Active Glycolaldehyde Research Articles

The mechanism of a one-substrate transketolase reaction.

Transketolase catalyzes the transfer of a glycolaldehyde residue from ketose (the donor substrate) to aldose (the acceptor substrate). In the absence of aldose, transketolase catalyzes a one-substrate reaction that involves only ketose. The mechanism of this reaction is unknown. Here, we show that hydroxypyruvate serves as a substrate for the one-substrate reaction and, as well as with the xylulose-5-phosphate, the reaction product is erythrulose rather than glycolaldehyde. The amount of erythrulose released into the medium is equimolar to a double amount of the transformed substrate. This could only be the case if the glycol aldehyde formed by conversion of the first ketose molecule (the product of the first half reaction) remains bound to the enzyme, waiting for condensation with the second molecule of glycol aldehyde. Using mass spectrometry of catalytic intermediates and their subsequent fragmentation, we show here that interaction of the holotransketolase with hydroxypyruvate results in the equiprobable binding of the active glycolaldehyde to the thiazole ring of thiamine diphosphate and to the amino group of its aminopyrimidine ring. We also show that these two loci can accommodate simultaneously two glycolaldehyde molecules. It explains well their condensation without release into the medium, which we have shown earlier.

Examination of Donor Substrate Conversion in Yeast Transketolase

The cleavage of the donor substrate d-xylulose 5-phosphate by wild-type and H263A mutant yeast transketolase was studied using enzyme kinetics and circular dichroism spectroscopy. The enzymes are able to catalyze the cleavage of donor substrates, the first half-reaction, even in the absence of any acceptor substrate yielding d-glyceraldehyde 3-phosphate as measured in the coupled optical test according to Kochetov (Kochetov, G. A. (1982) Methods Enzymol. 90, 209-223) and compared with the H263A variant. Overall, the H263A mutant enzyme is less active than the wild-type. However, an increase in the rate constant of the release of the enzyme-bound glycolyl moiety was observed and related to a stabilization of the "active glycolaldehyde" (alpha-carbanion) by histidine 263. Chemically synthesized dl-(alpha,beta-dihydroxyethyl)thiamin diphosphate is bound to wild-type transketolase with an apparent K(D) of 4.3 +/- 0.8 microm (racemate) calculated from titration experiments using circular dichroism spectroscopy. Both enantiomers are cleaved by the enzyme at different rates. In contrast to the enzyme-generated alpha-carbanion of (alpha,beta-dihydroxyethyl)thiamin diphosphate formed by decarboxylation of hydroxylactylthiamin diphosphate after incubation of transketolase with beta-hydroxypyruvate, the synthesized dl-(alpha,beta-dihydroxyethyl)thiamin diphosphate did not work as donor substrate when erythrose 4-phosphate is used as acceptor substrate in the coupled enzymatic test according to Sprenger (Sprenger, G. A., Schörken, U., Sprenger, G., and Sahm, H. (1995) Eur. J. Biochem. 230, 525-532).

Effect of ring substituents on the transketolase-catalyzed conversion of nitroso aromatics to hydroxamic acids

Transketolase catalyzed the conversion of eight different aromatic C-nitroso compounds into the corresponding N-glycolyl derived hydroxamic acids. Three of the nitroso compounds were also found to be converted in part to the arylhydroxylamines by a reductive process. A correlation was found for the rates of production of these metabolites with the electronegatives of substituent groups that were present on the aromatic ring. The rates of reaction of these substituted nitroso substrates with transketolase and d-fructose-6-phosphate were found to decrease in the order 4-NO 2 > 4-CF 3 > 3-CF 3, unsubstituted > 4-Cl > 4-CH 3, 4-phenyl > 4-OC 2H 5. N, N-Dimethyl- p-nitrosoaniline was not metabolized by transketolase under the conditions employed for the other substrates. Those substrates possessing the strong electron-withdrawing groups 4-NO 2, 4-CF 3 and 3-CF 3 were the only substrates that were found to undergo enzymatic reduction to the hydroxylamines as a competing process. A mechanism was proposed that involves a redox reaction between the nitroso substrate and the enzymatic intermediate “active glycolaldehyde” at the active-site of transketolase.

