Biosynthesis and Degradation of Nucleotides:- De Novo Purine Nucleotide Synthesis Begins with PRPP
The two parent purine nucleotides of nucleic acids are adenosine 5-monophosphate (AMP; adenylate) and guano sine 5-monophosphate (GMP; guanylate), containing the purine bases adenine and guanine. Figure 1 shows the origin of the carbon and nitro gen 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. 2). 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 or ganisms, with the exception of one step that differs in higher eukaryotes as noted below.
FIGURE 1 Origin of the ring atoms of purines. This information was obtained from isotopic experiments with 14C- or 15N-labeled pre cursors. Formate is supplied in the form of N10-formyltetrahydrofolate.
The second step is the addition of three atoms from glycine (Fig. 2, step 2). 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 con tributed 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 5). 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 6). This carboxylation is unusual in that it does not re quire 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 7). Steps 6 and 7 are found only in bacteria and fungi. In higher eukaryotes, including humans, the 5-aminoimidazole ribonucleotide product of step 5 is carboxylated directly to carboxyaminoimidazole ribonucleotide in one step instead of two (step 6a). The enzyme catalyzing this re action is AIR carboxylase. Aspartate now donates its amino group in two steps (8 and 9): formation of an amide bond, followed by elimination of the carbon skeleton of aspartate (as fumarate). 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 11).
FIGURE 2 (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 1. After step 2, R symbolizes the 5-phospho-D-ribosyl group on which the purine ring is built. Formation of 5-phosphoribosylamine (step 1) is the first com mitted step in purine synthesis. Note that the product of step 9, AICAR, is the remnant of ATP released during histidine biosynthesis . Abbreviations are given for most intermediates to simplify the naming of the pathway enzymes. Step 6a is the alternative path from AIR to CAIR occurring in higher eukaryotes.
The first intermediate with a complete purine ring is inosinate (IMP). As in the tryptophan and histidine biosynthetic pathways, the enzymes of IMP synthesis appear to be organized as large, multienzyme complexes in the cell. Once again, evidence comes from the existence of single polypeptides with several functions, some catalyzing nonsequential steps in the pathway. In eukaryotic cells ranging from yeast to fruit flies to chickens, steps 1, 3, and 5 in Figure 2 are catalyzed by a multifunctional protein. An additional multifunctional protein catalyzes steps 10 and 11. In humans, a multifunctional enzyme combines the activities of AIR carboxylase and SAICAR synthetase (steps 6a and 8). In bacteria, these activities are found on separate proteins, but a large noncovalent complex may exist in these cells. The channeling of reaction intermediates from one enzyme to the next permitted by these complexes is probably especially important for unstable intermediates such as 5-phosphoribosylamine. Conversion of inosinate to adenylate requires the insertion of an amino group derived from aspartate (Fig. 3); this takes place in two reactions similar to those used to introduce N-1 of the purine ring (Fig. 2, steps 8 and 9). A crucial difference is that GTP rather than ATP is the source of the high-energy phosphate in synthesizing adenyl succinate. Guanylate is formed by the NAD-requiring oxidation of inosinate at C-2, followed by addition of an amino group derived from glutamine. ATP is cleaved to AMP and PPi in the final step (Fig. 3).
FIGURE 3 Biosynthesis of AMP and GMP from IMP.
