24.2. Pyrimidine Biosynthesis
Pyrimidine nucleotide biosynthesis is a much more straight forward process. C2 of the pyrimidine ring comes from HCO3– (CO2), N3 comes from glutamine, and the remainder of the pyrimidine molecule (N1, C4, C5 and C6) comes from a molecule of Aspartate.
The pyrimidine ring is synthesized in four steps and then joined to PRPP to form the nucleoside-5´-monophosphate. This is different from purine synthesis where the ring is built step by step on ribose-5´-phosphate starting with PRPP. The pathway for the formation of pyrimidine nucleotides begins with the formation of carbamoylphosphate. This reaction is catalyzed by Carbamoylphosphate Synthetase II. Carbamoyl phosphate synthetase II convert glutamine into carbamoyl phosphate. This is the regulated step in the pyrimidine biosynthesis. The first intermediate is carbamoylphosphate. The corresponding enzyme, carbamoylphosphate synthetase 2, uses glutamine as the nitrogen donor. In contrast, carbamoylphosphate synthetase 1, which occurs in the urea cycle, uses free ammonia directly. Carbamoylphosphate Synthetase II is a cytosolic enzyme whereas Carbamoylphosphate Synthetase I is a mitochondrial enzyme. In the second reaction, aspartate transcarbamylase transfers the carbamoyl group of carbamoylphosphate to the α-amino group of aspartate, which yields carbamoylaspartate. Dihydroorotase catalyzes a dehydration reaction that results in closure of the pyrimidine ring. Dihydroorotate is the product of this reaction. The first three enzymes of the pathway; Carbamoyl. Phosphate Synthetase II, Aspartate Transcarbamoylase, and Dihydroorotase; are contained on a single multifunctional protein present in the cytoplasm.
Orotate phosphoribosyltransferase (5) forms orotidine-5′-monophosphate (OMP), in a reaction that resembles purine salvage. Indeed, this enzyme has relatively broad specificity and also functions in the salvage of uracil and in the activation of its analogue 5-fluorouracil. The dihydroorotate is oxidized to orotate by Dihydroorotate Dehydrogenase. In eukaryotes the enzyme is bound to the inner mitochondrial membrane. The electrons are immediately accepted by a quinine of electron transport chain and produce ATP. Orotate is now coupled to PRPP to form Orotidine-5´-monophosphate (OMP). This reaction is catalyzed by Orotate phosphoribosyl Transferase. Cytidine nucleotides are synthesized from UMP. However, before the uridine base can be converted to cytidine the UMP must be phosphorylated to UTP. The UTP is then converted to Cytidine-5´-triphosphate (CTP) by CTP Synthetase.
24.2.1 Control of Pyrimidine Biosynthesis
Pyrimidine nucleotide biosynthesis is controlled at the step catalyzed by Carbamoyl phosphate Synthetase II. This is an allosteric enzyme. PRPP and ATP activate the enzyme and UDP and UTP are allosteric inhibitors of its activity.
The four ribonucleotides obtained from the biosynthesis pathways - AMP, GMP, UMP, and CTP are reduced to the deoxyribonucleotides needed for DNA synthesis. Before they can be reduced to deoxyribonucleotides they must all be converted to the nucleoside diphosphate forms. The addition or removal of phosphate from the various nucleotides is accomplished by the Nucleoside Monophosphate Kinases or Nucleoside Diphosphate Kinase.
The reduction reaction is catalyzed by the enzyme ribonucleotide reductase. This enzyme has three sites an Activity Site, a Specificity Site, and the catalytic site. The Activity Site turns the enzyme “ON” or “OFF”; the Specificity Site controls which nucleotide will be reduced; and the catalytic site performs the reduction. When the Activity Site is occupied by ATP the enzyme is turned “ON”. When the Activity Site is occupied by deoxy ATP the enzyme is turned “OFF”.
When the Specificity Site is occupied by ATP or deoxy ATP (dATP) then CDP or UDP is reduced.
When the Specificity Site is occupied by deoxyTTP (dTTP) then GDP is reduced. When the Specificity Site is occupied by deoxyGTP (dGTP) then ADP is reduced. The Specificity Site assures that the deoxyribonucleotides are synthesized in balanced and adequate amounts.
