Nucleotide Metabolism Notes
Nucleotides are important for reasons besides being precursors of nucleic acids. Most of them provide energy used to drive biochemical reactions. ATP is the most commonly used source, but GTP is used in protein synthesis as well as a few other reactions. UTP is the source of energy for activating glucose in glycogen synthesis. CTP is an energy source in lipid metabolism (phosphatidyl syntheses). AMP is part of the structure of coenzymes like NAD and Coenzyme A. Nucleotides also play important roles in signal transduction (cellular signaling).
Nucleotides contain three main components - a sugar, a base, and at least one phosphate. The related molecules called nucleosides contain only a sugar and a base.
Nucleotides contain one of two kinds of sugar. Deoxyribonucleotides derive their name from the fact that they contain the sugar deoxyribose whereas ribonucleotides contain the sugar ribose. The sugar ribose is a product of the pentose phosphate pathway. Deoxyribose is not synthesized, as such, in the cell but, as we shall see, is produced by action of the enzyme ribonucleotide reductase on ribonucleotide diphosphates.
There are two kinds of nitrogen-containing bases in nucleic acids: purines and pyrimidines. Purines consist of two fused nitrogen-containing rings with a total of nine ring atoms. Pyridmidines have only a six-membered nitrogen-containing ring. Purines and pyrimidines are "flat", hydrophobic, aromatic molecules that absorb ultraviolet light (260 nanometers). There are two purines and three pyrimidines that are of concern to us.
Adenine and guanine are found in both DNA and RNA.Pyrimidines
Cytosine is found in both DNA and RNA. Uracil is usually found only in RNA and thymine is normally only in DNA. There are exceptions, however. Sometimes transfer RNA molecules will contain some thymine as well as uracil (a few DNA molecules also have uracil instead of thymine).
I shall use the term "nucleotides" to refer to either ribonucleotides or deoxyribonucleotides, in general, and the terms ribonucleotides or deoxyribonucleotides to refer specifically to nucleotides containing ribose or deoxyribose, respectively. Similarly, the term nucleosides shall mean either ribonucleosides or deoxyribonucleosides in general. Covalently linking carbon #1 of a sugar (ribose or 2-deoxyribose) with a nitrogen base (nitrogen 9 of a purine base or nitrogen 1 of a pyrimidine base) creates a nucleoside.
Purine nucleoside names end in -osine and pyrimidine nucleoside names end in -idine. The convention is to number the ring atoms of the base normally and to use l', etc. to distinguish the ring atoms of the sugar. Unless otherwise specificed, the sugar is assumed to be ribose. To indicate that the sugar is 2'-deoxyribose, a d- is placed before the name.
Nucleosides with phosphate(s) linked on the sugar portion of the molecule are called nucleotides. Nucleotides with one phosphate are also called nucleoside monophosphates (NMP); those with two phosphates are nucleoside diphosphates (NDP); and those with three phosphates are nucleoside triphosphates (NTP). Thus, ATP is a nucleotide and it is also called a nucleoside triphosphate. Generally, the phosphate is in ester linkage to carbon 5' of the sugar.
We synthesize nucleotides by both de novo (new synthesis from scratch) and salvage pathways and reuse those we already have. Before considering de novo synthesis of nucleotides, it is important to remember that the bases used in nucleotides can come both from new synthesis (de novo) or from salvage of bases previously made. Note that nucleotides are synthesized FIRST as ribonucleotides (whether by de novo or salvage) and then later converted to deoxyribonucleotides as needed to make DNA.
