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A hitherto unknown transketolase—catalyzed reaction
BBRC Biochemical and Biophysical Research Communications 313 (2004) 771–774 www.elsevier.com/locate/ybbrc
A hitherto unknown transketolase—catalyzed reactionq Irina A. Sevostyanova, Olga N. Solovjeva, and German A. Kochetov* A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119992 Moscow, GSP-2, Russia Received 25 November 2003
Abstract Yeast transketolase, in addition to catalyzing the transferase reaction through utilization of two substrates—the donor substrate (ketose) and the acceptor substrate (aldose)—is also able to catalyze a one-substrate reaction with only aldose (glycolaldehyde) as substrate. The interaction of glycolaldehyde with holotransketolase results in formation of the transketolase reaction intermediate, dihydroxyethyl-thiamin diphosphate. Then the glycolaldehyde residue is transferred from dihydroxyethyl-thiamin diphosphate to free glycolaldehyde. As a result, the one-substrate transketolase reaction product, erythrulose, is formed. The specific activity of transketolase was found to be 0.23 U/mg and the apparent Km for glycolaldehyde was estimated as 140 mM. Ó 2003 Published by Elsevier Inc. Keywords: Transketolase; One-substrate reaction; Thiamin diphosphate; Circular dichroism
Transketolase (EC 2.2.1.1) catalyzes the cleavage of a carbon–carbon bond adjacent to a carbonyl group in ketose and transfer of a two-carbon unit, a glycolaldehyde residue, to aldose [1]. The enzyme can utilize sugars such as D -xylulose 5-phosphate, D -fructose 6-phosphate, and D -sedoheptulose 7-phosphate, as well as hydroxypyruvate as donors of the transferred glycolaldehyde group. As acceptor substrates serve D -ribose 5-phosphate, D -glyceraldehyde 3-phosphate, D -erythrose 4-phosphate, and glycolaldehyde. Transketolase plays an important part in the rearrangement system, since it creates, together with transaldolase, a reversible link between the pentose phosphate pathway and glycolysis. Transketolase from the Saccharomyces cerevisiae is a homodimer with a molecular mass of 148.4 kDa [2], and it has two active sites of equal catalytic activity [3–5]. Its cofactors are thiamin diphosphate and divalent metal ions. Thiamin diphosphate binds at the interface between the subunits and interacts with residues from both subunits [5]. Interaction of ThDP with apotransketolase results in the appearance of a new band in the circular dichroism (CD) spectrum with a maximum at 315– 320 nm, this band being absent from the spectra of the q
Abbreviations: TK, transketolase; ThDP, thiamin diphosphate. Corresponding author. Fax: +7-095-939-3181. E-mail address: [email protected] (G.A. Kochetov). *
0006-291X/$ - see front matter Ó 2003 Published by Elsevier Inc. doi:10.1016/j.bbrc.2003.11.164
initial compounds [6,7]. The intensity of the band strictly correlates to the amount of the catalytically active holoenzyme and depends on the substrate concentrations [8–11]. This enables to study the interaction of coenzyme with apotransketolase [4,12] as well as to separate the investigation of the first and second steps of the transketolase reaction—the binding and cleavage of the donor substrate and transfer of the two-carbon fragment to the acceptor substrate, respectively [8,11,13–16]. Transketolase, while being a typical transferase, requires the presence of two substrates, ketose and aldose, for its action. Apart from that, the enzyme, as has recently been shown, is able to effect the catalytic transformation of ketose even in the absence of aldose, i.e., to catalyze the so-called one-substrate transketolase reaction [15,16]. In this work the possibility of aldose (glycolaldehyde) transformation by transketolase from S. cerevisiae was studied by CD spectroscopy and kinetic measurements.
