Thymidylate synthase, reaction catalyzed

Thymidylate synthase also catalyzes an exchange of tritium of [5- H]dUMP for water protons in the absence of CH2H4folate. The turnover number for this exchange reaction is about 1/45,000 that of dTMP formation, and the Kjn is 1.2 X 10 M, about the same as the of the enzyme-dUMP complex estimated by equilibrium dialysis. The exchange reaction provided compelling evidence that the enzymic reaction involves attack of an enzyme nucleophile on the 6 position of dUMP to pro-... 

FIGURE 22-43 Biosynthesis of thymidylate (dTMP). The pathways are shown beginning with the reaction catalyzed by ribonucleotide reductase. Figure 22-44 gives details of the thymidylate synthase reaction. 

In 1995, Horie et al. described a polymorphic tandem repeat found in the 5 -un-translated region of the thymidylate synthase gene [70]. Thymidylate synthase (TS TYMS) catalyzes the intracellular transfer of a methyl group to deoxyuridine-5-monophosphate (dUMP) to form deoxythymidine-5-monophosphate (dTMP), which is anabolized in cells to the triphosphate (dTTP). This pathway is the only de- novo source of thymidine, an essential precursor for DNA synthesis and repair. The methyl donor for this reaction is the folate cofactor 5,10-methylenetetrahydro-folate (CH2-THF) (Figure 24.4). 

Agrawal, N., Hong, B., Mihai, C. and Kohen, A. (2004). Vibrationally enhanced hydrogen tunneling in the Escherichia coli thymidylate synthase catalyzed reaction. Biochemistry 43, 1998-2006... 

Conversion of dUMP to dTMP is catalyzed by thy-midylate synthase. A one-carbon unit at the hydroxymethyl (—CH2OH) oxidation level (see Fig. 18-17) is transferred from Af5,Af10-methylenetetrahydrofolate to dUMP, then reduced to a methyl group (Fig. 22-44). The reduction occurs at the expense of oxidation of tetrahydrofolate to dihydrofolate, which is unusual in tetrahydrofolate-requiring reactions. (The mechanism of this reaction is shown in Fig. 22-50.) The dihydrofolate is reduced to tetrahydrofolate by dihydrofolate reductase—a regeneration that is essential for the many processes that require tetrahydrofolate. In plants and at least one protist, thymidylate synthase and dihy-drofolate reductase reside on a single bifunctional protein. 

Phosphorylation of dCDP to dCTP (step k, Fig. 25-14) completes the biosynthesis of the first of the pyrimidine precursors of DNA. The uridine nucleotides arise in two ways. Reduction of UDP yields dUDP (step), Fig. 25-14). However, the deoxycytidine nucleotides are more often hydrolytically deaminated (reactions / and / ) 274 Methylation of dUMP to form thymidylate, dTMP (step n, Fig. 25-14), is catalyzed by thymidylate synthase. The reaction involves transfer of a 1-carbon unit from methylene tetrahydrofolic acid with subsequent reduction using THF as the electron donor. A probable mechanism is shown in Fig. 15-21. See also Box 15-E. Some bacterial transfer RNAs contain 4-thiouridine (Fig. 5-33). The sulfur atom is introduced by a sulfurtransferase (the Thil gene product in E. coli). The same protein is essential for thiamin biosynthesis (Fig. 25-21)274a... 

A5, A1 °-Methylcnctctrahydrofolate is a hydroxymethyl-group donor substrate for several enzymes and a methyl-group donor substrate for thymidylate synthase (fig. 10.15). It arises in living cells from the reduction of A5,A10-methenyltetrahydrofolate by NADPH and also by the serine hydroxymethyltransferase-catalyzed reaction of serine with tetrahydrofolate. 

Methylation of dUMP to give thymidylate is catalyzed by thymidylate synthase and utilizes 5,10-methylenetetra-hydrofolate as the source of the methyl group. This reaction is unique in the metabolism of folate derivatives because the folate derivative acts both as a donor of the one-carbon group and as its reductant, using the reduced pteridine ring as the source of reducing potential. Consequently, in this reaction, unlike any other in folate metabolism, dihydrofolate is a product (fig. 23.16). Since folate derivatives are present in cells at very low concentrations, continued syn-... 