The first step in pyrimidine biosynthesis is the synthesis of carbamoyl phosphate from bicarbonate and ammonia. This reaction, which is catalyzed by the enzyme carbamoyl phosphate synthetase (CPS), involves multiple steps. The enzyme contains multiple active sites for catalysis. They include two separate ATP binding sites and a site to remove ammonia from glutamine. To catalyze these multiple reactions, the enzyme must move the substrate through a channel in the enzyme. In the second step of pyrimidine biosynthesis, carbamoyl phosphate and aspartate are joined by the enzyme aspartate transcarbamoylase (ATCase) to form carbamoylaspartate. As noted last term, ATCase is the textbook example of an allosterically regulated enzyme. CTP inhibits and ATP stimulates ATCase activity. E. coli also controls pyrimidine biosynthesis by regulating the transcription of the ATCase operon. With transcriptional regulation, levels of ATCase protein can vary by as much as 150-fold. High concentration of UTP lowers the rate of transcription of these genes. We will study genetic regulation later in this course.
The first pyrimidine ring (orotate) is created, then joined with a phosphorylated form of ribose called phosphoribosylpyrophosphate (PRPP) to form a pyrimidine nucleotide that your book calls orotidylate (also called OMP). Decarboxyation of orotidylate yields uridylate (also known as UMP). PRPP plays an important role in both purine and pyrimidine biosynthesis.
UTP is made from UMP by the sequential action of 1) a nucleoside monophosphate kinase and 2) nucleoside diphosphokinase (NDPK), as shown below:
UTP is then converted, using energy from ATP, to CTP. Note that this reaction also uses ammonia from glutamine and requires ATP energy.
Note that this pathway for synthesis of pyrimidines does not account for synthesis of (d)TTP, a pyrimidine used only in DNA. (d)TTP is not made by the de novo pyrimidine synthesis pathway. (d)TTP is made only after UDP or CDP is converted to dUDP or dCDP by action of the enzyme ribonucleoside diphosphate reductase. This enzyme plays an essential role in synthesis of deoxyribonucleotides, and is discussed below after description of synthesis of purine nucleotides.
In summary, for pyrimidine de novo biosynthesis,
Salvage pathways (synthesis of nucleotides from pre-existing pieces) are important ways of making purines, besides the de novo pathways. The most important pathway for salvaging purines uses the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT).
Guanine + PRPP <=> Guanylate (GMP) + PPi (Hypoxanthine-guanine phosphoribosyltransferase - HGPRT)
Hypoxanthine + PRPP <=> Inosinate (IMP) + PPi (HGPRT)
This enzyme is exceptionally important and it is inhibited by both IMP and GMP. HGPRT salvages guanine directly and adenine indirectly because AMP is generated primarily from IMP, not from free adenine. In addition, adenine can be salvaged by the enzyme adenine phosphoribosyl transferase in the reaction below
Adenine + PRPP <=> Adenylate + PPi (Adenine phosphoribosyl transferase)
De Novo Synthesis of Purine Nucleotides
Note in de novo purine biosynthesis that the base is "built" on the ribose sugar, whereas in pyrimidine de novo biosynthesis, the base was "built" first and the ring was added later. Note the intermediates involved in synthesis of the purine ring. They include the amino acid glycine, carbons from folate derivatives (THF), aspartic acid, and an amine group from glutamine. In addition, the citric acid cyle intermediate fumarate is produced as a byproduct of the pathway.
Inosinate (IMP) is a branch point between synthesis of AMP and GMP. Interestingly, energy from GTP is used to make AMP from IMP, whereas energy from ATP is used to make GMP from IMP. This "balancing" act is important for keeping the relative amounts of AMP and GMP in the proper proportions. In addition, AMP allosterically inhibits the branch from IMP to AMP and GMP allosterically inhibits the branch from IMP to GMP. As for the pyrimidine nucleotides, conversion of monophosphates to diphosphates is catalyzed by specific kinases in reactions as follows:
AMP + ATP <=> 2ADP (adenylate kinase)
GMP + ATP <=> GDP + ADP (guanylate kinase)
Purine diphosphates are converted to triphosphates in reactions catalyzed by nucleoside diphosphokinase (NDPK). This is the same enzyme that catalyzed conversions of pyrimidine diphosphates to triphosphates. Thus, NDPK catalyzes conversion of all nucleoside diphosphates to nucleoside triphosphates.