Materials and methods Materials. Thiamin diphosphate, glycyl-glycine, and glycolaldehyde were from Serva; Sephadex G-50 was from Pharmacia (Sweden); and other chemicals were of the highest quality commercially available. Transketolase purification. Transketolase was isolated from the S. cerevisiae according to the method described earlier [17] with some
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I.A. Sevostyanova et al. / Biochemical and Biophysical Research Communications 313 (2004) 771–774
modifications [18]. The crystalline enzyme was stored at 4 °C in ammonium sulfate solution of 0.5 saturation, pH 7.6. Transketolase (specific activity in transferase reaction, 18 U/mg) was homogeneous by SDS–PAGE. TK concentration was determined using the absor1% bance coefficient E280 ¼ 14:5 [19]. Prior to use, the TK solution was passed through a Sephadex G-50 column to remove ammonium sulfate. TK activity. The enzyme’s catalytic activity in the transferase reaction was measured with xylulose 5-phosphate and ribose 5-phosphate as substrates [1]. The enzyme’s catalytic activity in the onesubstrate reaction was measured with glycolaldehyde as substrate from the change in ellipticity at 278 nm (Mark V, “Jobin Ivon,” France, spectropolarimeter; path length 1 cm). The reaction mixture (final volume 1 ml) contained: 50 mM glycyl-glycine, pH 7.6, 2.5 mM CaCl2 , 60 lM ThDP, and 400 mM glycolaldehyde. The reaction was started by the addition of TK. Erythrulose determination. Erythrulose was determined colorimetrically [20] after the removal of protein by perchloric acid from the reaction mixture and, also, from the change in ellipticity, using the molar coefficient of CD absorption of erythrulose at 278 nm ¼ 0.5 M1 cm1 [21]. Circular dichroism spectra. The CD spectra were rerecorded in 50 mM glycyl-glycine, pH 7.6, at 25 °C (using a Mark V, “Jobin Ivon,” France spectropolarimeter; path length of 1 cm), and RDM software with standard parameters for smoothing the spectra.
Results and discussion Binding of ThDP in the transketolase active center is accompanied by emergence in the CD spectrum of the negative absorption band (with a maximum at 315– 320 nm) which was missing from initial components. Its presence in the CD spectrum of holoTK characterizes the holoenzyme as being catalytically active. Upon addition to holoTK of the donor substrate, ketose, the intensity of the negative band is diminished (Fig. 1A, curve 2); the higher the concentration of the added substrate, the greater is the intensity decrease (Fig. 1A, curves 2–4). The decrease of the negative band intensity is caused by formation of the transketolase reaction intermediate, dihydroxyethyl-ThDP, i.e., of the glycolaldehyde residue, covalently bound to ThDP within the holoenzyme (which is also named “active glycolaldehyde”) [11]. The intensity of the band with a maximum at 315–320 nm is likewise diminished upon the addition to holoTK of aldose (glycolaldehyde) (Fig. 1B, curve 2). Of note, the CD difference spectrum is analogous to the difference spectrum of holoTK in the presence of ketose (cf. insets in Figs. 1A and B). This was taken to mean that the transketolase reaction intermediate, dihydroxyethyl-ThDP, may be formed not only after ketose splitting but also upon direct interaction of the holoenzyme with free glycolaldehyde. In the presence of excess glycolaldehyde, the CD spectrum in the 300–360-nm range does not change for a sufficiently long time period while in the 260–290-nm range the gradual increase of its intensity is observed (Fig. 1B, curves 2–6). The latter phenomenon is explained by formation of the optically active compound
Fig. 1. (A) Near ultraviolet CD spectra of holoTK (2.7 lM) (1) and holoTK + xylulose 5-phosphate (2.5, 5, and 12.5 lM, respectively) (2–4). Inset: near ultraviolet difference CD spectrum (holoTK + xylulose 5-phosphate (spectrum 4) minus holoTK (spectrum 1)). (B) CD spectral changes occurring in TK upon addition of glycolaldehyde. ApoTK at a concentration of 3.1 lM was dissolved in 50 mM glycyl-glycine buffer, pH 7.6, containing 2.5 mM CaCl2 and 60 lM ThDP. After scanning the initial CD spectrum (1), to the enzyme solution was added 285 mM glycolaldehyde and the spectra were recorded after 0 (2), 5 (3), 10 (4), 15 (5), and 20 (6) min. Increase of positive ellipticity at 278 nm monitors erythrulose synthesis. Inset: near ultraviolet difference CD spectrum, obtained by subtraction of spectrum 1 (holoTK) from spectrum 2 (holoTK + glycolaldehyde).
