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in Chapter 18, in animals the carbamoyl phosphate required in urea synthesis is made in mitochondria by car-bamoyl 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 synthesis of arginine and pyrimidines. The bacterial enzyme has three separate active sites, spaced along a channel nearly 100 A long (Fig. 22-37). 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 A-carbamoylaspartate in the first committed step of pyrimidine biosynthesis (Fig. 22-36). 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 A-carbamoylaspartate, 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 dihy-droorotase—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. 22-36). Orotidylate is then decarboxy-lated to uridylate, which is phosphorylated to UTP. CTP is formed from UTP by the action of cytidylate syn-thetase, 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.

Pyrimidine Nucleotide Biosynthesis Is Regulated by Feedback Inhibition

Regulation of the rate of pyrimidine nucleotide synthesis in bacteria occurs in large part through aspartate transcarbamoylase (ATCase), which catalyzes the first reaction in the sequence and is inhibited by CTP, the end product of the sequence (Fig. 22-36). The bacterial ATCase molecule consists of six catalytic subunits and six regulatory subunits (see Fig. 6-27). The catalytic subunits bind the substrate molecules, and the allosteric subunits bind the allosteric inhibitor, CTP. The entire ATCase molecule, as well as its subunits, exists in two conformations, active and inactive. When CTP is

FIGURE 22-37 Channeling of intermediates in bacterial carbamoyl phosphate synthetase. (Derived from PDB ID 1M6V.) The reaction catalyzed 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 A 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).

FIGURE 22-37 Channeling of intermediates in bacterial carbamoyl phosphate synthetase. (Derived from PDB ID 1M6V.) The reaction catalyzed 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 A 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).

not bound to the regulatory subunits, the enzyme is maximally active. As CTP accumulates and binds to the regulatory subunits, they undergo a change in conformation. This change is transmitted to the catalytic sub-units, which then also shift to an inactive conformation. ATP prevents the changes induced by CTP. Figure 22-38 shows the effects of the allosteric regulators on the activity of ATCase.

Nucleoside Monophosphates Are Converted to Nucleoside Triphosphates

Nucleotides to be used in biosynthesis are generally converted to nucleoside triphosphates. The conversion pathways are common to all cells. Phosphorylation of AMP to ADP is promoted by adenylate kinase, in the reaction a.

Normal activity (no CTP)

Normal activity (no CTP)

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