Uridine enzymic synthesis

N. K. Kochetkov and V. N. Shibaev, Analogs of glucose uridine diphosphate Synthesis and reactions with enzymes, Russian Chem. Bull., 23 (1974) 1106-1121. 

Starting from ceramide D-glucoside, enzymic synthesis has been studied for producing gangliosides222,223 and for the conversion of Tay-Sachs ganglioside into monosialoganglioside by the uridine 5 -pyrophosphate of brain.224... 

Barber, G.A. The enzymic synthesis of uridine diphosphate L-rhamnose Fed. Am. Soc. Exptl. Biol. (1963). Baron, R.L. and F.E. Guthrie Study of sugars found in tobacco as affected by green peach aphid, Myzus per-sicae (Sulzer) Ann. Entomol. Soc. America 52 (1959) 257. 

Gygax D, Spies P, Winkler T, Pfaar U. Enzymic synthesis of p-D-glucuro-nides with in situ regeneration of uridine 5 -diphosphoglucuronic acid. Tetrahedron 1991 47 5119-5122. 

Hexokinase catalysed syntheses of 6-amino-6-deoxy-D-glucose and -D-gluconate 6-phosphates, 6-amino-6-deoxy-D-fructose 6-phosphate and 1,6-bisphosphate, and 5-amino-5-deoxy-D-ribulose 5-phosphate have been reported. From a study of the properties and reactivity in enzymic reactions of these compounds, it was concluded that they were excellent isosteric analogues of the normal metabolic intermediates except for reactions catalysed by kinases. A preparative enzymic synthesis of uridine 5 -diphospho-2-acetamido-2-deoxy-D-glucose used chemically synthesized 2-acetamido-2-deoxy-D-glucose 1-phosphate and uridine triphosphate as reactants, and a calf liver enzyme extract containing uridine-5 -phospho-A-acetylglucosamine pyrophosphorylase. ... 

While mammahan cells reutilize few free pyrimidines, salvage reactions convert the ribonucleosides uridine and cytidine and the deoxyribonucleosides thymidine and deoxycytidine to their respective nucleotides. ATP-dependent phosphoryltransferases (kinases) catalyze the phosphorylation of the nucleoside diphosphates 2 "-de-oxycytidine, 2 -deoxyguanosine, and 2 -deoxyadenosine to their corresponding nucleoside triphosphates. In addition, orotate phosphoribosyltransferase (reaction 5, Figure 34-7), an enzyme of pyrimidine nucleotide synthesis, salvages orotic acid by converting it to orotidine monophosphate (OMP). 

Figure 36-4. Illustration of the tight correlation between the presence of RNA polymerase II and RNA synthesis. A number of genes are activated when Chirono-mus tentans larvae are subjected to heat shock (39 °C for 30 minutes). A Distribution of RNA polymerase II (also called type B) in isolated chromosome IV from the salivary gland (at arrows). The enzyme was detected by immunofluorescence using an antibody directed against the polymerase. The 5C and BR3 are specific bands of chromosome IV, and the arrows indicate puffs. B Autoradiogram of a chromosome IV that was incubated in H-uridine to label the RNA. Note the correspondence of the immunofluorescence and presence of the radioactive RNA (black dots). Bar = 7 pm. (Reproduced, with permission, from Sass H RNA polymerase B in polytene chromosomes. Cell 1982 28 274. Copyright 1982 by the Massachusetts Institute of Technology.)...

Synthesis of sucrose 6 -phosphate by an enzymic method using uridine 5 -(a-D-glucopyranosyl diphosphate) plus D-glucose 6-phosphate has been reported.126-131 The first, unambiguous, chemical synthesis of sucrose 6 -phosphate was achieved by Buchanan and coworkers.18 The reaction of 2,3,4,6,l, 3, 4 -hepta-0-acetylsucrose, prepared by five steps of synthesis, with cyanoethyl phosphate in pyridine gave a crude product from which sucrose 6 -phosphate was isolated as the barium salt. 

Figure 20.9 The positions in the pathway for de novo pyrimidine nucleotide synthesis where GLUCOSE provides the ribose molecule and GLUTAMINE provides nitrogen atoms. Glucose forms ribose 5-phosphate, via the pentose phosphate pathway (see chapter 6), which enters the pathway, after phosphorylation, as 5-phospho-ribosyl 1-pyrophosphate. Glutamine provides the nitrogen atom to synthesise carbamoylphos-phate (with formation of glutamate), and also to form cytidine triphosphate (CTP) from uridine triphosphate (UTP), catalysed by the enzyme CTP synthetase. It is the amide nitrogen of glutamine that is the nitrogen atom that is provided in these reactions.

During the past 15 years data from experiments with different types of animal tissues and micro-organisms, using intact cells, crude extracts or purified enzymes, have firmly established the general occurrence of nucleotide reductases and have stressed their importance for DNA synthesis in essentially all types of rapidly growing cells [54]. It has been proposed that ribonucleotide diphosphates lose a hydroxide ion from C-2 to form a carbonium ion which is then stero-specifically reduced by a hydride ion derived from thioredoxin [54]. Adenosine diphosphate and guanosine diphosphate (as well as uridine and cytidine diphosphates) are reduced in this manner. 

Carbamoyl phosphate synthetase formation in liver taken from tadpoles treated with thyroxine is enhanced by the addition of orotic acid, uracil or uridine (cytosine and adenosine had no effect). The synthesis of this enzyme is not affected by these pyrimidines in untreated animals. This indicates that there is a relative pyrimidine deficiency during thyroxine-induced metamorphosis [140]. 

The enzyme is also responsible for converting cytidine diphosphate (CDP) to 2 -dCDP and uridine diphosphate (UDP) to 2 -dUDP for use in making nucleotides for DNA synthesis. 

Mutation of one of the two enzyme activities of UMP synthase leads to orotic aciduria, characterized by accumulation of its first substrate orotic acid and insufficient levels of the product UMP, which reduces availability of uridine triphosphate (UTP) and cytidine triphosphate (CTP) for use in nucleic acid synthesis. 

Several possible mechanisms of resistance to 5-fluo-rouracU have been identified, including increased synthesis of the target enzyme, altered affinity of thymidy-late synthetase for FdUMP, depletion of enzymes (especially uridine kinase) that activate 5-fluorouracil to nucleotides, an increase in the pool of the normal metabolite deoxyuridylic acid (dUMP), and an increase in the rate of catabolism of 5-fluorouracil. 

It was previously thought that 5-FU inhibits the enzyme by classical competitive inhibition. However, it was found that 5-FU is a transition-state substrate, and it forms a covalent complex with tetrahydrofolate and the enzyme in the same way that the natural substrate does. The reaction, however, will not go to completion, since the fluoro-uridine derived from the antimetabolite remains attached to the enzyme, and the latter becomes irreversibly deactivated. Recovery can occur only through the synthesis of new enzyme. Fluorouracil is used in the treatment of breast cancer and has found limited use in some intestinal carcinomas. Unfortunately, this drug has the side effects usually associated with antimetabolites. Its prodrug, fluorocytosine (8.35, which is also an antifungal agent) is better tolerated. 

The same procedure was applied212 for the preparation of uridine 5 -(a-D-glucopyranosyl pyrophosphate-4"-t, or -5"-f. A cell-free extract of Phytophthora cinnamoni was used for the synthesis of the 6"-t derivative,213 as well as yeast pyrophosphorylase.2133 An enzyme from Salmonella typhimurium was found quite satisfactory214 for obtaining this and other labeled nucleotide sugars. ... 

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