Fig. 2. CD spectra of the reaction mixture containing TK, and glycolaldehyde. TK (2.4 lM) was preincubated for 30 min with glycolaldehyde (350 mM) in 50 mM glycyl-glycine buffer, pH 7.6, containing 2.5 mM CaCl2 and 60 lM ThDP. CD spectra were recorded after protein precipitation.
I.A. Sevostyanova et al. / Biochemical and Biophysical Research Communications 313 (2004) 771–774 Table 1 The erythrulose amount formed in the course of incubation of TK with glycolaldehyde (other experimental conditions as in Fig. 2) Time of preincubation of holoTK (10 lM) with glycolaldehyde (2 M), hours
Erythrulose amounts revealed (mM) Colorimetrically
From the change in ellipticity
2 4 20
6 11 13
6.7 10.5 15
773
140 mM and by the Vmax ¼ 0:23 lmol min1 mg1 (data not shown). The rate of the one-substrate transketolase reaction with aldose (glycolaldehyde) as substrate is much lower than the two-substrate transketolase reaction rate with ketose and aldose as substrates (0.23 and 18 U/mg, respectively). In both cases the active glycolaldehyde (dihydroxyethyl-ThDP) is formed as an intermediate. Then the glycolaldehyde residue is transferred from dihydroxyethyl-ThDP to the acceptor substrate. The only difference is that in the second case the intermediate is formed as a result of splitting of donor substrate (ketose) while in the first case the formation of the intermediate occurs through direct interaction of glycolaldehyde with the coenzyme. Thus, the low rate of the one-substrate (compared to the two-substrate) reaction is caused by the lower rate of the intermediate formation.
Acknowledgment This work was supported by grant from the Russian Foundation for Basic Research 03-04-49025. Fig. 3. Kinetics of erythrulose formation by TK at different enzyme concentrations. TK, 0.7 (1), 1.4 (2), and 2.4 (3) lM, was dissolved in 50 mM glycyl-glycine buffer, pH 7.6, containing 2.5 mM CaCl2 and 60 lM ThDP. Reaction was started by addition of 400 mM glycolaldehyde and was monitored via the change in CD signal at 278 nm. Inset: the enzyme concentration dependence of the transketolase reaction rate.
