Cytidine enzymic synthesis

It was snbseqnently discovered that the first enzyme in the pathway for isoleucine synthesis, which is threonine deaminase, was inhibited by isoleucine in an extract of E. coli. No other amino acid caused inhibition of the enzyme. Threonine deaminase is, in fact, the rate-limiting enzyme in the pathway for isoleucine synthesis, so that this was interpreted as a feedback control mechanism (Fignre 3.13(a)). Similarly it was shown that the hrst enzyme in the pathway for cytidine triphosphate synthesis, which is aspartate transcarbamoylase, was inhibited by cytidine triphosphate (Fignre 3.13(b)). Since the chemical structures of isoleucine and threonine, or cytidine triphosphate and aspartate, are completely different, the qnestion arose, how does isolencine or cytidine triphosphate inhibit its respective enzyme The answer was provided in 1963, by Monod, Changenx Jacob. 

Scheme 19.—Enzymic Synthesis of Cytidine Monophosphate JV-Acetylneuraminic Acid Starting from CMP.

Although no recent activity has been reported in this field, it remains possible that more potent inhibitors of this enzyme have potential applications as anticancer therapeutics. It is also possible that the combination of salvage uridine-cytidine monophosphate synthesis inhibitors with inhibitors of de novo uridine-cytidine monophosphate synthesis (such as PALA or pyrazofurin) may result in more efficacious cancer therapeutics. 

It is an enzyme used in the early stages of cytidine nucleotide synthesis. 

Whitesides and coworkers have carried out a comparison of enzymic and chemical routes to CTP, GTP and UTP on a 10-gram scale. They concluded that CTP and GTP were best made enzymically, and UTP by reaction of CTP with nitrous acid. The triphosphates were then employed for the enzymic synthesis of UDP-Glucose, UDP-Glucuronic acid, and GDP-Mannose.i94 Cytidine diphosphate sugars have been prepared from the 3,6-dideoxyhexoses paratose and abequose,193 and all four nucleoside diphosphate sugars of 6-sulpho-a-D-quinovose have been synthesized for studies of sulpholipid biosynthesis in chloroplasts.196 The stable analogue (138) of CMP-KDO has been prepared by a triester approach, but was only a weak inhibitor of KDO incorporation into lipopolysaccharides.197 A reference to acetylated forms of UDPGlc is mentioned in Chapter 7. 

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.

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. 

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. 

Cidofovir (Figure 24.4) is an antiviral cytidine nucleotide analog with inhibitory activity against HCMV and other herpes viruses. Cidofovir is first converted to an active diphosphate form by cellular enzymes. Antiviral effects of cidofovir are due to inhibition of viral DNA polymerase by the diphosphate metabolite (Neyts and De Clercq, 1994 Plosker and Noble, 1999 Scholar and Pratt, 2000). The diphosphate probably interacts with DNA polymerase either as an alternate substrate (incorporation at the 3 end or within the interior of the DNA chain) or as a competitive inhibitor (with respect to the normal substrate dCTP). Cidofovir inhibits HCMV DNA synthesis at intracellular concentrations 1000-fold lower than are required to inhibit cellular DNA synthesis (Neyts and De Clercq, 1994). For HSV-1 and HSV-2 corresponding concentrations are at least 50-fold lower. 

The utilization of ammonia resulting from the combination of carbamyl phosphate with aspartic acid, the initial reaction for the synthesis of the pyrimidine nucleotides, continues only as long as there is a requirement for them (Fig. 3). Regulation of this biosynthetic pathway is probably by way of feedback inhibition of aspartate transcarbamylase. The rat liver enzyme is inhibited by uridine, cytidine or thymidine or such derivatives as CMP, UTP, or TMP, all intermediates or products of this pathway (B8). This is not the only enzyme of the pathway which may be subject to feedback regulation. Dihydroorotase from rat liver is also inhibited by some pyrimidines and purines (B9). 

Scheme L Synthesis of a2,64inked sialyl-N-acetyllactosamine using a one-pot multi-enzyme system with in situ regeneration of CMP-Neu5Ac. Abbreviations for enzymes CSS, CMP-sialic acid synthetase NMK, nucleoside monophosphate kinase PK, pyruvate kinase PPase, pyrophosphatase. Abbreviations for compounds PEP, phosphoenolpyruvate ADP, adenosine 5 -diphosphate ATP, adenosine 5 -triphosphate CMP, cytidine 5-monophosphate CDP, cytidine 5 -diphosphate CTP, cytidine 5-triphosphate LacNAc, N-acetyllactosamine NeuSAc, N-acetylneuraminic acid PPi, inorganic pyrophosphate.

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