Pyrithiamine as a substrate for thiamine pyrophosphokinase

Thiamine pyrophosphokinase transfers a pyrophosphate group from a nucleoside triphosphate, such as ATP, to the hydroxyl group of thiamine to produce thiamine pyrophosphate. Deficiencies in thiamine can result in the development of the neurological disorder Wernicke-Korsakoff Syndrome as well as the potentially fatal cardiovascular disease wet beriberi. Pyrithiamine is an inhibitor of thiamine metabolism that induces neurological symptoms similar to that of Wernicke-Korsakoff Syndrome in animals. However, the mechanism by which pyrithiamine interferes with cellular thiamine phosphoester homeostasis is not entirely clear. We used kinetic assays coupled with mass spectrometry of the reaction products and x-ray crystallography of an equilibrium reaction mixture of thiamine pyrophosphokinase, pyrithiamine, and Mg2+/ATP to elucidate the mechanism by which pyrithiamine inhibits the enzymatic production of thiamine pyrophosphate. Three lines of evidence support the ability of thiamine pyrophosphokinase to form pyrithiamine pyrophosphate. First, a coupled enzyme assay clearly demonstrated the ability of thiamine pyrophosphokinase to produce AMP when pyrithiamine was used as substrate. Second, an analysis of the reaction mixture by mass spectrometry directly identified pyrithiamine pyrophosphate in the reaction mixture. Last, the structure of thiamine pyrophosphokinase crystallized from an equilibrium substrate/product mixture shows clear electron density for pyrithiamine pyrophosphate bound in the enzyme active site. This structure also provides the first clear picture of the binding pocket for the nucleoside triphosphate and permits the first detailed understanding of the catalytic requirements for catalysis in this enzyme.     

A positive regulatory gene, THI3, is required for thiamine metabolism in Saccharomyces cerevisiae.

We have isolated a thiamine auxotrophic mutant carrying a recessive mutation which lacks the positive regulatory gene, THI3, which differs in the regulation of thiamine transport from the THI2 (PHO6) gene described previously (Y. Kawasaki, K. Nosaka, Y. Kaneko, H. Nishimura, and A. Iwashima, J. Bacteriol. 172:6145-6147, 1990) for expression of thiamine metabolism in Saccharomyces cerevisiae. The mutant (thi3) had a markedly reduced thiamine transport system as well as reduced activity of thiamine-repressible acid phosphatase and of several enzymes for thiamine synthesis from 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-beta-hydroxyethylthiazole. These results suggest that thiamine metabolism in S. cerevisiae is subject to two positive regulatory genes, THI2 (PHO6) and THI3. We have also isolated a hybrid plasmid, pTTR1, containing a 6.2-kb DNA fragment from an S. cerevisiae genomic library which complements thiamine auxotrophy in the thi3 mutant. This gene was localized on a 3.0-kb ClaI-BglII fragment in the subclone pTTR5. Complementation of the activities for thiamine metabolism in the thi3 mutant transformed by some plasmids with the THI3 gene was also examined.     

Phosphorylation of some thiamine analogs by yeast thiamine pyrophosphokinase]

A rapid efficient method of separation of the thiamine pyrophosphokinase reaction products (ATP: thiamine pyrophosphotransferase) on the column packed with DEAE-Sephadex A-25 and their subsequent identification by direct spectrophotometry is suggested. Phosphorylation of some thiamine analogs substituted at the second position of the pyrimidine ring was studied. It was shown that in addition to thiamine, the enzyme transfers the pyrophosphate group to some of its derivatives. The vitamin analogs devoid of quaternary nitrogen in the thiazole cycle, do not form pyrophosphate ethers (thus being unable to act as substrates), whereas 2'-phenoxythiamine, 2'-methoxythiamine and especially 2'-phenylthiamine are phosphorylated at a greater rate than does the "true" substrate, thiamine, under similar conditions.     

Pathway of thiamine pyrophosphate synthesis in Micrococcus denitrificans.