Remember that in DNA A pairs with T and G pairs with C.
- When the concentration of ATP is high, the cell is rich in energy, it has the energy to synthesize DNA and divide. ATP binds to the activity site to turn the enzyme “ON”. ATP also binds to the specificity site to stimulate the reduction of the pyrimidines, UDP and CDP. DeoxyUDP is the precursor of deoxythymidine (dTTP), the base pair partner of dATP in DNA high ATP concentrations stimulate the synthesis of its partner in DNA and the partner of deoxyguanosine.
- As dTTP concentrations build up it signals that the deoxy pyrimidines are present in adequate amounts for DNA replication. dTTP binds to the specificity site and stimulates the reduction of one of the purines, GDP to dGDP.
- As dGTP concentrations increase, it binds to the specificity site and stimulates the reduction of the other purine. ADP is reduced to dADP.
- As dATP concentrations increase they signal that all four deoxy nucleotide triphosphates are present in adequate amounts for DNA replication. dATP replaces ATP in the activity site and the enzyme is turned “OFF”.
Formation of DeoxyTMP from DeoxyUMP
Deoxyuridylate nucleotides are never incorporated into DNA. Two mechanisms assure that the deoxyuridylate nucleotides are not incorporated into DNA. First, the enzyme Deoxyuridine Triphosphate. Diphosphohydrolase rapidly converts any deoxyUTP that is formed to deoxyUMP. Second, the deoxyUMP is rapidly and quantitatively converted to deoxyTMP.
The conversion of deoxyUMP (dUMP) to deoxyTMP (dTMP) is catalyzed by the enzyme thymidylate Synthase. The enzyme, thymidylate synthase catalyzes the transfer of the one carbon methylene fragment from TH4 to C5 of uridine and it simultaneously reduces the one carbon fragment to a methyl group. The products of this reaction are dTMP and dihydrofolate.
Dihydrofolate is useless to the cell. It must be reduced to tetrahydrofolate if cellular metabolism is to be maintained. The reduction process is catalyzed by the enzyme Dihydrofolate Reductase. NADPH and a hydrogen ion (H+) donates the hydrogens and electrons necessary for the reaction. Once the TH4 is reformed it accepts a one carbon fragment from serine or glycine and it is ready for the next cycle of reactions.
The enzyme dihydrofolate reductase was the first target for cancer chemotherapeutic agents. dTMP (dTTP) is needed for DNA replication, inhibiting the formation of dTTP would inhibit DNA replication.
With DNA synthesis inhibited, cancer cells would cease to divide, and the tumor would stop growing. In fact all rapidly dividing cells cease to multiply. The drug methotrexate is a specific competitive inhibitor of the enzyme Dihydrofolate Reductase. This enzyme inhibitor was the first cancer chemotherapeutic agent.
24.4. Purine Salvage Pathways
The synthesis of purines is an energy expensive pathway and only a small amount of energy is recovered during their degradation. To save energy the cell recycles as many of the purine nucleotides as possible using the Purine Salvage Pathways.
During the digestion of food stuffs and cellular metabolism, the purine nucleotides are broken down to phosphate, ribose (deoxyribose) and the bases adenine, guanine, and/or hypoxanthine. Hypoxanthine is the purine base present on Inosine-5´-monophosphate, its the base on IMP.
The purine bases are salvaged by the action of two enzymes. Adenine phosphoribosyl Transferase couples the adenine base to 5-phosphoribosyl-α1-pyrophosphate (PRPP) to form AMP. Hypoxanthine-Guanine phosphoribosyl Transferase joins the hypoxanthine base to PRPP to form IMP and/or it attaches guanine to PRPP to form GMP. Bacteria have a salvage pathway for the pyrimidine bases.
Excess purines and pyrimidines originating from ingested nucleotides or from routine turnover of cellular nucleic acids are catabolized. Most intracellular purine bases are salvaged and pyrimidine salvage probably occurs. Purine breakdown yields only waste products that must be excreted, whereas the pyrimidines yields molecules that can enter metabolism for energy generation.
