); (2) holoTK in the presence of 2.5 m M
hydroxypyruvic acid (HPA) after subtraction of the spectra of apoTK, thiamin diphosphate (ThDP) and HPA; (3) complex of TK with N3¢-pyridyl-ThDP after subtraction of the spectra of apoTK and N3¢-pyridyl-ThDP.
Trang 3was studied when apoenzyme was incubated in the presence
of 2.5 mMHPA and 2.5 mMMgCl2prior to the addition of
ThDP
Reconstitution of the TK–N3¢-pyridyl-ThDP (an inactive
analogue of ThDP) complex, was monitored by a change in
the optical density at 345 nm (absorption maximum of the
band induced as a result of formation of the complex
3 in Fig 1) Spectrophotometric titration was carried out
similarly to the experiments with ThDP, using a
two-wavelength mode (k¼ 345 nm, k ¼ 420 nm), on an
AMINCO DW 2000 spectrophotometer in 50 mM
glycyl-glycine buffer, pH 7.6, containing 2.5 mMMgCl2 Binding
of N3¢-pyridyl-ThDP (Ki¼ 1.3 nM[19]) is impaired in the
presence of 20 mMinorganic 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 presence of 2.5 mM HPA
and 2.5 mMMgCl2, prior to the addition of analogue
Determination ofKDfor 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 saturating
concentra-tion of ThDP, the maximum alteraconcentra-tion in the absorbance at
320 nm corresponds to 100% formation of holoTK The
KDfor ThDP was calculated using the programSCIENTIST
Calculation
12 based on the model for two active centers,
according to Dixon’s method [20]
½holoTK ¼0:5 ½TK ½ThDPfree
½ThDPfree þ K1
D
þ0:5 ½TK ½ThDPfree
½ThDPfree þ K2
D :
The concentration of free ThDP was determined according
to the following equation:
½ThDPfree ¼ ½ThDPtotal ½ThDPbound where [ThDPbound] is equivalent to the concentration of the active centers occupied by ThDP
Determination ofKdfor ThDP in the presence and absence of X5P
The apoTK (1–2 lgÆmL)1) was preincubated at 25C in
50 mM glycyl-glycine buffer, pH 7.6, containing 2.5 mM MgCl2 and 0.1% BSA (for TK stabilization) at different concentrations of ThDP (0.5–120 lM) in the presence or absence of 0.5 mM X5P The reconstitution reaction was allowed to proceed 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 (1 mM sodium arsenate, 0.37 mM NAD+,
3 U glyceraldehyde 3-phosphate dehydrogenase, 3.2 mM dithiothreitol, 1 mM ribose 5-phosphate) The changes in optical density at 340 nm were measured as described above
Based on the data, the Kd for ThDP in the presence and absence of X5P was determined using the program SCIENTIST Calculation
m¼0:5 Vmax ½ThDP
½ThDP þ K1 þ0:5 Vmax ½ThDP
½ThDP þ K2 :
Results
Influence of the donor substrate on the reconstitution
of apoTK with ThDP The influence of the donor substrate on the binding of ThDP to apoTK in the presence of Mg2+, as investigated
by the spectrophotometric titration method, is 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
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 , at 25 C.
Trang 4the same in both cases, revealing K1D¼ K2D¼ 5.2 lM
(Table 1) The addition of HPA (an artificial donor
substrate for TK, cleaved in an irreversible manner) caused
a significant increase in the affinities of the two active centers
to ThDP (Fig 3, curve 2 and Table 1); moreover, these
affinities were exhibited to different degrees: for one active
center, the dissociation constant (K2
D) decreased to 1.6 lM, while the affinity of the other increased to such an extent
that K1Dcould not be determined under the experimental
conditions used The 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 added
to the sample was stoichiometrically bound to the first active
center Thus, in the presence of HPA, the affinity of apoTK
to ThDP increased and 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 cleaved by the enzyme in reversible 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
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 Kd of 4.6 lM (curve 1, Fig 4) In the presence of X5P (curve 2, Fig 4) the values K1d¼ 0.22 lM and K2d¼ 4.4 lM for ThDP were determined (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 In contrast, the acceptor substrates (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¢-pyridyl-ThDP is an inactive analogue 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 binding of this analogue
to the apoenzyme using the spectrophotometric titration method Figure 5 shows the formation of an inactive complex of TK with N3¢-pyridyl-ThDP in the presence or
Table 1 Dissociation constants of thiamin diphosphate (ThDP) of the
two active sites of transketolase 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 ) The data were
cal-culated
17 using the program SCIENTIST K D and K d were determined
based on the data presented in Figs 3 and 4, respectively.