The pathway of thiamine pyrophosphate (TPP) biosynthesis, which is formed either from exogeneously added thiamine or from the pyrimidine and thiazole moieties of thiamine, in Micrococcus denitrificans was investigated. The following indirect evidence shows that thiamine pyrophosphokinase (EC 2.7.6.2) catalyzes the synthesis of TPP from thiamine: (i) [35S]thiamine incubated with cells of this microorganism was detected in the form of [35S]thiamine; (ii) thiamine gave a much faster rate of TPP synthesis than thiamine monophosphate (TMP) when determined with the extracts; and (iii) a partially purified preparation of the extracts can use thiamine, but not TMP, as the substrate. The activities of the four enzymes involved in TMP synthesis from pyrimidine and thiazole moieties of thiamine were detected in the extracts of M. denitrificans. The extracts contained a high activity of the phosphatase, probably specific for TMP. After M. denitrificans cells were grown on a minimal medium containing 3 mM adenosine, which causes derepression of de novo thiamine biosynthesis in Escherichia coli, the activities of the four enzymes involved with TMP synthesis, the TMP phosphatase, and the thiamine pyrophosphokinase were enhanced two- to threefold. These results indicate that TPP is synthesized directly from thiamine without forming TMP as an intermediate and that de novo synthesis of TPP from the pyrimidine and thiazole moieties involves the formation of TMP, followed by hydrolysis to thiamine, which is then converted to TPP directly. Thus, the pathway of TPP synthesis from TMP synthesized de novo in M. denitrificans is different from that found in E. coli, in which TMP synthesized de novo is converted directly to TPP without producing thiamine.     

Enzymatic conversion of the antibiotic metronidazole to an analog of thiamine

We propose that adverse effects of the antibiotic metronidazole may be due, wholly or in part, to its conversion to a thiamine analog and consequent vitamin B1 antagonism. Consistent with this hypothesis, the drug is accepted as a substrate for the thiaminase (EC 2.5.1.2) elaborated as an exoenzyme by the human gut flora constituent Bacillus thiaminolyticus and is also a substrate for the intracellular thiaminase of the mollusk Venus mercenaria. The product, identified as the 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-hydroxyethyl)-2-methyl-4 - nitroimidazolium cation, is a close structural analog of thiamine and is an effective inhibitor of thiamine pyrophosphokinase in vitro. Due to its susceptibility to nucleophilic attack, the analog is unstable, releasing inorganic nitrite under mild conditions. Enzymatic alkylation reactions such as that effected by thiaminase may have general pharmacological significance as a route of increasing the electrophilicity and/or reduction potential of drugs which are heterocyclic weak bases.     

A rapid and sensitive radiometric assay procedure for thiamine pyrophosphokinase activity using anion-exchange paper discs

A radiochemical method for the direct measurement of thiamine pyrophosphokinase (ATP: thiamine pyrophosphotransferase, EC 2.7.6.2) activity was described earlier (1,2). It avoided the difficulties associated with assay systems based on the coenzyme nature of thiamine pyrophosphate in TPP-dependent¹ enzyme reactions using apopyruvate decarboxylase (3) (2-oxoacid carboxylase, EC 4.1.1.1) or apotransketolase (4) (sedoheptulose-7-phosphate: d-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1). Since the chromatographic isolation of TPP is time-consuming, a procedure for the rapid determination of thiamine pyrophosphokinase activity was desirable.The simplified method described here takes advantage of the anionic character of TPP. The assay is carried out with [¹⁴C]thiamine as substrate. After incubation with the enzyme in the presence of Mg²⁺-ATP, the reaction mixture is applied to a DEAE-cellulose paper disc. The disc is extensively washed with sodium acetate resulting in the quantitative elution of [¹⁴C]thiamine and partial retention of [¹⁴C]TPP. This is quantitatively measured using the liquid scintillation counting technique.A similar procedure has been described for the determination of glycerol kinase (ATP: glycerol phosphotransferase, EC 2.7.1.30) and hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) activities (5).     

Mechanisms of rat liver thiamine pyrophosphokinase activation by magnesium ions]

The role of Mg2+ in the activation of thiamine pyrophosphokinase from rat liver was studied. The dependence of the thiamine pyrophosphokinase reaction rate on total and free ATP concentrations suggests that the role of the metal comes down to optimization of conditions of the active enzyme-substrate complex formation due to incorporation of the reactions of Mg2+ and ATP-4 consecutive acception, the affinity of the latter for the enzyme being higher than that of Mg-ATP-2. The kinetic parameters of the reaction were determined. In the absence of ATP-4 free Mg2+ ions were shown to compete with the Mg-ATP-2 complex (Ki = 18 . 10(-3) M). Thiamine pyrophosphokinase is also inhibited by free ATP-4 with Ki = 3 . 10(-3) M.     

