Biosynthesis and Degradation of Nucleotides:- Pyrimidine Nucleotides Are Made from Aspartate, PRPP, and Carbamoyl Phosphate
The common pyrimidine ribonucleotides are cytidine 5 monophosphate (CMP; cytidylate) and uridine 5 monophosphate (UMP; uridylate), which contain the pyrimidines cytosine and uracil. De novo pyrimidine nu cleotide biosynthesis (Fig. 1) proceeds in a somewhat different manner from purine nucleotide synthesis; the six-membered pyrimidine ring is made first and then attached to ribose 5-phosphate. Required in this process is carbamoyl phosphate, also an intermediate in the urea cycle . However, as we noted in animals the carbamoyl phosphate re quired in urea synthesis is made in mitochondria by carbamoyl phosphate synthetase I, whereas the carbamoyl phosphate required in pyrimidine biosynthesis is made in the cytosol by a different form of the enzyme, carbamoyl phosphate synthetase II. In bacteria, a single enzyme supplies carbamoyl phosphate for the syn thesis of arginine and pyrimidines. The bacterial enzyme has three separate active sites, spaced along a channel nearly 100 Å long (Fig. 2). Bacterial carbamoyl phosphate synthetase provides a vivid illustration of the channeling of unstable reaction intermediates between active sites. Carbamoyl phosphate reacts with aspartate to yield N-carbamoyl aspartate in the first committed step of pyrimidine biosynthesis (Fig. 1). This reaction is catalyzed by aspartate transcarbamoylase. In bacteria, this step is highly regulated, and bacterial aspartate transcarbamoylase is one of the most thoroughly studied allosteric enzymes (see below). By removal of water from N-carbamoyl aspartate, a reaction catalyzed by dihydroorotase, the pyrimidine ring is closed to form L-dihydroorotate. This compound is oxidized to the pyrimidine derivative orotate, a reaction in which NAD is the ultimate electron acceptor. In eukaryotes, the first three enzymes in this pathway—carbamoyl phosphate synthetase II, aspartate transcarbamoylase, and dihydroorotase—are part of a single trifunctional protein. The protein, known by the acronym CAD, contains three identical polypeptide chains (each of Mr 230,000), each with active sites for all three reactions. This suggests that large, multienzyme complexes may be the rule in this pathway. Once orotate is formed, the ribose 5-phosphate side chain, provided once again by PRPP, is attached to yield orotidylate (Fig. 1). Orotidylate is then decarboxylated to uridylate, which is phosphorylated to UTP. CTP is formed from UTP by the action of cytidylate synthetase, by way of an acyl phosphate intermediate (consuming one ATP). The nitrogen donor is normally glutamine, although the cytidylate synthetases in many species can use NH4 directly.
FIGURE 1 De novo synthesis of pyrimidine nucleotides: biosyn thesis of UTP and CTP via orotidylate. The pyrimidine is constructed from carbamoyl phosphate and aspartate. The ribose 5-phosphate is then added to the completed pyrimidine ring by orotate phosphori-bosyltransferase. The first step in this pathway is the synthesis of carbamoyl phosphate from CO2 and NH4, catalyzed in eukaryotes by carbamoyl phosphate synthetase II.
FIGURE 2 Channeling of intermediates in bacterial carbamoyl phosphate synthetase. (Derived from PDB ID 1M6V.) The reaction cat alyzed by this enzyme is illustrated in Figure 18–11a. The large and small subunits are shown in gray and blue, respectively; the channel between active sites (almost 100 Å long) is shown as a yellow mesh. A glutamine molecule (green) binds to the small subunit, donating its amido nitrogen as NH4 in a glutamine amidotransferase–type reaction. The NH4 enters the channel, which takes it to a second active site, where it combines with bicarbonate in a reaction requiring ATP (bound ADP in blue). The carbamate then reenters the channel to reach the third active site, where it is phosphorylated to carbamoyl phosphate (bound ADP in red).
