Pyrophosphokinases

Biosynthesis of pyrophosphate (5) from pyrimidine phosphate (47) and thia2ole phosphate (48) depends on the activity of five en2ymes, four of them kinases (87). In yeasts and many other organisms, including humans, pyrophosphate (5) can be obtained from exogenous thiamine in a single step cataly2ed by thiamine pyrophosphokinase (88). 

Mouillon, J.M. et al., Folate synthesis in higher-plant mithocondria coupling between the dihydropterin pyrophosphokinase and the dihydropteroate synthase activities, Biochem. J., 363, 313, 2002. 

In brain, as in most mammalian cells, thiamine occurs predominantly in the form of TDP, the remainder being made up of thiamine monophosphate (10%), thiamine triphosphate (5-10%) and trace amounts of free thiamine. Thiamine is transported into brain and phosphory-lated by the action of thiamine pyrophosphokinase, and inhibition of this enzyme by thiamine antagonists such as pyrithiamine results in decrease synthesis of TDP. Treatment of experimental animals with pyrithiamine results in a generalized reduction of TDP concentrations and an early selective loss in activity of a-KGDH in regions... 

This enzyme [EC 2.7.6.3], also known as 6-hydroxy-methyl-7,8-dihydropterin pyrophosphokinase and 7,8-dihydro - 6 - hydroxymethylpterin pyrophosphokinase, catalyzes the reaction of ATP with 2-ammo-4-hydroxy-... 

The final control mechanism is the inhibition of PRPP synthesis by the allosteric regulation of ribose phosphate pyrophosphokinase. This enzyme is inhibited... 

PRPP is an "activated pentose" that participates in the synthesis of purines and pyrimidines, and in the salvage of purine bases (see p. 294). Synthesis of PRPP from ATP and ribose 5-phosphate is catalyzed by PRPP synthetase (ribose phosphate pyrophosphokinase, Figure 22.6). This enzyme is activated by inorganic phosphate (Pi) and inhibited by purine nucleotides (end-product inhibition). [Note The sugar moiety of PRPP is ribose, and therefore ribonucleotides are the end products of de novo purine synthesis. When deoxy-ribonucleotides are required for DNA synthesis, the ribose sugar moiety is reduced (see p. 295).]... 

The formation of PRPP from ribose-5-phosphate and ATP is catalyzed by ribose-5-phosphate pyrophosphokinase (fig. 23.7). This is an unusual kinase because the pyrophos-phoryl group is transferred rather than the phosphoryl group. 

Intracellular protozoa of the phylum Apicomplexa such as plasmodium, toxoplasma, and eimeria have long been known to respond to sulfonamides and sulfones. This has led to the assumption that Apicomplexa must synthesize their own folate in order to survive. The reaction of 2-amino-4-hydroxy-6-hydroxymethyl-dihydropteridine diphosphate with />aminobenzoate to form 7,8-dihydropteroate has been demonstrated in cell-free extracts of the human malaria parasite Plasmodium falciparum. 2-Amino-4-hydroxy-6-hydroxymethyl-dihydropteridine pyrophosphokinase and 7,8-dihydropteroate synthase have also been identified. Sulfathiazole, sulfaguanidine, and sulfanilamide act as competitive inhibitors of p-aminobenzoate. It has not been possible to demonstrate dihydrofolate synthase activity in the parasites, which raises the possibility that 7,8-dihydropteroate may have substituted for dihydrofolate in malaria parasites. Similar lack of recognition of folate as substrate was also observed in the dihydrofolate reductase of Eimeria tenella, a parasite of chickens. 

The gene encoding 7,8-dihydropteroate synthase was cloned from P falciparum and found to encode a bifunctional enzyme that includes the pyrophosphokinase at the amino terminal of the protein. Discrepancies were observed in the sequences of 7,8-dihydropteroate synthase portion of the genes from sulfadoxine-sensitive versus sulfadoxine-resistant P falciparum, thus confirming that this enzyme is the target for the antimalarial sulfonamide drugs. 

The synthesis of functionalized 7,7-dialkyl-7,8-dihydropterins and a 7-spirocyclopropane derivative have been performed to find new types of inhibitors for 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase and dihydrofolate reductase <85JCS(Pl)2133, 85JCS(P1)2145, 89JCS(P1)1297>. An informative review on the chemical and biological reactions of reduced pterins is also available <84MI 718-08). 

Both free thiamin and thiamin monophosphate circulate in plasma about 60% of the total is the monophosphate. Under normal conditions, most is bound to albumin when the albumin binding capacity is saturated, the excess is rapidly filtered at the glomerulus and excreted in the urine. Although a significant amount of newly absorbed thiamin is phosphorylated in the Uver, aU tissues can take up both thiamin and thiamin monophosphate, and are able to phosphorylate them to thiamin diphosphate and thiamin triphosphate. In most tissues, it is free thiamin that is the immediate precursor of thiamin diphosphate, which is formed by a pyrophosphokinase both the p-and y-phosphates of ATP are incorporated. Thiamin monophosphate arises mainly as a result of sequential hydrolysis of thiamin triphosphate and thiamin diphosphate. 

Genetic defects of the tissue thiamin transport protein and thiamin pyrophosphokinase cause megaloblastic anemia, presumahly as a result of impaired synthesis of pentoses for DNA synthesis from low activity of trans-ketolase (Section 6.3.2). In many cases, this megaloblastic anemia is thiamin-responsive, suggesting that the defect must be because of low af nity of either the transport protein or thiarnin pyrophosphokinase for its substrate (Neufeld et al., 2001). 

Thiamine uptaken into the cell is phosphorylated to TDP by the enzyme thiamine pyrophosphokinase. TDP is then further phosphorylated to thiamine triphosphate (TTP) or is dephosphorylated to thiamine monophosphate (TMP). 

Fig. 2 Intercellular trafficking and thiamine and thiamine esters in brain. TMP thiamine monophosphate, TDP thiamine diphosphate, TTP thiamine triphosphate, TPKinase thiamine pyrophosphokinase...

V The prs gene (coding for ribose phosphate pyrophosphokinase ) was isolated and integrated into the chromosome... 

Ribose phosphate pyrophosphokinase catalyzes the phosphorylation of ribose-5-phosphate to give PRPP. 

Thiamine is absorbed by a pathway that is saturable at concentrations of 0.5-1.0 jumol/L. Oral doses in excess of 10 mg do not significantly increase blood or urine concentrations of vitamin Bi. In the human, absorption occurs predominantly in the jejunum and ileum. Some ferns, shellfish, fish, and species of bacteria contain thiami-nase, which cleaves the pyrimidine ring from the thiazole ring. This enzyme causes thiamine deficiency in cattle. In plasma, thiamine is transported bound to albumin and, to a small extent, other proteins. TPP is synthesized in the liver by thiamine pyrophosphokinase. 

Three novel bisubstrate analogues (133a-c) acting as potent inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase have been synthesised and used in the co-crystallisation of the kinase. These compounds were synthesised by coupling adenosine nucleotides (AMP, ADP and ATP) to 6-hy-... 

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