Ribonucleotide Reductase - Metabolic Pathways

Metabolic Pathways

Several major pathways lead to the generation of precursors for the de novo synthesis of nucleotides. These pathways involve the generation of ribose 5-phosphate, carbon dioxide, amino acids and ammonia. Ribose 5-phosphate generation begins with a molecule of glucose that is oxidized via the pentose phosphate pathway. The pentose phosphate pathway produces NADPH for reducing power involved in the catalysis of NTPs to dNTPs, and to produce ribose 5-phosphate necessary for the synthesis of ribonucleotides. Carbon dioxide is always available for biosynthesis because its concentration in the blood is kept nearly constant via the bicarbonate buffer system. An important co-factor for ribonucleotide synthesis is tetrahydrofolate, which is the major mediator for carbon transfers. Its derivative, folate (a vitamin), cannot be synthesized in mammals. Many forms of tetrahydrofolate follow pathways that are interconnected. For ribonucleotide synthesis, the N10-formyl-tetrahydrofolate molecule is necessary for the transfer of formyl groups to the purine ring. Amino groups or ammonia are donated from the catabolism of amino acids beginning with a dietary protein molecule. The free ammonia is combined with glutamate by a reaction involving adenosine 5’-triphosphate (ATP) and the activity of glutamine synthetase, which produces a nontoxic molecule of glutamine that can be transported in the bloodstream. Glutamine synthetase is present in nearly all organisms and is allosterically regulated by end products of glutamine metabolism. During synthesis of purines, amino groups are removed from glutamine for purine rings. Purine ribonucleotides are attached to ribose 5-phosphate during assembly of intermediate inosinate (IMP) from precursors in the purine pathway, including glutamine, glycine, N10-formyl-tetrahydrofolate, bicarbonate, aspartate and ATP. Synthesis is catalyzed by large multienzyme complexes. Purine ribonucleotides are adenosine 5’-monophosphate (AMP) and guanosine 5’-monophosphate (GMP). AMP is formed from IMP by aspartate donating an amino group (leaving as fumarate) and guanosine 5’-triphosphate (GTP) providing a phosphate. GMP is formed by the oxidation of IMP at C-2 requiring NAD+. Following oxidation, glutamine donates an amino group (leaving as glutamate) then ATP provides a phosphate.

Pyrimidine ribonucleotides are formed from an orotate molecule that is assembled from aspartate to form the pyrimidine ring. Subsequently, orotate is attached to ribose 5-phosphate to yield orotidylate. These two steps are catalyzed by a large multienzyme complex (CAD). Pyrimidine ribonucleotides are cytidine 5’-monophosphate (CMP) and uridine 5’-monophosphate (UMP). Orotidylate is decarboxylated to form UMP. UMP and two ATPs are transferred by kinases to form uridine 5’-triphosphate (UTP). Cytidine 5’-triphosphate (CTP) is formed from UTP by glutamine donating an amino group (leaving as glutamate) and ATP providing a phosphate. In some species, ammonia can donate an amino group instead of glutamine.

Generation of 2’-deoxythymidine 5’-monophosphate (dTMP) occurs by conversion of 2’-deoxyuridine 5’-monophosphate (dUMP). Thymidylate synthase catalyzes the reaction in which dTMP is formed from dUMP; to provide the carbon atom N5, N10-methylene-tetrahydrofolate is oxidized to 7, 8-dihydrofolate. Dihydrofolate reductase (DHFR) is an essential enzyme that regenerates tetrahydrofolate at the expense of NADPH. Ribonucleoside monophosphates (AMP, GMP, CMP, and UMP) are phosphorylated to ribonucleoside diphosphates for their particular base by specific kinases. Ribonucleoside diphosphates are phosphorylated a second time to ribonucleoside triphosphates by nucleoside-diphosphate kinase, which is not specific for their base or for their 2’-carbon of ribose 5-phosphate and its 2’-deoxy derivative. The activity of nucleoside diphosphate kinase is sequential based on which class of RNR is used. These metabolic pathways generate the ribonucleotides (ATP, GTP, CTP, and UTP) that are precursors for dNTPs. Thus, RNR reduces the corresponding NTPs to dNTPs for DNA synthesis. Cellular concentration of dNTP is much lower than required for DNA replication, and RNR is essential for adequate precursors during DNA synthesis. After RNR reduces NDP or NTP the enzyme becomes inactive because a disulfide bond is formed in the active site. An exchange reaction occurs that reduces the disulfide bond of RNR catalyzed by thioredoxin or glutaredoxin. RNR gains electrons on the active-site dithiol groups necessary for its activity.