Tetrahydrofolate cofactors participate in one-carbon transfer reactions. As described above in the section on vitamin B12, one of these essential reactions produces the dTMP needed for DNA synthesis. In this reaction, the enzyme thymidylate synthase catalyzes the transfer of the one-carbon unit of N 5,N 10-methylenetetrahydrofolate to deoxyuridine monophosphate (dUMP) to form dTMP (Figure 33-2, reaction 2). Unlike all of the other enzymatic reactions that utilize folate cofactors, in this reaction the cofactor is oxidized to dihydrofolate, and for each mole of dTMP produced, one mole of tetrahydrofolate is consumed. In rapidly proliferating tissues, considerable amounts of tetrahydrofolate can be consumed in this reaction, and continued DNA synthesis requires continued regeneration of tetrahydrofolate by reduction of dihydrofolate, catalyzed by the enzyme dihydrofolate reductase. The tetrahydrofolate thus produced can then reform the cofactor N 5,N 10-methylenetetrahydrofolate by the action of serine transhydroxy- methylase and thus allow for the continued synthesis of dTMP. The combined catalytic activities of dTMP synthase, dihydrofolate reductase, and serine transhydroxymethylase are often referred to as the dTMP synthesis cycle. Enzymes in the dTMP cycle are the targets of two anticancer drugs methotrexate inhibits dihydrofolate reductase, and a metabolite of 5-fluorouracil inhibits thymidylate synthase (see Chapter 55 Cancer Chemotherapy). 

A highly unusual feature of DHFR in Apicomplexa and Kinetoplastida is its association with thymidylate synthase in the same protein. DHFR activity is always located at the amino terminal portion, while the thymidylate synthase activity resides in the carboxyl terminal. The two enzyme functions do not appear to be interdependent eg, the DHFR portion of the P falciparum enzyme molecule was found to function normally in the absence of the thymidylate synthase portion. It is likely that since the protozoan parasites do not perform de novo synthesis of purine nucleotides, the primary function of the tetrahydrofolate produced by DHFR is to provide 5,10-methylenetetrahydrofolate only for the thymidylate synthase-catalyzed reaction. Physical association of the two enzymes may improve efficiency of TMP synthesis. If an effective means of disrupting the coordination between the two activities can be developed, this bifunctional protein may qualify as a target for antiparasitic therapy. 

Cells making DNA must also be able to make deoxythymidine triphosphate (dTTP). The key step in the synthesis of dTTP is the conversion of dUMP to dTMP via thymidylate synthase. The reaction requires a source of N5,Nw-methylene tetrahydrofolate (see Sec. 15.7, Fig. 15-19) to provide the methyl group. In this reaction, the tetrahydrofolate is oxidized to dihydrofolate. Dihydrofolate must be reduced to tetrahydrofolate via the enzyme dihydrofolate reductase so that more Af5,A,l0-methylene tetrahydrofolate can be made from serine in a reaction catalyzed by serine hydroxymethyltransferase. These three reactions, which are essential for the formation of dTMP, are shown below. 

The chemical structures of four commonly used anticancer drugs are shown in Fig. 15-17. Methotrexate was the first "true anticancer drug, synthesized in 1949, and has been in clinical use for treatment of a variety of cancers since the early 1950s. Methotrexate is a potent inhibitor of dihydrofolate reductase with an inhibition constant (Kt) for interaction with the enzyme of 10 9Af. Inhibition of this enzyme in a cell leads to major accumulation of DHF to concentrations of 2.5 fiM, and minor decreases in THF. Marked decreases in THF may not be seen due to the release of bound THF in methotrexate-treated cells. The high levels of DHF are toxic to the cell, inhibiting the reaction catalyzed by thymidylate synthase,... 

Dihydrofolate Reductase. The reduced form of folate (tetrahydrofolate) acts as a one-carbon donor in a wide variety of biosynthetic transformations. This includes essential steps in the synthesis of purine nucleotides and of thymidylate, essential precursors to EHA and I A. For this reason, folate-dependent enzymes have been useful targets for the development of anticancer and anti-inflammatory drugs Ce.g., methotrexate) and anti-infectives (trimethoprim, pyrimethamine). During the reaction catalyzed by thymidylate synthase (TS), tetrahydrofolate also acts as a reducltant and is converted stoichiometrically to dihydrofolate. The regeneration of tetrahydrofolate, required for the continuous fimc-tioning of this cofactor, is catalyzed by dihydrofolate reductase (DHFR). 

Uracil, produced by the pyrimidine synthesis pathway, is not a component of DNA. Rather, DNA contains thymine, a methylated analog of uracil. Another step is required to generate thymidylate from uracil. Thymidylate synthase catalyzes this finishing touch deoxyuridylate (dUMP) is methylated to thymidylate (TMP). As will be discussed in Chapter 27. the methylation of this nucleotide facilitates the identification of DNA damage for repair and, hence, helps preserve the integrity of the genetic information stored in DNA. The methyl donor in this reaction is N, N methylenetetrahydrofolate rather than -S-adenosylmethionine. 

Figure 25.13. Thymidylale Synthesis. Thymidylate synthase catalyzes the addition of a methyl group (derived from TV 5 N 10-methylenetertahydrofolate to dUMP to form TMP. The addition of a thiolate from the enzyme activates dUMP. Opening the five-membered ring of the THF derivative prepares the methylene group for nucleophilic attack by the activated dUMP. The reaction is completed by the transfer of a hydride ion to form dihydrofolate.

