We turn now to the synthesis of deoxyribonucleotides. These precursors of DNA are formed by the reduction of ribonucleotides; specifically, the 2′-hydroxyl group on the ribose moiety is replaced by a hydrogen atom. The substrates are ribonucleoside diphosphates, and the ultimate reductant is NADPH. The enzyme ribonucleotide reductase is responsible for the reduction reaction for all four ribonucleotides. The ribonucleotide reductases of different organisms are a remarkably diverse set of enzymes. Yet detailed studies have revealed that they have a common reaction mechanism, and their three-dimensional structural features indicate that these enzymes are homologous. We will focus on the best understood of these enzymes, that of E. coli living aerobically.

The ribonucleotide reductase of E. coli consists of two subunits: R1 (an 87-kDa dimer) and R2 (a 43-kDa dimer). The R1 subunit contains the active site as well as two allosteric control sites (Section 25.4). This subunit includes three conserved cysteine residues and a glutamate residue, all four of which participate in the reduction of ribose to deoxyribose (Figure 25.9). The R2 subunit’s role in catalysis is to generate a free radical in each of its two chains. Each R2 chain contains a tyrosyl radical with an unpaired electron delocalized onto its aromatic ring, generated by a nearby iron center consisting of two ferric (Fe³⁺) ions bridged by an oxide (O²⁻) ion (Figure 25.10). This very unusual free radical is remarkably stable, with a half-life of 4 days at 4^°C. By contrast, free tyrosine radicals in solution have microsecond lifetimes.

In the synthesis of a deoxyribonucleotide, the OH bonded to C-2′ of the ribose ring is replaced by H, with retention of the configuration at the C-2′ carbon atom (Figure 25.11).

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The electrons for this reduction come from NADPH, but not directly. One carrier of reducing power linking NADPH with the reductase is thioredoxin, a 12- kDa protein with two exposed cysteine residues near each other. These sulfhydryls are oxidized to a disulfide in the reaction catalyzed by ribonucleotide reductase itself. In turn, reduced thioredoxin is regenerated by electron flow from NADPH. This reaction is catalyzed by thioredoxin reductase, a flavoprotein. Electrons flow from NADPH to bound FAD of the reductase, to the disulfide of oxidized thioredoxin, and then to ribonucleotide reductase and finally to the ribose unit.

Ribonucleotide reductases that do not contain tyrosyl radicals have been characterized in other organisms. Instead, these enzymes contain other stable radicals that are generated by other processes. For example, in one class of reductases, the coenzyme adenosylcobalamin (vitamin B₁₂) is the radical source (Section 22.3). Despite differences in the stable radical employed, the active sites of these enzymes are similar to that of the E. coli ribonucleotide reductase, and they appear to act by the same mechanism, based on the exceptional reactivity of cysteine radicals. Thus, these enzymes have a common ancestor but evolved a range of mechanisms for generating stable radical species that function well under different growth conditions. The primordial enzymes appear to have been inactivated by oxygen, whereas enzymes such as the E. coli enzyme make use of oxygen to generate the initial tyrosyl radical. Note that the reduction of ribonucleotides to deoxyribonucleotides is a difficult reaction chemically, likely to require a sophisticated catalyst. The existence of a common protein enzyme framework for this process strongly suggests that proteins joined the RNA world before the evolution of DNA as a stable storage form for genetic information.

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). Recall that thymidylate synthase also functions in the thymine salvage pathway. As will be described in Chapter 28, the methylation of this nucleotide marks sites 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 N⁵, N¹⁰-methylenetetrahydrofolate rather than S-adenosylmethionine (Section 24.2).

The methyl group becomes attached to the C-5 atom within the aromatic ring of dUMP, but this carbon atom is not a good nucleophile and cannot itself attack the appropriate group on the methyl donor. Thymidylate synthase promotes methylation by adding a thiolate from a cysteine side chain to this ring to generate a nucleophilic species that can attack the methylene group of N⁵, N¹⁰-methylenetetrahydrofolate (Figure 25.12). This methylene group, in turn, is activated by distortions imposed by the enzyme that favor opening the five-membered ring. The activated dUMP’s attack on the methylene group forms the new carbon–carbon bond. The intermediate formed is then converted into product: a hydride ion is transferred from the tetrahydrofolate ring to transform the methylene group into a methyl group, and a proton is abstracted from the carbon atom bearing the methyl group to eliminate the cysteine and regenerate the aromatic ring. The tetrahydrofolate derivative loses both its methylene group and a hydride ion and, hence, is oxidized to dihydrofolate. For the synthesis of more thymidylate, tetrahydrofolate must be regenerated.

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Tetrahydrofolate is regenerated from the dihydrofolate that is produced in the synthesis of thymidylate. This regeneration is accomplished by dihydrofolate reductase with the use of NADPH as the reductant.

A hydride ion is directly transferred from the nicotinamide ring of NADPH to the pteridine ring of dihydrofolate. The bound dihydrofolate and NADPH are held in close proximity to facilitate the hydride transfer.

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Rapidly dividing cells require an abundant supply of thymidylate for the synthesis of DNA. The vulnerability of these cells to the inhibition of TMP synthesis has been exploited in the treatment of cancer. Thymidylate synthase and dihydrofolate reductase are choice targets of chemotherapy (Figure 25.13).

Fluorouracil, an anticancer drug, is converted in vivo into fluorodeoxyuridylate (F-dUMP). This analog of dUMP irreversibly inhibits thymidylate synthase after acting as a normal substrate through part of the catalytic cycle. Recall that the formation of TMP requires the removal of a proton (H⁺) from C-5 of the bound nucleotide (Figure 25.12). However, the enzyme cannot abstract F⁺ from F-dUMP, and so catalysis is blocked at the stage of the covalent complex formed by F-dUMP, methylenetetrahydrofolate, and the sulfhydryl group of the enzyme (Figure 25.14). We see here an example of suicide inhibition, in which an enzyme converts a substrate into a reactive inhibitor that halts the enzyme’s catalytic activity (Section 8.5).

The synthesis of TMP can also be blocked by inhibiting the regeneration of tetrahydrofolate. Analogs of dihydrofolate, such as aminopterin and methotrexate (amethopterin), are potent competitive inhibitors (K_i < 1 nM) of dihydrofolate reductase.

Methotrexate is a valuable drug in the treatment of many rapidly growing tumors, such as those in acute leukemia and choriocarcinoma, a cancer derived from placental cells. However, methotrexate kills rapidly replicating cells whether they are malignant or not. Stem cells in bone marrow, epithelial cells of the intestinal tract, and hair follicles are vulnerable to the action of this folate antagonist, accounting for its toxic side effects, which include weakening of the immune system, nausea, and hair loss.

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Folate analogs such as trimethoprim have potent antibacterial and antiprotozoal activity. Trimethoprim binds 10⁵-fold less tightly to mammalian dihydrofolate reductase than it does to reductases of susceptible microorganisms. Small differences in the active-site clefts of these enzymes account for the highly selective antimicrobial action. The combination of trimethoprim and sulfamethoxazole (an inhibitor of folate synthesis) is widely used to treat infections such as bronchitis, traveler’s diarrhea, and urinary tract infections.