whose spectrum (Fig. 2) is analogous to that of erythrulose [21]. Erythrulose formation upon incubation of holoTK with glycolaldehyde was confirmed by its direct chemical identification. The quantitative determination of erythrulose by two methods (colorimetrically and from the change in ellipticity) gave virtually identical results (Table 1). Thus, it was found that holotransketolase is capable of interacting with glycolaldehyde with formation of dihydroxyethyl-ThDP, a typical transketolase reaction intermediate. The glycolaldehyde residue is then transferred to the free glycolaldehyde where upon erythrulose is formed. The rate of transketolase reaction with the use of glycolaldehyde as substrate is measurable by the increase of ellipticity in the 260–290-nm range—which is determined by formation of the reaction product, erythrulose. The reaction rate remains constant for a sufficiently long time period (Fig. 3) and appears to be proportional to the TK concentration in the range 0.7– 2.4 lM (inset in Fig. 3). Glycolaldehyde-dependent erythrulose production by TK is hyperbolic and is characterized by the Km ¼
References [1] G.A. Kochetov, Transketolase from yeast, rat liver, and pig liver, Methods Enzymol. 90 (1982) 209–223. [2] M. Sundstr€ om, Y. Lindqvist, G. Schneider, U. Hellman, H. Ronne, Yeast TKL1 gene encodes a transketolase that is required for efficient glycolysis and biosynthesis of aromatic amino acids, J. Biol. Chem. 268 (1993) 24346–24352. [3] G.A. Kochetov, L.E. Meshalkina, R.A. Usmanov, The number of active sites in a molecule of transketolase, Biochem. Biophys. Res. Commun. 41 (1970) 1134–1140. [4] L.E. Meshalkina, G.A. Kochetov, The functional identity of the active centers of transketolase, Biochim. Biophys. Acta 571 (1979) 218–223. [5] M. Nikkola, Y. Lindqvist, G. Schneder, Refined structure of resolution, J. transketolase from Saccharomyces cerevisiae at 2.0 A Mol. Biol. 238 (1994) 387–404. [6] G.A. Kochetov, R.A. Usmanov, V.P. Merzlov, Thiaminepyrophosphate induced changes in the optical activity of baker’s yeast transketolase, FEBS Lett. 9 (1970) 265–266. [7] G.A. Kochetov, R.A. Usmanov, Charge transfer interactions in transketolase-thiamine pyrophosphate complex, Biochem. Biophys. Res. Commun. 41 (1970) 1134–1140. [8] R.A. Usmanov, G.A. Kochetov, Interaction of baker’s yeast transketolase with substrates, Biochem. Int. 5 (1982) 727–734. [9] R.A. Usmanov, G.A. Kochetov, Binding of substrates to transketolase from baker’s yeast. Function of the anionic group of the donor substrate, Biokhimiya (in Russian) 48 (1983) 550–558. [10] M.G. Pustynnikov, H. Neef, R.A. Usmanov, A. Schellenberger, G.A. Kochetov, Functional groups of thiamin pyrophosphate in holotransketolase, Biokhimiya (in Russian) 51 (1986) 1003–1016. [11] O.N. Solov’eva, I.A. Bykova, L.E. Meshalkina, M.V. Kovina, G.A. Kochetov, Cleaving of ketosubstrates by transketolase and the nature of the products formed, Biochemistry (Moscow) 66 (2001) 932–936.
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[12] M.V. Kovina, V.A. Selivanov, N.V. Kochevova, G.A. Kochetov, Kinetic mechanism of active site non-equivalence in transketolase, FEBS Lett. 418 (1997) 11–14. [13] C.P. Heinrich, K. Noack, O. Wiss, A circular dichroism study of transketolase from backer’s yeast, Biochem. Biophys. Res. Commun. 44 (1971) 275–279. [14] G.A. Kochetov, R.A. Usmanov, A.T. Mevkh, The role of the charge transfer complex in the transketolase catalyzed reaction, Biochem. Biophys. Res. Commun. 54 (1973) 275–279. [15] I.A. Bykova, O.N. Solov’eva, L.E. Meshalkina, M.V. Kovina, G.A. Kochetov, One-substrate transketolase-catalyzed reaction, Biochem. Biophys. Res. Commun. 280 (2001) 845–847. [16] E. Fiedler, R. Golbik, G. Schneider, K. Tittman, H. Neef, S. K€ onig, G. H€ ubner, Examination of donor substrate conversion in yeast transketolase, J. Biol. Chem. 276 (2001) 16051–16058.
