Uridine kinase reaction

The purine and pyrimidine bases can be converted to then-respective nncleotides by reaction with 5-phosphoribosyl 1-pyrophosphate. Since these bases are not very soluble, they are not transported in the blood, so that the reactions are only of qnantitative significance in the intestine, where the bases are produced by degradation of nucleotides. In contrast, in some cells, nucleosides are converted back to nucleotides by the activity of kinase enzymes. In particular, adenosine is converted to AMP, by the action of adenosine kinase, and uridine is converted to UMP by a uridine kinase... 

Fluorouracil (5-FU) requires enzymatic conversion to the nucleotide (ribosylation and phosphorylation) in order to exert its cytotoxic activity. Several routes are available for the formation of floxuridine monophosphate (FUMP). 5-FU may be converted to fluorouridine by uridine phos-phorylase and then to FUMP by uridine kinase, or it may react directly with 5-phosphoribosyl-l-pyrophosphate (PRPP), in a reaction catalyzed by orotate phosphoribosyl transferase, to form FUMP. Many metabolic pathways are available to FUMP. As the triphosphate FUTP, it may be incorporated into RNA. An alternative reaction sequence... 

Once inside the cell, nucleosides are converted to the corresponding NMP adenosine by adenosine kinase and uridine by uridine kinase. The reaction is as follows ... 

Two routes are known by which the free base, uracU, can enter the ribonucleotide pool. One proceeds by the sequential actions of uridine phosphorylase and uridine-cytidine kinase (reactions 5 and 6, Fig. 12-1) this route is discussed below. The other route is by way of a single-step phosphoribosyltransferase reaction specific for uracil (reaction 4, Fig. 12-1) ... 

The kinase step enables cells to utilize nucleosides from their milieu for the incorporation into nucleotide coenzymes and polynucleotides, and has the obvious value that synthesis de novo is spared. The kinase reaction, operating in sequence with the readily reversible uridine phosphorylase reaction, also provides a route by which uracil may enter into the pyrimidine nucleotide pool. 

Possible hypotheses for the defect in E.v.d.B. are 1. Decreased reutilisation of uracil at the level of uridine phosphorylase or uridine kinase. At low uracil levels reutilisation will predominate over catabolism via the diHPyDH reaction and at high uracil concentrations the reverse will be the case presumably. 2. Increased synthesis of pyrimidines due to a fluctuating regulation at the level of aspartate transcarbamylase and/or ornithine trans-carbamylase. It has to be presumed that attacks of periodic hyperammonemia have not occurred during the limited time of investigations, with the phenomena of patient A.G. in mind. 

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). 

Some of the reactions of PO3- parallel enzymatic reactions promoted by adenosine triphosphate (ATP). Pyruvate kinase catalyzes the equilibration of ATP and pyruvate with adenosine diphosphate (ADP) and phosphoenol pyruvate (11,12). In a formal sense, this reaction resembles the preparations of enol phosphate (eqs. 6 and 7). Cytidine triphosphate synthetase catalyzes the reaction of uridine triphosphate with ammonia to yield cytidine triphosphate (13). In a formal sense, this reaction resembles the replacement of the ester carbonyl group of ethyl acetate by the nitrogen of aniline (eq. 8). 

Some biosynthetic reactions are driven by the hydrolysis of nucleoside triphosphates that are analogous to ATP—namely, guanosine triphosphate (GTl ), uridine triphosphate (UTP), and cytidine triphosphate (CTP). The diphosphate forms of these nucleotides are denoted by GDP, UDP, and CDP, and the monophosphate forms are denoted by GMP, UMP, and CMP. Enzymes catalyze the transfer of the terminal phosphoryl group from one nucleotide to another. The phosphorylation of nucleoside monophosphates is catalyzed by a family of nucleoside monophosphate kinases, as discussed in Section 9.4. The phosphorylation of nucleoside diphosphates is catalyzed by 7iucleoside diphosphate kinase, an enzyme with broad... 

The answer is e. (Murray, pp 375-401. Scriver, pp 2663-2704. Sack, pp 121-138. Wilson, pp 287—320.) Orotic aciduria is the buildup of orotic acid due to a deficiency in one or both of the enzymes that convert it to UMP Either orotate phosphoribosyltransferase and orotidylate decarboxylase are both defective, or the decarboxylase alone is defective. UMP is the precursor of UTP, CTP, and TMP All of these end products normally act in some way to feedback-inhibit the initial reactions of pyrimidine synthesis. Specifically, the lack of CTP inhibition allows aspartate transcarbamoylase to remain highly active and ultimately results in a buildup of orotic acid and the resultant orotic aciduria. The lack of CTP, TMP, and UTP leads to a decreased erythrocyte formation and megaloblastic anemia. Uridine treatment is effective because uridine can easily be converted to UMP by omnipresent tissue kinases, thus allowing UTP, CTP, and TMP to be synthesized and feedback-inhibit further orotic acid production. 

Liacouras, A.S. Garvey, T.Q. Millar, F.K. Anderson, E.P. Uridine-cytidine kinase. Kinetic studies and reaction mechanism. Arch. Biochem. Biophys., 168, 74-80 (1975)... 

UMP is phosphorylated by two kinases to uridine triphosphate (UTP) via two sequential reactions with ATP. First, the diphosphate form UDP is produced, and further phosphorylation leads to UTP. Both steps are fueled by ATP hydrolysis. CTP (cytidine triphosphate) is subsequently formed by the amination of UTP by the catalytic activity of CTP synthetase. Glutamine is the NH3 donor, and also in this case the reaction is fueled by ATP hydrolysis. Cytidine monophosphate (CMP) is derived from CTP with subsequent loss of two phosphates. 

Fio. 12-1. Metabolism of uracil, cytosine, and their tibonucleosides. Solid lines, enzyme activity in both animal and microbial cells large-dashed line, enzyme activity in microbial cells only small-dashed lines, reaction not known. Reactions (1) CTP synthetase (2) cytosine deaminase (3) cytidine deaminase (4) uracil phosphoribosyl transferase (5) uridine phosphorylase (6) uiidine-i tidine kinase (7) 5 -nucleotidase. 

Recently, a universal enzyme-coupled fluorescence assay for glycosyl transferases was developed. This method is extremely cost-effective and is based on the quantification of nucleotides produced in the glycosyl transfer reaction. The guanosine diphosphate (GDP), uridine diphosphate (UDP), and cytidine monophosphate (CMP) are phos-phorylated with nucleotide kinase in the presence of excess of ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase,glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for the conversion of resazurin to resorufin, which is then quantified by fluorescence measurement. 

A study of the mechanism of uracil incorporation into uridine phosphates was carried out in the Ehrlich ascites tumor, a tissue which utilized uracil as well as small molecule precimsors for nucleic acid formation (312). Uridine 5 -phosphate (UMP) was formed from uracil, ATP, and ribose 1-phosphate (R-l-P). Uridine was an intermediate in the formation of the nucleotide and was formed by the reaction of uracil and R-l-P with pyrimidine nucleoside phosphorylase (313, 314). Nucleoside kinase reacted the nucleoside with ATP to form UMP. The sequence is ... 

In most of these reactions adenine nucleotides are employed although reactions involving nucleotides of the uridine, cytidine, and guanosine series are well known. It should be noted that nucleosides themselves and nucleotide monophosphates are surprisingly inactive in biosynthetic processes. Thus, nucleoside kinases, monophosphate kinases, and diphosphate kinases, which convert nucleoddes and nucleoside monophosphates to the di- and triphosphate stage are incUqiensable for cellular bio yntheds. 

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. ... 

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