Molecular-kinetic parameters of thiamine enzymes and the mechanism of antivitamin action of hydroxythiamine in animal organisms]

The molecula-kinetic parameters (Km, Ki) of three thiamine enzymes, e. g. thiamine pyrophosphokinase (EC 2.7.6.2), pyruvate dehydrogenase (EC 1.2.4.1) and transketolase (EC 2.2.1.1) with respect to the effects of the thiamine antimetabolite hydroxythiamine in the whole animal organism have been compared. It has been shown that only the first two enzymes, which interact competitively with the vitamin, antivitamin or their pyrophosphate ethers, obey the kinetic parameters obtained for the purified enzymes in vitro. The anticoenzymic effect of hydroxythiamine pyrophosphate with respect to transketolase is not observed in vivo at maximal concentration of the anticoenzyme in tissues due to the absence of competitive interactions with thiamine pyrophosphate. The incorporation of the true and false coenzymes into transketolase occurs only during de novo transketolase synthesis (the apoform is absent in tissues, with the exception of erythrocytes) and proceeds slowly with a half-life time equal to 24--30 hrs. After a single injection of hydroxythiamine at a large dose (70--400 mg/kg) the maximal inhibition of the transketolase activity in tissues (liver, heart, kidney, muscle, spleen, lungs adrenal grands) manifests itself by the 48th--72nd hour, when the concentration of free hydroxythiamine and its pyrophosphate is minimal and the whole anticoenzyme is tightly bound to the protein, forming the false holoenzyme. The use of hydroxythiamine for inhibition of pyruvate dehydrogenase or transketolase in animal organism is discussed.     

The role of bound thiamine pyrophosphate in the synthesis of thiamine triphosphate in rat liver.

Thiamine pyrophosphate-ATP phosphoryltransferase, the enzyme that catalyzes the synthesis of thiamine triphosphate, has been found in the supernatant fraction of rat liver. The substrate for the enzyme is endogenous, bound thiamine pyrophosphate, since the addition of exogenous thiamine pyrophosphate had no effect. Thus, when a rat liver supernatant was incubated with gamma-labelled [32P]ATP, thiamine [32P]triphosphate was formed whereas the incubation of thiamine [32P]pyrophosphate with ATP did not produce thiamine [32P]triphosphate. The endogenous thiamine pyrophosphate was found to be bound to a high molecular weight protein which comes out in the void volume of Sephadex G-75, and is not dialyzable. The activity that catalyzes the formation of thiamine triphosphate has an optimum pH between 6 and 6.5, a linear time course of thiamine triphosphate synthesis up to 30 min, and is not affected by Ca2+, cyclic GMP and sulfhydryl reagents.     

Anthranilate synthase can generate sufficient phosphoribosyl amine for thiamine synthesis in Salmonella enterica

In bacteria, the biosynthetic pathway for the hydroxymethyl pyrimidine moiety of thiamine shares metabolic intermediates with purine biosynthesis. The two pathways branch after the compound aminoimidazole ribotide. Past work has shown that the first common metabolite, phosphoribosyl amine (PRA), can be generated in the absence of the first enzyme in purine biosynthesis, PurF. PurF-independent PRA synthesis is dependent on both strain background and growth conditions. Standard genetic approaches have not identified a gene product singly responsible for PurF-independent PRA formation. This result has led to the hypothesis that multiple enzymes contribute to PRA synthesis, possibly as the result of side products from their dedicated reaction. A mutation that was able to restore PRA synthesis in a purF gnd mutant strain was identified and found to map in the gene coding for the TrpD subunit of the anthranilate synthase (AS)-phosphoribosyl transferase (PRT) complex. Genetic analyses indicated that wild-type AS-PRT was able to generate PRA in vivo and that the P362L mutant of TrpD facilitated this synthesis. In vitro activity assays showed that the mutant AS was able to generate PRA from ammonia and phosphoribosyl pyrophosphate. This work identifies a new reaction catalyzed by AS-PRT and considers it in the context of cellular thiamine synthesis and metabolic flexibility.     