Two steps in purine de novo biosynthesis require carbons from tetrahydrofolate derivatives. One product of these reactions is tetrahydrofolate (THF). As shall be seen later, THF and its derivatives must be recycled to keep purine synthesis going. Interruption of the recycling is a strategy of chemotherapy.
In summary for purine de novo synthesis,
Deoxyribonucleotides are synthesized from ribonucleoside diphosphosphates via catalysis by the enzyme ribonucleotide reductase. Ribonucleotide reductase catalyzes the following reactions (not all intermediates shown):
ADP <=> dADP
GDP <=> dGDP
CDP <=> dCDP
UDP <=> dUDP
Note that all conversions involve diphosphates (not triphosphates) and that thymidine nucleotides are NOT direct products of action by ribonucleotide reductase. They are produced by reactions described below. The reaction catalyzed by ribonucleotide reductase replaces the hydroxyl group of carbon #2 of ribose with a hydrogen atom. As seen in the mechanism, three cysteines and a glutamic acid residue in the active site play roles in the catalysis. The process is initiated by transfer of an electron from a cysteine residue on R1 to a tyrosine radical on R2, generating a highly reactive thiyl radical (step 1) that abstracts a hydrogen atom from carbon 3 of the ribose (step 2). This, in turn, stimulates removal of the hydroxyl from carbon 2 of ribose. It combines with a hydrogen from a cysteine to form water (step 3). The radical at carbon 2 from step three resolves in step 4 by taking a hydrogen from a cysteine, creating a disulfide bond (your book incorrectly calls the hydrogen transferred from cysteine a hydroxide). Next the radical on carbon 3 recovers the hydrogen it originally lost (step 5) and an electron is transferred from R2 to reduce the thiyl radical (step 6). In the last step (not shown in book), the disulfide that was formed in the active site must be reduced to a sulfhydryl before another catalytic cycle can begin. Reduction of the disulfide is accomplished using disulfide-containing proteins, such as thioredoxin or glutaredoxin. Thioredoxin, in turn, must be reduced by NADPH in a reaction catalyzed by the enzyme thioredoxin reductase.
The enzyme catalyzing formation of deoxyribonucleotides (ribonucleotide reductase) is allosterically regulated as described below.
In summary for deoxyribonucleotide biosynthesis,
Thymidine Nucleotide Synthesis
Thymidine nucleotides are produced as products of "roundabout" synthetic pathways from corresponding deoxyuridine nucleotides. The roundabout nature of thymidine nucleotide synthesis is apparent from the following scheme showing the reactions going from UMP to dTMP.
UMP -> UDP -> dUDP -> dUTP -> dUMP -> dTMP
Note that deoxyuridine nucleotides are synthesize solely for the purpose of making deoxythymidine nucleotides. Cells convert dUDP to dUTP and then dUTP to dUMP. This is curious for two reasons. First, it requires extra energy compared to simply directly converting dUDP to dUMP. Second, dUTP can be recognized by DNA polymerase and occasionally results in incorporatioin of uracil into DNA in place of thymine. To minimize this possibility, cells have a lot of the enzyme dUTPase, which catalyzes the conversion of dUTP to dUMP.
Synthesis of dTMP (often confusingly called TMP - I will use the dTMP designation) requires transfer of a methyl group to dUMP from N5N10-Methylenetetrahydrofolate (a THF derivative). This unusual reaction, catalyzed by the enzyme thymidylate synthase, results in production of molecule dihydrofolate (DHF). As described below, DHF must ultimately be converted to THF in order to continue the recycling of THF derivatives for synthesis of purines and dTMP. As with the other monophosphate nucleotides, dTMP is converted to dTDP by specific kinases and then the dTDP is converted to dTTP by NDPK.
