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pathways are important in cellular metabolism and both are presented in this section.

The de novo pathways for purine and pyrimidine biosynthesis appear to be nearly identical in all living organisms. Notably, the free bases guanine, adenine, thymine, cytidine, and uracil are not intermediates in these pathways; that is, the bases are not synthesized and then attached to ribose, as might be expected. The purine ring structure is built up one or a few atoms at a time, attached to ribose throughout the process. The pyrimidine ring is synthesized as orotate, attached to ribose phosphate, and then converted to the common pyrimidine nucleotides required in nucleic acid synthesis. Although the free bases are not intermediates in the de novo pathways, they are intermediates in some of the salvage pathways.

Several important precursors are shared by the de novo pathways for synthesis of pyrimidines and purines.

Phosphoribosyl pyrophosphate (PRPP) is important in both, and in these pathways the structure of ribose is retained in the product nucleotide, in contrast to its fate in the tryptophan and histidine biosynthetic pathways discussed earlier. An amino acid is an important precursor in each type of pathway: glycine for purines and aspartate for pyrimidines. Glutamine again is the most important source of amino groups—in five different steps in the de novo pathways. Aspartate is also used as the source of an amino group in the purine pathways, in two steps.

Two other features deserve mention. First, there is evidence, especially in the de novo purine pathway, that the enzymes are present as large, multienzyme complexes in the cell, a recurring theme in our discussion of metabolism. Second, the cellular pools of nucleotides (other than ATP) are quite small, perhaps 1% or less of the amounts required to synthesize the cell's DNA. Therefore, cells must continue to synthesize nucleotides during nucleic acid synthesis, and in some cases nu-cleotide synthesis may limit the rates of DNA replication and transcription. Because of the importance of these processes in dividing cells, agents that inhibit nu-cleotide synthesis have become particularly important to modern medicine.

We examine here the biosynthetic pathways of purine and pyrimidine nucleotides and their regulation, the formation of the deoxynucleotides, and the degradation of purines and pyrimidines to uric acid and urea. We end with a discussion of chemotherapeutic agents that affect nucleotide synthesis.

De Novo Purine Nucleotide Synthesis Begins with PRPP

The two parent purine nu-cleotides of nucleic acids are adenosine 5'-monophosphate (AMP; adenylate) and guano-sine 5'-monophosphate (GMP; guanylate), containing the purine bases adenine and gua-nine. Figure 22-32 shows the origin of the carbon and nitrogen atoms of the purine ring system, as determined by John Buchanan using isotopic tracer experiments in birds. The detailed pathway of purine biosynthesis was worked out primarily by Buchanan and G. Robert Greenberg in the 1950s.

In the first committed step of the pathway, an amino group donated by glutamine is attached at C-1 of PRPP (Fig. 22-33). The resulting 5-phosphoribosylamine is highly unstable, with a half-life of 30 seconds at pH 7.5. The purine ring is subsequently built up on this structure. The pathway described here is identical in all organisms, with the exception of one step that differs in higher eukaryotes as noted below.

John Buchanan

Aspartate

Glycine

Formate

Amide N of glutamine

FIGURE 22-32 Origin of the ring atoms of purines. This information was obtained from isotopic experiments with 14C- or 15N-labeled precursors. Formate is supplied in the form of N10-formyltetrahydrofolate.

The second step is the addition of three atoms from glycine (Fig. 22-33, step ©). An ATP is consumed to activate the glycine carboxyl group (in the form of an acyl phosphate) for this condensation reaction. The added glycine amino group is then formylated by N10-formyltetrahydrofolate (step (3)), and a nitrogen is contributed by glutamine (step (4), before dehydration and ring closure yield the five-membered imidazole ring of the purine nucleus, as 5-aminoimidazole ribonucleotide (AIR; step ©).

At this point, three of the six atoms needed for the second ring in the purine structure are in place. To complete the process, a carboxyl group is first added (step ©). This carboxylation is unusual in that it does not require biotin, but instead uses the bicarbonate generally present in aqueous solutions. A rearrangement transfers the carboxylate from the exocyclic amino group to position 4 of the imidazole ring (step ©). Steps © and (7) are found only in bacteria and fungi. In higher eukary-otes, including humans, the 5-aminoimidazole ribonucleotide product of step © is carboxylated directly to carboxyaminoimidazole ribonucleotide in one step instead of two (step The enzyme catalyzing this reaction is AIR carboxylase.

Aspartate now donates its amino group in two steps (© and ©): formation of an amide bond, followed by elimination of the carbon skeleton of aspartate (as fu-marate). Recall that aspartate plays an analogous role in two steps of the urea cycle (see Fig. 18-10). The final carbon is contributed by N10-formyltetrahydrofolate (step 10), and a second ring closure takes place to yield the second fused ring of the purine nucleus (step

FIGURE 22-33 (facing page) De novo synthesis of purine nucleotides: construction of the purine ring of inosinate (IMP). Each addition to the purine ring is shaded to match Figure 22-32. After step R symbolizes the 5-phospho-D-ribosyl group on which the purine ring is built. Formation of 5-phosphoribosylamine (step d) is the first committed step in purine synthesis. Note that the product of step AICAR, is the remnant of ATP released during histidine biosynthesis (see Fig. 22-20, step ©). Abbreviations are given for most intermediates to simplify the naming of the pathway enzymes. Step (6^ is the alternative path from AIR to CAIR occurring in higher eukaryotes.

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