Effect of thiamine and its derivatives on acetylcholinesterase activity of the blood and brain of albino mice

Thiamine, thiaminepyrophosphate and 4-methyl-5-beta-oxyethylthiazole are studied for their effect on the acetylcholinesterase activity in the brain, blood plasma and cells. The activity of acetylcholinesterase in blood cells is shown to be inhibited most of all by thiamine and thiazole. Acetylcholinesterase of the brain has been efficiently inhibited only by thiamine pyrophosphate.     

Interaction of thiamine pyrophosphokinase from brewer's yeast with thiamine and adenosine-5'-triphosphate]

The interaction of thiamine pyrophosphokinase (Thiaminkinase EC 2.7.6.2) with thiamine and ATP was studied. It was shown that the mechanism of the thiaminokinase reactions is Rapid Equilibrium Random Bi -- Bi. The enzyme binds 8 moles ATP and 1 mole thiamine per mole protein Ks = 0,8-10(-3) and 4.10(-6) M for ATP and thiamine respectively.     

Partial purification and properties of thiamine pyrophosphokinase from pig brain.

Pig brain thiamine pyrophosphokinase (ATP: thiamine pyrophosphotransferase, EC 2.7.6.2) was purified 260-fold over extracts of brain acetone powder. A direct, radiometric assay was used to follow the purification. By isoelectric focusing, the purified enzyme appeared to have an isoionic point of approx. pH 4.2, but these preparations were still not homogeneous by disc-gel electrophoresis nor by analytical ultracentrifugation. The purified enzyme has a broad pH optimum extending from pH 8.3 to 9.3 in 0.028 M phosphate/glycylglycine buffers. For optimal enzymatic activity, the ratio of magnesium to ATP must be fixed at 0.6, which suggests that for this ATP-pyrophosphoryl transfer reaction, the enzymatically preferred reactant may be Mg(ATP)6-/2. A preliminary study of the kinetics of the reaction reveals that the enzyme may function via a partial "ping-pong" mechanism; on this basis, dissociation constants for ATPt and for thiamine were evaluated. Pyrithiamine, butylthiamine, ethylthiamine, and oxythiamine appeared to be competitive inhibitors with respect to thiamine as the variable substrate, and their inhibitor dissociation constants were calculated. The relatively poor affinity of oxythiamine to the enzyme emphasizes the 4-amino group in the pyrimidine ring as one of the specificity requirements for thiamine pyrophosphokinase. Preliminary values for the apparent equilibrium coefficient of the thiamine pyrophosphokinase-catalyzed reaction, in terms of total species, has been approximated at several initial concentrations of reactants: e.g. K'eq,app = (see article) 9.66 - 10(-3) M; and [Th]initial - 1 - 10(-6) and 2 - 10(-6) M, respectively, where TDP, Th, t and eq represent thiamine diphosphate, thiamine, total concentration and equilibrium concentration, respectively.     

Purification and properties of thiamine pyrophosphokinase in Paracoccus denitrificans

The existence of thiamine pyrophosphokinase [EC 2.7.6.2] in procaryotic cells was first demonstrated in Paracoccus denitrificans (J. Bacteriol, (1976) 126, 1030-1036). The enzyme was therefore purified from this organism to determine its molecular structure and properties. Thiamine pyrophosphokinase which was purified 620-fold from P. denitrificans showed a single band on both polyacrylamide and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the molecular weight in the latter case was calculated to be 23,000. Gel filtration analysis using Sephadex G-150 gave a molecular weight of 44,000, indicating that this enzyme contains at least two identical subunits. Although sedimentation equilibrium analysis gave a molecular weight of 96,000, indirect evidence suggests that the form having this molecular weight is an aggregate of the functional dimer. The activity of the purified enzyme required thiamine, ATP, and Mg2+, and the enzyme catalyzed thepyrophosphorylation of thiamine by ATP. Km values for thiamine and ATP were 10 microM and 0.38 mM, respectively. The activity was competitively inhibited by pyrithiamine, giving a Ki value of 19 microM. Oxythiamine and chloroethylthiamine were very weak inhibitors of the enzyme. The activity was also inhibited by the product, TPP.     

Regulation of thiamine biosynthesis in Saccharomyces cerevisiae.