A more complete and refined diagram of 1-carbon metabolism is given by Figure 9,6, The cycle of reactions that regenerate methionine is shown at the center, as in the previous diagrams, Thymidylate synthase (TS), at the left, catalyzes the conversion of deoxyuridylic acid (dUMF) to thymidylic acid (dTMP),... 

All cells, especially rapidly growing cells, must synthesize thymidylate (dTMP) for DNA synthesis. The difference between (T) and (U) is one methyl gronp at the carbon-5 position. Thymidylate is synthesized by the methyla-tion of uridylate (dUMP) in a reaction catalyzed by the enzyme thymidylate synthase. This reaction reqnires a methyl donor and a source of reducing eqnivalents, which are both provided by N, N °-methylene THF (Figure 3-2). For this reaction to continue, the regeneration of THF from dihydrofolate (DHF) must occur. 

Figure 3-2. Thymidylate synthesized by the methylation of uridylate (dUMP) in a reaction catalyzed by the enzyme thymidylate synthase.

Thymidylate (dTMP) is formed intracellularly either de novo, in a process of the C(5) methylation of 2 -deoxyuridylate (dUMP), catalyzed by the enzyme thymidylate synthase (TS), or as a product of thymidine salvage via phosphorylation, catalyzed by the enzyme thymidine kinase. The dUMP methylation reaction involves a concerted transfer and reduction of the one-carbon group of... 

Enzyme Assays Activities of thymidylate synthase (Rode et al. 1990), thymidine kinase (Tsukamoto et al. 1991) and dihydrofolate reductase (Mathews et al. 1963) were assayed according to previously published procedures. dUTP-ase activity was determined by coupling with the thymidylate synthase-catalyzed reaction and measuring tritium released from [ H]dUMP (Golos and Rode 1999). The activity unit was defined as the enzyme amount required to convert 1 pmol of substrate per 1 min at 37°C. 

The deoxyuridylate (dUMP) produced by dephosphorylation of the dUDP product of ribonucleotide reductase is not a component of DNA, but its methylated derivative deoxythymidylate (dTMP) is. The methylation of dUMP is catalyzed by thymidylate synthase, which utilizes N5,N10-methylene THF. As the methylene group is transferred, it is reduced to a methyl group, while the folate coenzyme is oxidized to form dihydrofolate. THF is regenerated from dihydrofolate by dihydrofolate reductase and NADPH. (This reaction is the site of action of some anticancer drugs, such as methotrexate.) Deoxyuridylate can also be synthesized from dCMP by deoxycytidylate deaminase. 

Relatively recently, it was found that certain bacteria including Chlamydia trachomatis use thymidylate synthases requiring flavocoenzymes (ThyX protein, EC 2.1.1.148) as donors of reduction equivalents. Although both enzymes, ThyA and ThyX, catalyze the formation of thymidylate in vitro, their reductive mechanisms are dramatically different. ThyX catalysis results in the formation of THE, and not dihydrofolate, as the product of the methylation reaction. ... 

Thymidylate synthase catalyzes the conversion of 2 -deoxyuridine 5 -phosphate to thymidine 5 -phosphate, and it is the sole source of this essential component of DNA. The reaction proceeds by transfer of a one-carbon fragment from the cofactor, 5,10-methylene tetrahydrofolate (CH2H4folate), of the... 

Note that conversion of dUMP to dTMP requires transfer of a single carbon from 5,10-methylenetetrahydrofolate in the reaction catalyzed by thymidylate synthase. The relationship between thymidylate synthase and the enzymes of tetrahydrofolate metabolism is shown in Figure... 

The reaction catalyzed by thymidylate synthase is the only one known in the cell in which tetrahydrofolate is not regenerated. Dihyrofolate reductase, thus, plays an essential role in the ultimate regeneration of 5,10-methylene tetrahydrofolate. This enzyme, which can be inhibited by the drug methotrexate, is a target of some anticancer treatments. 

Thymidylate synthase is an enzyme that catalyzes the reaction that follows (Figure 22.18) dUMP + 5,10-Methylene-THF <=> dTMP + Dihydrofolate... 

Fluorodeoxyuridine monophosphate (FdUMP) is a molecule that is a mechanism-based inhibitor. Irreversible binding of the substance to thymidylate synthase occurs only in the presence of 5,10-methylenetetrahydrofolate, a cofactor for the reaction catalyzed by thymidylate synthase (Figure 22.18). Crystallographic analysis of thymidylate synthase with dUMP and an analog of 5,10-methylenetetrahydrofolate (that could not be acted on by the enzyme) revealed that thymidylate synthase normally makes a transient covalent bond in the process of catalysis of the reaction. Apparently FdUMP s structure traps the enzyme-substrate covalent bond and prevents it from breaking down. 

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