Cytidine, phosphorylation

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 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.

Lamivudine is a synthetic cytidine analogue used in the treatment of HIV (see Chapter 51) and HBV. Its activation requires phosphorylation by cellular enzymes. Lamivudine triphosphate competitively inhibits HBV DNA polymerase and HIV reverse transcriptase and causes chain termination. It inhibits the activity of mammalian DNA polymerases with a much lower potency. 

The last three sources are transported into the cell via inositol transporter [T]. Inositol combines with cytidine monophosphate phosphatidic acid [GMPPA] to be converted to PI, which is then phosphorylated to phos-phatidylinositol phosphate [PIP] and to PIP2 to be reused to form the PI cycle-derived second messengers IP3 and DAG [Kofman and Belmaker 1993]. 

Few pyrimidine bases are salvaged in human cells. However, the pyrimidine nucleosides uridine and cytidine can be salvaged by uri-dine-cytidine kinase, deoxycytidine can be salvaged by deoxycytidine kinase, and thymidine can be salvaged by the enzyme thymidine kinase. Each of these enzymes catalyzes the phosphorylation of a nucleoside(s) utilizing ATP, and forming UMP, CMP, dCMP, and TMP. 

Choline and ethanolamine are activated in much the same way as are sugars. For example, choline can be phosphorylated using ATP (Eq. 17-58, step a) and the phosphocholine formed can be further converted (Eq. 17-58, step b) to cytidine diphosphate choline. Phosphocholine is transferred from the latter onto a suitable acceptor to form the final product (Eq. 17-58, step c). Tire polymerization pattern differs from that for polysaccharide synthesis. When the sugar nucleotides react, the entire nucleoside diphosphate is eliminated (Eq. 17-56), but CDP-choline and CDP-ethanolamine react with elimination of CMP (Eq. 

Available evidence (14,15) favors the pathway for pyruvate kinase by way of phosphorylation of pyruvate enol. Furthermore, J. Knowles and his coworkers (16,17), using chiral thiophosphates and chiral (160,170,180) phosphate have shown that pyruvate kinase transfers phosphate from phosphoenolpyruvate to ADP with stereochemical inversion at phosphorus. Since monomeric metaphosphate is presumably planar, a chemical reaction by way of that ion should proceed with racemization. In the active site of an enzyme, however, all components might be held so rigidly that racemization need not occur. Furthermore, no information is yet available on the detailed mechanism of reactions catalyzed by cytidine synthetase our own experiments, designed to distinguish among the mechanisms here discussed, are as yet incomplete. 

An important difference between the two compounds is that azacytidine (2) incorporates into RNA, while decitabine (1) acts on DNA. Within cells, decitabine (1) is phosphorylated by deoxycytidine kinase, and after conversion to decitabine triphosphate, it is incorporated into DNA in place of deoxycytidine triphosphate. Azacitidine (2) is phosphorylated by uridine-cytidine kinase and eventually incorporated into RNA, inhibiting the processing of ribosomal RNA and ultimately protein synthesis. Azacitidine (2) can also inhibit DNMTs when azacytidine diphophate is reduced to decitabine diphosphate, which is further phosporylated by kinases to dcitabine triphosphate and incorporated into DNA. Because of the inefficiency of these extra steps, the hypomethylating potency of 2 is believed to be one fifth to one tenth that of l.6-8... 

JCS(P1)1171] and Sung (81CC1089 82JOC3623) found that these 1,2,4-triazole derivatives of uridine can be readily prepared at room temperature with excess 1,2,4-triazole and phosphoryl chloride and triethylamine or with 0-chlorophenyldi-(l//-l,2,4-triazol-l-yl)phosphine oxide in acetonitrile. These stable, often crystalline 1,2,4-triazole derivatives react readily with aqueous ammonia and primary or secondary amines at room temperature to afford the corresponding cytidines in high yields. 

The catalytic activity of enzymes is controlled in several ways. Reversible allosteric control is especially important. For example, the first reaction in many biosynthetic pathways is allosterically inhibited by the ultimate product of the pathway. The inhibition of aspartate trans carbamoyl as e by cytidine triphosphate (Section 10.1) is a well-understood example offeedback inhibition. This type of control can be almost instantaneous. Another recurring mechanism is reversible covalent modification. For example, glycogen phosphorylase, the enzyme catalyzing the breakdovm of glycogen, a storage form of sugar, is activated by phosphorylation of a particular serine residue when glucose is scarce (Section 21.2.1). 

How is the other major pyrimidine ribonucleotide, cytidine, formed It is synthesized from the uracil base of UMP, but UMP is converted into UTP before the synthesis can take place. Recall that the diphosphates and triphosphates are the active forms of nucleotides in biosynthesis and energy conversions. Nucleoside monophosphates are converted into nucleoside triphosphates in stages. First, nucleoside monophosphates are converted into diphosphates by specific nucleoside monophosphate kinases that utilize ATP as the phosphoryl-group donor (Section 9.4). For example, UMP is phosphorylated to UDP by UMP kinase. 

Phosphorylation of trityl-cytidine, followed by detritylation, allegedly gives cytidylic acid, but this synthesis merely indicates that cytidylic... 

Gulland and Jackson performed some experiments with 5-nucleotidase, a highly specific enzyme which dephosphorylates 5-phospho-adenosine and -inosine but not" 5-phospho-guanosine and -uridine it is apparently not yet known whether the enzyme dephosphorylates 5-phos-pho-cytidine. They found that a mixture of phosphodiesterase with 5-nucleotidase liberates 35% of the total phosphorus as inorganic phosphate, and therefore decided that two or more of the phosphoryl groups may be attached at position (5) of the ribose units. The 35% dephosphorylation, intermediate between 25 and 50%, was explained as the result of simultaneous, competitive diesterase action at A and B, on two or more phosphoryl groups ... 

Cytidine undergoes eyclization to 2,2 -cyclocytidine under mild Vilsmeier conditions (Scheme 13), phosphoryl chloride being preferred to thionyl chloride.37... 

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