A pho6 mutant of Saccharomyces cerevisiae, lacking a regulatory gene for the synthesis of periplasmic thiamine-repressible acid phosphatase activity, was found to be auxotrophic for thiamine. The activities of four enzymes involved in the synthesis of thiamine monophosphate were hardly detectable in the crude extract from the pho6 mutant. On the other hand, the activities of these enzymes and thiamine-repressible acid phosphatase in a wild-type strain of S. cerevisiae, H42, decreased with the increase in the concentration of thiamine in yeast cells. These results suggest that thiamine synthesis in S. cerevisiae is subject to a positive regulatory gene, PHO6, whereas it is controlled negatively by the intracellular thiamine level.     

Synthesis, in vitro and in vivo activity of thiamine antagonist transketolase inhibitors

Tumor cells extensively utilize the pentose phosphate pathway for the synthesis of ribose. Transketolase is a key enzyme in this pathway and has been suggested as a target for inhibition in the treatment of cancer. In a pharmacodynamic study, nude mice with xenografted HCT-116 tumors were dosed with 1 ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of thiamine, the co-factor of transketolase. Transketolase activity was almost completely suppressed in blood, spleen, and tumor cells, but there was little effect on the activity of the other thiamine-utilizing enzymes alpha-ketoglutarate dehydrogenase or glucose-6-phosphate dehydrogenase. Synthesis and SAR of transketolase inhibitors is described.     

Nucleotide specificity of rat liver thiamine pyrophosphokinase]

The nucleotide specificity of thiamine pyrophosphokinase from rat liver was studied. The enzyme was found to possess a sufficiently wide substrate specificity. Any of the nucleotides can be a donor of the pyrophosphate groups for TDP biosynthesis at two pH optima of the enzyme in the T-kinase reaction under the Mg2++/NTP optimal ratio. The minimal requirement for the substrate structure allowing to predict the position of the split nucleotide phosphoester bond was postulated.     

Evidence that rseC, a gene in the rpoE cluster, has a role in thiamine synthesis in Salmonella typhimurium.

In Salmonella typhimurium, the genetic loci and biochemical reactions necessary for the conversion of aminoimidazole ribotide (AIR) to the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine remain unknown. Preliminary genetic analysis indicates that there may be more than one pathway responsible for the synthesis of HMP from AIR and that the function of these pathways depends on the availability of AIR, synthesized by the purine pathway or by the purF-independent alternative pyrimidine biosynthetic (APB) pathway (L. Petersen and D. Downs, J. Bacteriol. 178:5676-5682, 1996). An insertion in rseB, the third gene in the rpoE rseABC gene cluster at 57 min, prevented HMP synthesis in a purF mutant. Complementation analysis demonstrated that the HMP requirement of the purF rseB strain was due to polarity of the insertion in rseB on the downstream rseC gene. The role of RseC in thiamine synthesis was independent of rpoE.     

Some properties of the thiamine uptake system in isolated rat hepatocytes

A kinetic study of [14C]thiamine uptake over a concentration range from 0.1 microM to 4 mM was performed in isolated rat hepatocytes. The results showed that two processes contribute to the entry in rat hepatocytes: a low affinity process with a Kt of 34.1 microM and Vmax of 20.8 pmol/10(5) cells per 30 s and a high affinity process with a Kt of 1.26 microM and Vmax of 1.21 pmol/10(5) cells per 30 s. The uptake of thiamine by the high affinity process was concentrative and reduced in a betaine medium or K+ medium. Both ouabain and 2,4-dinitrophenol decreased the thiamine uptake by the high affinity process. These findings indicate that the transport of thiamine via a high affinity process is dependent on Na+ and biological energy. The uptake of thiamine was strongly inhibited by thiamine analogs such as dimethialium and chloroethylthiamine. Among quarternary ammonium compounds other than thiamine derivatives, choline and acetylcholine significantly inhibited thiamine uptake by rat liver cells, whereas betaine and carnitine did not. A kinetic study of thiamine uptake by rat hepatocytes preloaded with pyrithiamine, a potent inhibitor of thiamine pyrophosphokinase, revealed that the biphasic property of thiamine uptake disappeared and a single carrier system for thiamine with a Kt of 40.5 microM, which was similar to the Kt value of the low affinity process, was retained. These results strongly suggest that thiamine transport system in rat liver cells is closely connected with thiamine pyrophosphokinase, which accelerates the uptake rat of thiamine by pyrophosphorylation at physiological concentrations of thiamine.