| FORMATION OF DEOXYNUCLEOTIDES
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| Because DNA uses deoxyribonucleotides instead of the ribonucleotides found in RNA, cells require pathways to convert ribonucleotides into the deoxy forms. The adenine, guanine and uracil deoxyribonucleotides are synthesized from their corresponding ribonucleotides by direct reduction of the 2'-hydroxyl by the enzyme ribonucleotide reductase.
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| SOME CHEMOTHERAPEUTIC AGENTS BLOCK PYRIMIDINE BIOSYNTHESIS |
When DNA synthesis is blocked, cells cannot divide. Because of this, several important anticancer drugs that block the synthesis of TMP are widely used as chemotherapeutic agents (see Fig. 29.6). These include pyrimidine analogs such as fluorouridine and fluorocytosine, as well as the antifolates: aminopterin, methotrexate, and trimethoprim . |
| One of the unique steps in DNA biosynthesis is the formation of TMP from dUMP by the enzyme thymidylate synthase. During the process of catalysis, thymidylate synthase forms a covalent bond with its substrate, dUMP. The enzyme-substrate complex then forms a complex with N5,N10-methylene-THF. Once this ternary complex is formed, the methyl transfer occurs. |
| Fluorodeoxyuridylate (FdUMP) is a specific, suicide inhibitor of thymidylate synthase. In FdUMP, a highly electronegative fluorine replaces the carbon-5 proton of uridine. This compound can begin the enzymatic conversion into dTMP by forming the enzyme-FdUMP covalent complex; however, the covalent intermediate cannot accept the donated methyl group from methylene THF, nor can it be broken down to release the active enzyme. The result is a suicide complex in which the substrate is covalently locked at the active site of thymidylate synthase. The drug is frequently administered as fluorouridine (Adrucil, Efudex, Fluracil), and the body's normal metabolism converts the fluorouridine into FdUMP. Fluorouridine is used against breast, colorectal, gastric, and uterine cancers. |
| Fluorocytosine (Flucytosine, Alcobon, Ancotil) is a potent antimicrobial agent. Its mechanism of action is similar to that of FdUMP; however, it must first be converted into fluorouracil by the action of cytosine deaminase. The fluorouracil is subsequently converted into FdUMP, which blocks thymidylate synthase as above. While cytosine deaminase is present in most fungi and bacteria, it is absent in animals and plants. Therefore, in humans, fluorocytosine is not converted into fluorouracil and is nontoxic, while in the microbes, metabolism of fluorocytosine results in cell death. |
| Aminopterin and methotrexate are folic acid analogs that bind about 1000-fold more tightly to dihydrofolate reductase (DHFR) than does dihydrofolate. See Figure 29.6 for the sites of action of these drugs. Antifolates effectively block the synthesis of tetrahydrofolate, which in turn limits the formation of N5,N10-methylene-THF. In this manner, they block the synthesis of dTMP. Further, these compounds are competitive inhibitors of other THF-dependent enzyme reactions used in the biosynthesis of purines, histidine, and methionine. Trimethoprim binds to DHFR, and binds more tightly to bacterial DHFRs than it does to mammalian enzymes, making it an effective antibacterial agent. Folate analogs are relatively nonspecific chemotherapeutic agents. They poison rapidly dividing cells, including those in hair follicles and gut endothelia, causing the loss of hair and gastrointestinal side-effects of chemotherapy. |
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| The nucleotide deoxy-TMP, abbreviated as TMP because it is unique to DNA, is synthesized by a special pathway involving methylation of the deoxyribose form of uridylate, dUMP. However, reduction of ribonucleotides to deoxyribonucleotides occurs only as diphosphates, not as monophosphates. Thus, the TMP biosynthetic pathway leads from UMP to UDP to dUDP (see Fig. 29.6). dUDP is then phosphorylated to dUTP, which creates an unexpected biochemical problem. DNA polymerase does not effectively discriminate between dUTP and TTP and can incorporate dUTP into DNA, which would lead to high rates of mutagenesis. Therefore, cells limit the concentration of dUTP by rapidly hydrolyzing dUTP to dUMP with the enzyme dUTP diphosphohydrolase. This enzyme releases pyrophosphate, which is rapidly hydrolyzed
to phosphate, pulling the reaction towards the formation of dUMP. The dUMP then serves as the substrate for thymidylate synthase, which transfers a methyl group from N5,N10-methylene tetrahydrofolate to make TMP. The folate is released as dihydrofolate. Two rounds of phosphorylation using ATP as phosphate donor yield TDP and subsequently TTP. The synthesis of TTP is a roundabout pathway, but does provide opportunities for chemotherapy through inhibition of TMP biosynthesis.
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| Regeneration of the methyl donor is accomplished in two steps. First, dihydrofolate is reduced to tetrahydrofolate, a step that is inhibited by several important chemotherapeutic agents. Then the amino acid, serine, is degraded to produce formaldehyde and glycine. The formaldehyde reacts with the N5,N10-imine groups of tetrahydrofolate to yield N5,N10-methylene tetrahydrofolate.
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