Báo cáo khoa học: Donor substrate regulation of transketolase pptx

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- 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; 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 à -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 of transketolase (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 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 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 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 , 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 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 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] ½holoTK¼ 0:5 ½TK½ThDP free  ½ThDP free þK 1 D þ 0:5 ½TK½ThDP 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  V max ½ThDP ½ThDPþK 1 d þ 0:5  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 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 Regulation of transketolase (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 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 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 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 ). 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 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 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 donor substrate 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 of substrate 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 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, 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 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 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 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 regulation of 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 donor substrate on the formation of an inactive complex of transketolase (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 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). References 1. Kochetov, G.A. ( 1982) Transketolase from yeast, rat liver and pig liver. Methods Enzymol. 90, 209–223. 2. Kochetov, G.A., Tikhomirova, N.K. & P hilippov, P.P. (1975) The binding of t hiamine pyrophosphate with transketolase in equili- brium conditions. Biochem. Biophys. Res. Commun. 63, 924–930. 3. Kochetov, G.A., Meshalkina, L.E. & U smanov, R.A. (19 76) The number of active sites in a molec ule of transketolase. Biochem. Biophys. Res. Commun. 69, 836–843. 4. 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