In summary for thymidine biosynthesis,
Recycling of Folates
To recycle DHF back to THF, action of the enyzme dihydrofolate reductase is necessary. This reduction reaction requires electrons form NADPH. If this reaction is inhibited, all of the folate derivatives in a cell will end up as DHF, causing synthesis of purines and thymidine to cease. For this reason, dihydrofolate reductase derivatives, such as aminopterin and methotrexate are used as chemotherapeutic agents. Another folate analog called trimethoprim has potent antibacterial and antiprotozoal activity. Trimethoprim and sulfamethoxazole (an inhibitor of folate synthesis) are used together to treat infections.
Regulation of Nucleotide Biosynthesis
Nucleotide biosynthesis is one of the most tightly regulated metabolic pathways in cells. This is apparently important because imbalances in the relative amounts of nucleotides in cells is linked with increased mutation. You will recall (hopefully) from BB 450 (and from above) that the enzyme aspartate transcarbamoylase (ATCase) is a model of allosteric control, being inhibited by an end-product of pyrimidine biosynthesis (CTP) and activated by an end-product of purine biosynthesis (ATP). Thus, when pyrimidines are present in too high of a concentration, the entire pathway of pyrimidine de novo biosynthesis is turned off, whereas when purines appear in too high of a concentration, pyrimidine synthesis is stimulated to keep the relative ratios constant.
Note that synthesis of PRPP and conversion of PRPP to phosphoribosylamine are BOTH inhibited by the purine nucleotides IMP, AMP, and GMP. The more important enzyme affected is the second one, gluatmine phosphoribosyl amidotransferase, which catalyzes the conversion of PRPP to phosphoribosylamine. As noted in purine biosynthesis, conversion of IMP to AMP is inhibited by AMP and conversion of IMP to GMP is inhibited by GMP and AMP synthesis requires GTP energy while GMP synthesis requires AMP energy.
Synthesis of deoxyribonucleotides from ribonucleotides is regulated carefully by allosteric interactions on ribonucleotide reductase. The enzyme has different regions of it that act differently on allosteric regulators. At the activity site of ribonucleotide reductase, dATP is a general inhibitor for all substrates (signals an abundance of deoxyribonucleotides) and ATP is an activator. Binding of nucleotides at the specificity site further controls the activity of the enzyme towards different substrates in order to maintain an appropriate balance of all deoxynucleotides for DNA synthesis. For example, binding of dATP or ATP (purines) to the specificity site enhances formation of dUDP and dCDP (pyrimidines). Binding of dTTP (pyrimidine) to the specificity site favors formation of dGDP (purine) and inhibits formation of deoxypyrimidines. Note that the allosteric effectors are TRIPHOSPHATES whereas the enzyme's products are DIPHOSPHATES. Thus the enyzme plays an active role in balancing the appropriate amount of deoxypyrimidines and deoxypurines for DNA synthesis. Remember that the dNDPs produced by ribonucleotide reductase are phosphorylated to dNTPs by kinases.
Catalysis by nucleotidases converts nucleotides to nucleosides. Nucleoside phosphorylases catalyze reactions on nucleosides to yield ribose-1-phosphate and free bases (uracil, for example). (Note that all of these components can be converted, if necessary back into nucleotides via salvage pathways. Ribose-1-phosphate, for example can be converted to ribose-5-phosphate by phosphoribomutase). Xanthine, which is an intermediate in breakdown of both AMP and guanine, yields uric acid upon catalysis by xanthine oxidase. Uric acid (urate) is the causative agent in gout. It arises from the low solubility of urate in water, causing painful joints (usually manifested in the big toe) and can damage kidneys. Urate is a very useful antioxidant and may have protective effects in humans.
A syndrome known as Lesch-Nyhan syndrome is linked to defects in purine metabolism. People with this syndrome exhibit compulsive self-destructive behavior, biting fingers and lips and may chew them off if not restrained. In these people, the enzyme HGPRT (see above) is absent, causing hypoxanthine levels to increase and preventing the salvage of purine nucleotides. Urate is produced in high amounts in these individuals as a byproduct.