[17] N.K. Tikhomirova, G.A. Kochetov, Purification of transketolase from baker’s yeast by an immunosorbent, Biochem. Int. 22 (1990) 33–36. [18] O.N. Solovjeva, Isolation and properties of noncovalent complex of transketolase with RNA, Biochemistry (Moscow) 67 (2002) 667–671. [19] C.P. Heinrich, K. Noak, O. Wiss, Chemical modification of tryptophan at the binding site of thiamine pyrophosphate in transketolase from baker’s yeast, Biochem. Biophys. Res. Commun. 49 (1972) 1427–1432. [20] K. Morii, S. Hosomi, T. Terada, T. Mizoguchi, Methods for enzymatic and colorimetric determinations of D -erythrulose (D -tetrulose), Anal. Biochem. 151 (1985) 188–191. [21] G.A. Kochetov, R.A. Usmanov, A.T. Mevkh, A new method of determination of transketolase activity by asymmetric synthesis reaction, Anal. Biochem. 88 (1978) 296–301.
A hitherto unknown transketolase—catalyzed reactionq Irina A. Sevostyanova, Olga N. Solovjeva, and German A. Kochetov* A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119992 Moscow, GSP-2, Russia Received 25 November 2003
Abstract Yeast transketolase, in addition to catalyzing the transferase reaction through utilization of two substrates—the donor substrate (ketose) and the acceptor substrate (aldose)—is also able to catalyze a one-substrate reaction with only aldose (glycolaldehyde) as substrate. The interaction of glycolaldehyde with holotransketolase results in formation of the transketolase reaction intermediate, dihydroxyethyl-thiamin diphosphate. Then the glycolaldehyde residue is transferred from dihydroxyethyl-thiamin diphosphate to free glycolaldehyde. As a result, the one-substrate transketolase reaction product, erythrulose, is formed. The specific activity of transketolase was found to be 0.23 U/mg and the apparent Km for glycolaldehyde was estimated as 140 mM. Ó 2003 Published by Elsevier Inc. Keywords: Transketolase; One-substrate reaction; Thiamin diphosphate; Circular dichroism
Transketolase (EC 2.2.1.1) catalyzes the cleavage of a carbon–carbon bond adjacent to a carbonyl group in ketose and transfer of a two-carbon unit, a glycolaldehyde residue, to aldose [1]. The enzyme can utilize sugars such as D -xylulose 5-phosphate, D -fructose 6-phosphate, and D -sedoheptulose 7-phosphate, as well as hydroxypyruvate as donors of the transferred glycolaldehyde group. As acceptor substrates serve D -ribose 5-phosphate, D -glyceraldehyde 3-phosphate, D -erythrose 4-phosphate, and glycolaldehyde. Transketolase plays an important part in the rearrangement system, since it creates, together with transaldolase, a reversible link between the pentose phosphate pathway and glycolysis. Transketolase from the Saccharomyces cerevisiae is a homodimer with a molecular mass of 148.4 kDa [2], and it has two active sites of equal catalytic activity [3–5]. Its cofactors are thiamin diphosphate and divalent metal ions. Thiamin diphosphate binds at the interface between the subunits and interacts with residues from both subunits [5]. Interaction of ThDP with apotransketolase results in the appearance of a new band in the circular dichroism (CD) spectrum with a maximum at 315– 320 nm, this band being absent from the spectra of the q
Abbreviations: TK, transketolase; ThDP, thiamin diphosphate. Corresponding author. Fax: +7-095-939-3181. E-mail address: [email protected] (G.A. Kochetov). *
0006-291X/$ - see front matter Ó 2003 Published by Elsevier Inc. doi:10.1016/j.bbrc.2003.11.164
initial compounds [6,7]. The intensity of the band strictly correlates to the amount of the catalytically active holoenzyme and depends on the substrate concentrations [8–11]. This enables to study the interaction of coenzyme with apotransketolase [4,12] as well as to separate the investigation of the first and second steps of the transketolase reaction—the binding and cleavage of the donor substrate and transfer of the two-carbon fragment to the acceptor substrate, respectively [8,11,13–16]. Transketolase, while being a typical transferase, requires the presence of two substrates, ketose and aldose, for its action. Apart from that, the enzyme, as has recently been shown, is able to effect the catalytic transformation of ketose even in the absence of aldose, i.e., to catalyze the so-called one-substrate transketolase reaction [15,16]. In this work the possibility of aldose (glycolaldehyde) transformation by transketolase from S. cerevisiae was studied by CD spectroscopy and kinetic measurements.