Substrate
K1D
(l M )
K2D
(l M )
K1d
(l M )
K2d
(l M )
No substrate 5.2 5.2 4.6 4.6
2.5 m M HPA a 1.6b – –
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.bIn 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 holoenzyme (holoTK), reconstituted
at different concentrations of 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 The activity of holoTK was determined as described in the Materials and methods: (1) reconstitution of holoTK without substrate; and (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)1and the ThDP concentrations used ranged from 0 to 20 l M and from 20 to
120 l M , respectively The data were fitted to the TK concentration 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 donor substrate on the formation of transketolase
holoenzyme (holoTK) from the transketolase apoenzyme (apoTK)
(0.7 mgÆmL)1) and 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 (1) in the
absence of substrate and (2) in the presence of 2.5 m M hydroxypyruvic
acid (HPA) The formation of holoenzyme was observed by the change
in absorbance at 320 nm, as described in the Materials and methods.
The points are obtained experimentally; the lines are calculated for a
set of parameters presented in Table 1 Insertion shows the initial part
of the curves.
Trang 5absence of 2.5 mMHPA 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 catalytically active holoenzyme, but not the formation of
the catalytically inactive complex of TK with
N3¢-pyridyl-ThDP
Discussion
In the presence of Mg2+, the two active centers of TK have
the same affinity for ThDP (Table 1) Donor substrates,
converted both reversibly (X5P) and irreversibly (HPA),
enhance the affinity of the coenzyme for apoTK In the
presence of any donor substrate during holoTK
reconsti-tution, the affinity for the cofactor ThDP increased together
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) on the
reconstitution of holoTK have shown that the acceptor
substrate, in contrast to the donor substrate, 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 of the TK reaction intermediate,
DHEThDP, which exhibits a higher affinity than ThDP to
TK [14] This conclusion was supported by the experiment
with the inactive coenzyme analogue N3¢-pyridyl-ThDP,
which is similar to the native coenzyme except for 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-dependent enzymes [22,23] The
inhi-bition constant of N3¢-pyridyl-ThDP for TK is 1.3 nM[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 points to the competent (correct) binding of this analogue to TK and indicates the same microenvironment
of the analogue in the active site, as in the case of ThDP The X-ray crystallography structure of the TK-N3¢-pyridyl-ThDP complex shows that after reconstitution, the ana-logue displays the same V-conformation typical of ThDP 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 for the inactivity of the analogue The donor substrate has no effect on the binding of this analogue to TK (Fig 5)
Conversion of HPA resulted in a significant increase in the affinities of the two active centers to ThDP; 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 of the 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 has previously been reported
result was the appearance of the negative cooperativity on the binding of ThDP to apoTK in the presence of the donor substrates
In the presence of X5P (a reversible donor substrate), the affinity of ThDP increases in one 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 half-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) The 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 regulation of TK by the donor substrate at a low concentration of coenzyme
The proposed mechanism explains various data reporting the coenzyme’s affinity to apoTK in the presence of magnesium ions in the literature [5–7,25] Some authors report a negative cooperativity, albeit slightly pronounced [5], while others call into question the nonequivalency of the enzyme’s active centers on ThDP binding [6,7] All of these
Fig 5.
21 Influence of the donor substrate on the formation of an inactive
complex of transketolase (TK) (0.7 mgÆmL)1) with N3¢-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 was measured in the
absence of substrate; curve 2 was measured in the presence of 2.5 m M
hydroxypyruvic acid (HPA) Owing to the high affinity of
N3¢-pyridyl-ThDP to the transketolase apoenzyme (apoTK) (K i ¼ 1.3 n M ) [20],
20 m M inorganic diphosphate was added as described in the Materials
and methods.
Trang 6data were received in the 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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