Competition by 4-chloronitrosobenzene for the active glycolaldehyde intermediate of transketolase

The incubation of 4-chloronitrosobenzene with yeast transketolase, Mg 2+, and thiamime pyrophosphate in the presence of excess xylulose-5-phosphate resulted in the formation of N-(4-chlorophenyl)glycolhydroxamic acid. This enzyme-catalyzed C 2 transfer displayed a K m of 0.92 m M and a V max of 5.2 × 10 −2 μmol min −1 unit enzyme −1. Conversion was inhibited by the normal acceptor sugar, ribose-5-phosphate, with a K i of 0.35 m M. Kinetic analysis showed inhibition was competitive in nature, reinforcing the proposed theory for similarity in catalytic formation of both the hydroxamic acid and sedoheptulose-7-phosphate. Most interesting about the conversion of this alternative substrate is that even at high concentrations of ribose-5-phosphate, a significant amount of the nitroso compound was converted to the hydroxamic acid, implying that 4-chloronitrosobenzene can successfully compete for active glycoaldehyde. Using the yeast enzyme as a model for transketolase in higher organisms, the adventitious conversion of such xenobiotics in vivo is proposed.

Glycolate synthesis by intact chloroplasts: Studies with inhibitors of photophosphorylation

Intact spinach chloroplasts, capable of evolving O 2 in response to CO 2 at rates greater than 70 μmol/h · mg of chlorophyll, synthesize glycolate from added dihydroxyacetone phosphate, ribose 5-phosphate, or xylulose 5-phosphate, when illuminated in the presence of O 2. The synthesis of glycolate from these compounds is dependent upon photophosphorylation and is inhibited by each of the three classes of photophosphorylation inhibitors [Izawa, S., and Good, N. E. (1972) in Methods in Enzymology, Vol. 24, Part B, pp. 355–377)]: an uncoupler, carbonylcyanide-4-trifluoromethoxyphenylhydrazone (FCCP), an energy transfer inhibitor, Dio-9, and a phosphate analog, arsenate. Neither arsenate nor Dio-9 causes the collapse of the light-generated proton gradient between thylakoid and stroma compartments of the chloroplasts, so that the inhibition of glycolate synthesis by these compounds cannot be ascribed to an inactivation of Calvin cycle enzymes thought to be associated with the collapse of such a proton gradient. The dependency of glycolate synthesis upon photophosphorylation indicates that an ATP-consuming reaction(s) is involved in the conversion of the substrates (triose and pentose monophosphates) to glycolate. The formation of dihydroxyethylthiamine pyrophosphate, the “active glycolaldehyde” intermediate of the transketolase reaction, from triose and pentose monophosphates has no known requirements for ATP. On the other hand, the conversion of both triose and pentose monophosphates to ribulose 1,5-bisphosphate, the substrate for the ribulose 1,5-bisphosphate oxygenase reaction, requires ATP. It is concluded that glycolate synthesis by intact isolated chloroplasts is primarily the result of ribulose 1,5-bisphosphate oxygenase activity. No substantial role in glycolate synthesis can be attributed to the oxidation of dihydroxyethylthiamine pyrophosphate, the intermediate of the transketolase reaction.

The urinary excretion of glycollic acid and threonic acid by xylitol-infused patients and their relationship to the possible role of 'active glycoladehyde' in the transketolase reaction in vivo.

Xylitol has been used in several countries as a parenteral nutrient. Adverse reactions to intravenous xylitol infusions have been reported with calcium oxalate crystal deposition within the walls of the intracranial arteries and extensively in the kidneys (Evans et al., 1973; Schroder et al., 1974). Similar but more widespread calcium oxalate deposits occur in primary hyperoxaluria (Scowen et al., 1959). The urinary excretion of organic acids by xylitol-infused patients was examined and compared to that by glucose-infused patients, in order to define any alterations that might be related to the organic acidurias observed in patients with primary hyperoxaluria types I and I1 (hyperoxaluria with hyperglycollic aciduria and hyperglyoxylic aciduria, and hyperoxaluria with hyper-Lglyceric aciduria respectively). Quantitative extraction and g.l.c., and g.1.c.-mass-spectrometric methods were used. Four out of five of the xylitol-infused patients showed grossly increased excretions of glycollic acid and of threonic acid [2,3,4trihydroxy(threo)butanoic acid]. There was no detectable increase in the excretion of oxalic acid, glyoxylic acid, or glyceric acid. S. Hauschildt, R. A. Chalmers, A. M. Lawson, K., Schulthis & R. W. E. Watts (unpublished work) found that xylitol infusion did not increase blood oxalate concentrations. Xylitol is metabolized in man to D-XylUlOSe, D-xylulose Sphosphate, and thence to glucose via the pentose phosphate pathway. Transketolase (sedoheptulose 7-phosphateD-glyceraldehyde 3-phosphate glycolaldehydetransferase, EC 2.2.1 .l) intervenes twice in the metabolism of pentose phosphates, once to form glyceraldehyde 3-phosphate and sedoheptulose 7-phosphate from xylulose 5-phosphate and ribose 5-phosphate, and again to form fructose 6-phosphate and glyceraldehyde 3-phosphate from another molecule of xylulose 5-phosphate and erythrose 4-phosphate (Fig. 1). In these reactions the enzyme catalyses the stereospecific reversible transfer of a glycolaldehyde moiety from a donor ketose to an acceptor aldose. Besides xylulose Sphosphate, suitable donors include hydroxypyruvate, L-erythrulose, D-fructose Gphosphate and D-sedoheptulose 7-phosphate. Suitable acceptors include, in addition to D-ribose 5-phosphate and D-erythrose +phosphate, glycolaldehyde (-+ L-erythrulose), D-glyceraldehyde 3-phosphate (-+ D-XylUlOSe 5-phosphate) and formaldehyde (-+ dihydroxyacet one).