During the catabolic process AMP can be converted to IMP by AMP Deaminase and then the IMP is converted to inosine (a nucleoside) by the enzyme 5´-Nucleotidase, or AMP is first dephosphorylated to adenosine (a nucleoside) by 5´-Nucleotidase and then the adenosine is converted to inosine by Adenosine Deaminase. The net result of these two pairs of reactions is the conversion of AMP (a nucleotide) to inosine (a nucleoside). The inosine is then phosphorolytically cleaved, phosphate is added across the N-glycosidic bond, to yield the base hypoxanthine and ribose-1-phosphate by the enzyme Purine Nucleoside Phosphorylase.
Hypoxanthine is converted to xanthine by the action of the enzyme Xanthine Oxidase. This enzyme uses molecular oxygen (O2) to oxidize hypoxanthine to xanthine and hydrogen peroxide (H2O2). Hydrogen peroxide is a very destructive compound to have within the cell. It is rapidly and quantitatively destroyed by the enzyme Catalase. Xanthine Oxidase resides in lysosomes and peroxisomes.
Xanthine is then converted to uric acid, the final excretory product in mammals, by a second reaction catalyzed by Xanthine Oxidase. GMP catabolism is similar. GMP is first dephosphorylated to guanosine (a nucleoside) by the action of 5´- Nucleotidase. The guanine base is then released from the nucleoside by Purine Nucleoside Phosphorylase.
The guanine base is converted to xanthine by the enzyme Guanase. Once formed, xanthine is converted to uric acid by the action of Xanthine Oxidase.
In Mammals the uric acid is usually oxidized to Allantoin by Urate Oxidase and the allantoin is the major secretory product.
24.5. Pyrimidine Catabolism
The pyrimidine nucleotides are converted to their respective nucleosides by the action of 5´-Nucleotidase.
Cytidine (nucleoside) is converted to uridine (nucleoside) by the action of Cytidine deaminase.
Ribose is removed from uridine by the enzyme Uridine Phosphorylase to release the free base uracil, and it is removed from thymidine by the action of thymidine phosphorylase to release the free base thymine.
The enzyme dihydrouracil dehydrogenase reduces the bases uracil and thymine to dihydrouracil and dihydrothymine, respectively.
These two compounds are then acted upon by the enzyme. Dihydropyrimidinase to form uridopropionate or uridoisobutyrate.
The enzyme Uridopropionase hydrolytically removes NH4+ and HCO3– from these compounds to form β-alanine (from uracil) and β-aminoisobutyrate (from thymine).
An aminotransferase (Transaminase) converts β-alanine into malonic semialdehyde and converts β-aminoisobutyrate into methylmalonic semialdehyde.
A dehydrogenase complex oxidizes malonic semialdehyde and couples it to coenzyme A to form malonyl-CoA. The malonyl-CoA can enter fatty acid biosynthesis or more likely it is decarboxylated by malonyl-CoA decarboxylase to acetyl-CoA. and the acetyl-CoA oxidized for energy (ATP).
The same or a similar dehydrogenase complex oxidizes methylmalonic semialdehyde and couples it to CoA forming D-methylmalonyl-CoA. D-methylmalonyl-CoA is an intermediate in the metabolism of odd chain length fatty acids and the amino acids, Met, Val, Thr, and Ile. D-methylmalonyl-CoA is ultimately converted to succinyl-CoA as described previously.
After the base is phosphorylytically released from the deoxyribose by nucleoside Phosphorylase or thymidine phosphorylase the phosphate is moved from C-1 to C-5 by phosphopentose mutase to form deoxyribose-5-phosphate. The deoxyribose-5-phosphate is cleaved to ethanal and glyceraldehyde-3-phosphate by 2-deoxyribose-5-phosphate Aldolase. Carbon 1 & 2 becomes the ethanal and 3, 4, &5 become glyceraldehyde-3-phosphate. The glyceraldehyde-3-phosphate enters glycolysis or gluconeogenesis depending upon the tissue and blood glucose levels. Ethanal is oxidized to acetate by aldehyde dehydrogenase and then the acetate is coupled to Coenzyme A by Acetyl-CoA Synthetase. Acetyl-CoA enters any of the pathways that utilizes Acetyl-CoA, most likely the TCA cycle.