BIOCHEMISTRY - L. Stryer - 1984

VOLUME 2

PART III. BIOSYNTHESIS OF MACROMOLECULAR PRECURSORS

CHAPTER 22. NUCLEOTIDE BIOSYNTHESIS

22.6. Purine Bases Can Be Reused Through Salvage Pathways Involving PRPP

Free purine bases are formed by the hydrolytic Cleavage of Nucleic Acids and NUCLEOTIDES. Purine nucleotides can be synthesized from these preformed bases via Salvage Pathways. These reactions are simpler than the de novo synthesis pathways discussed above and are much less costly to The Cell. In the salvage pathway, the ribose phosphate group of PRPP is transferred to a purine to form the corresponding nucleotide.

Two salvage Enzymes with different specificities are involved in these pathways. Adenine phosphoribosyltransferase catalyzes The formation of adenylate:

Adenine + PRPP → Adenylate + PPi

Hypoxanthine-guanine phosphoribosyltransferase catalyzes the formation of inosinate and guanylate:

Hypoxanthine + PRPP → Inosinate + PPi

Guanine + PRPP → Guanylate + PPi.

The diversity and efficiency of purine utilization pathways can also be demonstrated by The Biosynthesis of Histidine. A six-membered fragment of the purine ring of ATP is incorporated into the imidazole ring of histidine (Section 21.10). The remainder of the purine Skeleton is not wasted either: it is conserved as the ribonucleotide 5-aminoimidazole-4-carboxamide, an intermediate in the de novo biosynthesis of the purine ring.

22.7. AMP and GMP Are Feedback Inhibitors of Purine Nucleotide Biosynthesis

Purine nucleotide synthesis is regulated by feedback inhibition at several steps (Fig. 22.10).

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Fig. 22.10. Regulation of Purine Biosynthesis

1. The concentration of PRPP is regulated by the inhibition of 5-phosphoribosyl-1-pyrophosphate synthetase by purine nucleotides. This synthetase is inhibited by AMP, GMP, and IMP.

2. The committed step in purine nucleotide biosynthesis is The conversion of PRPP into phosphoribosylamine by The transfer of the amino group from the glutamine side chain. Glutamine-phosphoribosylpyrophosphate amidotransferase is feedback-inhibited by many purine ribonucleotides. Notably, the inhibitory effects of AMP and GMP on this enzyme are synergistic.

3. The formation of inosinate is the branch point in the synthesis of AMP and GMP. The pathway reactions leading from inosinate to AMP and GMP are feedback-inhibited. AMP inhibits the conversion of inosinate to adenylosuccinate, its immediate precursor. Similarly, GMP inhibits the conversion of inosinate to xanthylate, the immediate precursor of GMP.

4. GTP serves as a substrate for the synthesis of AMP, whereas ATP is the substrate for the synthesis of GMP. This reciprocal substrate relationship is essential for balancing the synthesis of adenine and guanine ribonucleotides.

22.8. The Pyrimidine Ring Is Synthesized from Carbamoyl Phosphate and Aspartate

In contrast to the de novo pathway of purine nucleotide synthesis, the pyrimidine ring is assembled first and only then attached to ribose phosphate to form a pyrimidine nucleotide. As in purine nucleotide synthesis, PRPP serves as the donor of the ribose phosphate group. The precursors of the pyrimidine ring are carbamoyl phosphate and aspartate.

Pyrimidine synthesis begins with the formation of carbamoyl phosphate, which also serves as an intermediate in urea synthesis (Section 18.4). The synthesis of this activated carbamoyl group donor is compartmentalized in Eukaryotic Cells. Carbamoyl phosphate used for pyrimidine synthesis is produced in the Cytosol, whereas carbamoyl phosphate used for urea synthesis is generated in the Mitochondria (Section 18.5). There are two distinct carbamoyl phosphate synthetases. Another major difference is that in cytosolic carbamoyl phosphate synthesis, glutamine, rather than NH₄⁺, serves as the nitrogen donor:

Glutamine + 2ATP + HCO₃^- → Carbamoyl phosphate + 2ADP + Pi + Glutamate.

The committed step in pyrimidine biosynthesis is the formation of N-carbamoylaspartate from aspartate and carbamoyl phosphate. This carbamoylation is catalyzed by aspartate transcarbamoylase (aspartate carbamoyltransferase, ATCase), an enzyme of particular regulatory interest (Section 22.14). The pyrimidine ring is formed in the next reaction, in which carbamoylaspartate cyclizes with the loss of a Water molecule to yield dihydroorotate. Dihydroorotate is then oxidized to form orotate.

22.9. Orotate acquires a ribose phosphate moiety from PRPP

The next step in the synthesis of pyrimidine nucleotides is the acquisition of a ribose phosphate group. Orotate (a free pyrimidine) reacts with PRPP to form orotidylate (a pyrimidine nucleotide). This reaction, catalyzed by orotidylate pyrophosphorylase, is driven by the Hydrolysis of pyrophosphate. Orotidylate is then decarboxylated to form uridylate (uridine monophosphate, UMP), a major pyrimidine nucleotide.

22.10. The first three enzymes of pyrimidine biosynthesis are synthesized as a single polypeptide chain

In E. coli, the six enzymes that synthesize UMP from simple precursors appear to be independent of one another. In contrast, in higher organisms, several of these enzymes form a multienzyme complex. The level of this complex in mammalian cells is greatly increased by N-(phosphonacetyl)-L-aspartate (PALA), a potent inhibitor of aspartate transcarbamoylase (ATCase). PALA binds tightly to ATCase (Ki = 10^-8 M) because it has a certain structural similarity to the Transition State arising during catalysis (Fig. 22.12). Surviving cells overcome the inhibitory effect of PALA by producing 100 times as much ATCase as do normal cells. The concentrations of carbamoyl phosphate synthetase and dihydroorotase are also increased 100-fold, whereas the activities of the enzymes catalyzing the subsequent steps of pyrimidine biosynthesis are virtually unchanged. These findings established the fact that carbamoyl phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase are covalently linked within a single polypeptide chain with a molecular mass of 200 kDa. The enzymes catalyzing the last two steps of pyrimidine biosynthesis, orotate phosphoribosyltransferase and orotidylate decarboxylase, form another com

plex. They, too, may be covalently linked. Recall that the Yeast fatty acid synthase complex consists of Two Types of polypeptide chains, each containing several enzymes (Section 17.20). In all probability, the covalent linkage of functionally related enzymes is a general phenomenon in eukaryotes. It may facilitate the assembly of multienzyme complexes. Another postulated advantage of joining several enzymes in one polypeptide chain is that they are synthesized in equimolar amounts.

Fig. 22.12. Structure of PALA, a potent inhibitor of ATCase