Materials and methods Materials. Thiamin diphosphate, glycyl-glycine, and glycolaldehyde were from Serva; Sephadex G-50 was from Pharmacia (Sweden); and other chemicals were of the highest quality commercially available. Transketolase purification. Transketolase was isolated from the S. cerevisiae according to the method described earlier [17] with some
772
I.A. Sevostyanova et al. / Biochemical and Biophysical Research Communications 313 (2004) 771–774
modifications [18]. The crystalline enzyme was stored at 4 °C in ammonium sulfate solution of 0.5 saturation, pH 7.6. Transketolase (specific activity in transferase reaction, 18 U/mg) was homogeneous by SDS–PAGE. TK concentration was determined using the absor1% bance coefficient E280 ¼ 14:5 [19]. Prior to use, the TK solution was passed through a Sephadex G-50 column to remove ammonium sulfate. TK activity. The enzyme’s catalytic activity in the transferase reaction was measured with xylulose 5-phosphate and ribose 5-phosphate as substrates [1]. The enzyme’s catalytic activity in the onesubstrate reaction was measured with glycolaldehyde as substrate from the change in ellipticity at 278 nm (Mark V, “Jobin Ivon,” France, spectropolarimeter; path length 1 cm). The reaction mixture (final volume 1 ml) contained: 50 mM glycyl-glycine, pH 7.6, 2.5 mM CaCl2 , 60 lM ThDP, and 400 mM glycolaldehyde. The reaction was started by the addition of TK. Erythrulose determination. Erythrulose was determined colorimetrically [20] after the removal of protein by perchloric acid from the reaction mixture and, also, from the change in ellipticity, using the molar coefficient of CD absorption of erythrulose at 278 nm ¼ 0.5 M1 cm1 [21]. Circular dichroism spectra. The CD spectra were rerecorded in 50 mM glycyl-glycine, pH 7.6, at 25 °C (using a Mark V, “Jobin Ivon,” France spectropolarimeter; path length of 1 cm), and RDM software with standard parameters for smoothing the spectra.
Results and discussion Binding of ThDP in the transketolase active center is accompanied by emergence in the CD spectrum of the negative absorption band (with a maximum at 315– 320 nm) which was missing from initial components. Its presence in the CD spectrum of holoTK characterizes the holoenzyme as being catalytically active. Upon addition to holoTK of the donor substrate, ketose, the intensity of the negative band is diminished (Fig. 1A, curve 2); the higher the concentration of the added substrate, the greater is the intensity decrease (Fig. 1A, curves 2–4). The decrease of the negative band intensity is caused by formation of the transketolase reaction intermediate, dihydroxyethyl-ThDP, i.e., of the glycolaldehyde residue, covalently bound to ThDP within the holoenzyme (which is also named “active glycolaldehyde”) [11]. The intensity of the band with a maximum at 315–320 nm is likewise diminished upon the addition to holoTK of aldose (glycolaldehyde) (Fig. 1B, curve 2). Of note, the CD difference spectrum is analogous to the difference spectrum of holoTK in the presence of ketose (cf. insets in Figs. 1A and B). This was taken to mean that the transketolase reaction intermediate, dihydroxyethyl-ThDP, may be formed not only after ketose splitting but also upon direct interaction of the holoenzyme with free glycolaldehyde. In the presence of excess glycolaldehyde, the CD spectrum in the 300–360-nm range does not change for a sufficiently long time period while in the 260–290-nm range the gradual increase of its intensity is observed (Fig. 1B, curves 2–6). The latter phenomenon is explained by formation of the optically active compound
Fig. 1. (A) Near ultraviolet CD spectra of holoTK (2.7 lM) (1) and holoTK + xylulose 5-phosphate (2.5, 5, and 12.5 lM, respectively) (2–4). Inset: near ultraviolet difference CD spectrum (holoTK + xylulose 5-phosphate (spectrum 4) minus holoTK (spectrum 1)). (B) CD spectral changes occurring in TK upon addition of glycolaldehyde. ApoTK at a concentration of 3.1 lM was dissolved in 50 mM glycyl-glycine buffer, pH 7.6, containing 2.5 mM CaCl2 and 60 lM ThDP. After scanning the initial CD spectrum (1), to the enzyme solution was added 285 mM glycolaldehyde and the spectra were recorded after 0 (2), 5 (3), 10 (4), 15 (5), and 20 (6) min. Increase of positive ellipticity at 278 nm monitors erythrulose synthesis. Inset: near ultraviolet difference CD spectrum, obtained by subtraction of spectrum 1 (holoTK) from spectrum 2 (holoTK + glycolaldehyde).