A circular dichroism study of transketolase from baker's yeast

The circular dichroism of transketolase from baker's yeast was examined in the 400 to 200 nm region in the presence and absence of its coenzyme and substrates. Coenzyme binding caused an extrinsic Cotton effect v pyrophosphate and a tryptophan moiety of the enzyme protein. Addition of donor substrates as D-fructose-6-phosphate and hydroxy-pyruvate abolished the ellipticity band at 235 nm. Steric hindrance might be responsible for the fact that 2-(1,2-dihydroxyethyl) thiamine pyrophosphate (“active glycolaldehyde”) can no longer form a charge transfer complex. On the other hand, acceptor substrates in the transketolase reaction, such as ribose-5-phosphate, did not influence the broad negative dichroism at 325 nm.

Zum wirkungsmechanismus der phosphoketolase I. Oxydation verschiedener substrate mit ferricyanid

1. 1. Purified phosphoketolase from Lactobacillus plantarum reduces ferricyanide with pentose 5-phosphate (probably xylulose 5-phosphate), hydroxypyruvate, and fructose 6-phosphate as substrates. The rates of the reaction with these three substrates are in the ratio of 100 : 56 : 14, respectively. As product of the oxidation one mole of glycolic acid is formed for every two moles of ferricyanide. 2. 2. In the presence of orthophosphate all three substrates yield acetyl phosphate. The rates of these reactions are the same as those of the ferricyanide reduction when compared on the basis of one mole acetyl phosphate being equal to two moles of ferrocyanide. 3. 3. The reduction of ferricyanide catalyzed by phosphoketolase is just as thiamine pyrophosphate-dependent as the formation of acetyl phosphate. Evidence is presented for the first intermediate, common to both reactions, being “active glycoladehyde” [2-(α,β-dihydroxyethyl)thiamine pyrophosphate]. Other intermediates and the kinetics of both reactions are discussed. A dehydration of “active glycolaldehyde” to α-hydroxyvinyl thiamine pyrophosphate is proposed to be an intermediate step in the formation of acetyl phosphate, and is discussed by analogy with known similar reactions. 4. 4. The dependence of the rate of ferricyanide reduction on the concentration of the substrates, ferricyanide, thiamine pyrophosphate, and on the pH is described.

A thiamine pyrophosphate-glycolaldehyde compound (“active glycolaldehyde”) as intermediate in the transketolase reaction

A thiamine pyrophosphate-glycolaldehyde compound (“active glycolaldehyde”) as intermediate in the transketolase reaction

Formaldehyde as an Acceptor Aldehyde for Transketolase, and the Biosynthesis of Triose

THE enzyme transketolase is biologically widely distributed, and, in presence of thiamine pyrophosphate and magnesium ions, transfers the ketol group or ‘active glycolaldehyde’ (CH2OH.CO…H) from a variety of ketose phosphates (sedoheptulose 7-phosphate, D-fructose 6-phosphate, D-xylulose 5-phosphate), from certain free ketoses (L-erythrulose, D-xylulose) and from hydroxypyruvic acid to certain ‘acceptor aldehydes’, thus synthesizing new ketoses. The ‘acceptor aldehydes’ hitherto recognized are D-glyceraldehyde 3-phosphate, D-ribose 5-phosphate, D-erythrose 4-phosphate and 2-deoxyribose 5-phosphate; also free glyceraldehyde and glycolaldehyde (for reviews see refs. 1–3). Acetaldehyde and formaldehyde are stated not to be acceptors4, but the former appears in our preliminary tests to be weakly active.