Fig. 2. CD spectra of the reaction mixture containing TK, and glycolaldehyde. TK (2.4 lM) was preincubated for 30 min with glycolaldehyde (350 mM) in 50 mM glycyl-glycine buffer, pH 7.6, containing 2.5 mM CaCl2 and 60 lM ThDP. CD spectra were recorded after protein precipitation.
I.A. Sevostyanova et al. / Biochemical and Biophysical Research Communications 313 (2004) 771–774 Table 1 The erythrulose amount formed in the course of incubation of TK with glycolaldehyde (other experimental conditions as in Fig. 2) Time of preincubation of holoTK (10 lM) with glycolaldehyde (2 M), hours
Erythrulose amounts revealed (mM) Colorimetrically
From the change in ellipticity
2 4 20
6 11 13
6.7 10.5 15
773
140 mM and by the Vmax ¼ 0:23 lmol min1 mg1 (data not shown). The rate of the one-substrate transketolase reaction with aldose (glycolaldehyde) as substrate is much lower than the two-substrate transketolase reaction rate with ketose and aldose as substrates (0.23 and 18 U/mg, respectively). In both cases the active glycolaldehyde (dihydroxyethyl-ThDP) is formed as an intermediate. Then the glycolaldehyde residue is transferred from dihydroxyethyl-ThDP to the acceptor substrate. The only difference is that in the second case the intermediate is formed as a result of splitting of donor substrate (ketose) while in the first case the formation of the intermediate occurs through direct interaction of glycolaldehyde with the coenzyme. Thus, the low rate of the one-substrate (compared to the two-substrate) reaction is caused by the lower rate of the intermediate formation.
Acknowledgment This work was supported by grant from the Russian Foundation for Basic Research 03-04-49025. Fig. 3. Kinetics of erythrulose formation by TK at different enzyme concentrations. TK, 0.7 (1), 1.4 (2), and 2.4 (3) lM, was dissolved in 50 mM glycyl-glycine buffer, pH 7.6, containing 2.5 mM CaCl2 and 60 lM ThDP. Reaction was started by addition of 400 mM glycolaldehyde and was monitored via the change in CD signal at 278 nm. Inset: the enzyme concentration dependence of the transketolase reaction rate.
whose spectrum (Fig. 2) is analogous to that of erythrulose [21]. Erythrulose formation upon incubation of holoTK with glycolaldehyde was confirmed by its direct chemical identification. The quantitative determination of erythrulose by two methods (colorimetrically and from the change in ellipticity) gave virtually identical results (Table 1). Thus, it was found that holotransketolase is capable of interacting with glycolaldehyde with formation of dihydroxyethyl-ThDP, a typical transketolase reaction intermediate. The glycolaldehyde residue is then transferred to the free glycolaldehyde where upon erythrulose is formed. The rate of transketolase reaction with the use of glycolaldehyde as substrate is measurable by the increase of ellipticity in the 260–290-nm range—which is determined by formation of the reaction product, erythrulose. The reaction rate remains constant for a sufficiently long time period (Fig. 3) and appears to be proportional to the TK concentration in the range 0.7– 2.4 lM (inset in Fig. 3). Glycolaldehyde-dependent erythrulose production by TK is hyperbolic and is characterized by the Km ¼
References [1] G.A. Kochetov, Transketolase from yeast, rat liver, and pig liver, Methods Enzymol. 90 (1982) 209–223. [2] M. Sundstr€ om, Y. Lindqvist, G. Schneider, U. Hellman, H. Ronne, Yeast TKL1 gene encodes a transketolase that is required for efficient glycolysis and biosynthesis of aromatic amino acids, J. Biol. Chem. 268 (1993) 24346–24352. [3] G.A. Kochetov, L.E. Meshalkina, R.A. Usmanov, The number of active sites in a molecule of transketolase, Biochem. Biophys. Res. Commun. 41 (1970) 1134–1140. [4] L.E. Meshalkina, G.A. Kochetov, The functional identity of the active centers of transketolase, Biochim. Biophys. Acta 571 (1979) 218–223. [5] M. Nikkola, Y. Lindqvist, G. Schneder, Refined structure of resolution, J. transketolase from Saccharomyces cerevisiae at 2.0 A Mol. Biol. 238 (1994) 387–404. [6] G.A. Kochetov, R.A. Usmanov, V.P. Merzlov, Thiaminepyrophosphate induced changes in the optical activity of baker’s yeast transketolase, FEBS Lett. 9 (1970) 265–266. [7] G.A. Kochetov, R.A. Usmanov, Charge transfer interactions in transketolase-thiamine pyrophosphate complex, Biochem. Biophys. Res. Commun. 41 (1970) 1134–1140. [8] R.A. Usmanov, G.A. Kochetov, Interaction of baker’s yeast transketolase with substrates, Biochem. Int. 5 (1982) 727–734. [9] R.A. Usmanov, G.A. Kochetov, Binding of substrates to transketolase from baker’s yeast. Function of the anionic group of the donor substrate, Biokhimiya (in Russian) 48 (1983) 550–558. [10] M.G. Pustynnikov, H. Neef, R.A. Usmanov, A. Schellenberger, G.A. Kochetov, Functional groups of thiamin pyrophosphate in holotransketolase, Biokhimiya (in Russian) 51 (1986) 1003–1016. [11] O.N. Solov’eva, I.A. Bykova, L.E. Meshalkina, M.V. Kovina, G.A. Kochetov, Cleaving of ketosubstrates by transketolase and the nature of the products formed, Biochemistry (Moscow) 66 (2001) 932–936.
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[12] M.V. Kovina, V.A. Selivanov, N.V. Kochevova, G.A. Kochetov, Kinetic mechanism of active site non-equivalence in transketolase, FEBS Lett. 418 (1997) 11–14. [13] C.P. Heinrich, K. Noack, O. Wiss, A circular dichroism study of transketolase from backer’s yeast, Biochem. Biophys. Res. Commun. 44 (1971) 275–279. [14] G.A. Kochetov, R.A. Usmanov, A.T. Mevkh, The role of the charge transfer complex in the transketolase catalyzed reaction, Biochem. Biophys. Res. Commun. 54 (1973) 275–279. [15] I.A. Bykova, O.N. Solov’eva, L.E. Meshalkina, M.V. Kovina, G.A. Kochetov, One-substrate transketolase-catalyzed reaction, Biochem. Biophys. Res. Commun. 280 (2001) 845–847. [16] E. Fiedler, R. Golbik, G. Schneider, K. Tittman, H. Neef, S. K€ onig, G. H€ ubner, Examination of donor substrate conversion in yeast transketolase, J. Biol. Chem. 276 (2001) 16051–16058.
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