Table of Contents
- Nucleotide Construction
- Nucleoside Construction
- Purine Nucleotide de Novo Synthesis Process
- Pyrimidine Nucleotide De Novo Synthesis Process
- Deoxy-forms Synthesis of Purine and Pyrimidine Nucleotides
- Degradation of Purine Nucleotide or Purine Bases
- Pyrimidine Nucleotide and Bases Degradation
- Recycle of Purine and Pyrimidine Nucleotides (Salvage-Pathways)
The four nucleotides serve as DNA (Deoxyribonucleic acid) components. A nucleotide is, in turn, composed of a deoxyribose (sugar), a phosphate group, and a nitrogen basis.
There are four different nitrogen bases, and, therefore, four sorts of nucleotides. The four bases are:
The resulting nucleotide is named accordingly. If the base is e.g., adenosine, it is named deoxyadenosine-5’-monophosphate. The respective nucleotides are therefore indicated, in short form, with the initial of their base, i.e. A, G, C, and T.
Nitrogen bases are alike two by two since their construction and are grouped into two categories. Thus, adenine and guanine constitute the purine category, while cytosine and thymine form the pyrimidine class.
A nucleoside is composed of deoxyribose and a nitrogen basis, i.e. contrary to a nucleotide, it doesn’t have a phosphate group anymore. This means that a nucleoside can come from a nucleotide through the removal of a phosphate group.
Purine Nucleotide de Novo Synthesis Process
Purine nucleotides are synthesized directly at the nucleotide’s ribose within their synthesis process. This represents an important difference from pyrimidine synthesis (see below), in which the ribose is only added subsequently to the complete formation of the pyrimidine ring.
The purine nucleotide synthesis takes place in multiple individual steps. During the first step, Ribose-5-Phosphate becomes phosphoribosylpyrophosphate (PRPP) – converting ATP to AMP. The enzyme involved is called PRPP-synthetase, in accordance with its final product.
Meanwhile, the rate-limiting reaction, the second step of the purine nucleotide synthesis, takes place. During this reaction, the combination of PRPP and glutamine with the separation of pyrophosphate (PP) results in 5-Phosphoribosylamine. The configuration on the C-1 atom of the ribose is converted from an α-structure to a β-structure.
The enzyme involved in this rate-limiting reaction is glutamine-phosphoribosylpyrophosphate-amidotransferase. Substances like IMP, GMP, and AMP hinder the enzyme and the reaction with it. The reaction can be re-driven through pyrophosphate hydrolysis.
During the third step of the purine nucleotide synthesis, which is represented by an ATP-dependent reaction, 5-phosphoribosylamine becomes a glycinamide-ribonucleotide. The glycinamide-kinosynthase enzyme mediates this reaction.
Through formylation, the glycinamide-ribonucleotide becomes a formyl-glycinamide-ribonucleotide. The necessary residual formyl comes from an N10-formyltetrahydrofolate.
In the following step, an N-atom with glutamine serves as a donor and a closed 5-membered ring is formed through an H2O separation. Glutamine thus becomes glutamate.
A CO2 molecule is formed in the closed 5-membered ring. Another N-atom is subsequently added through an ATP-dependent reaction, which, in this case, is provided by aspartate (similarly to the urea cycle).
Aspartate becomes fumarate through the loss of the N-atom. Alongside this process, another C-1 fragment is incorporated in the 5-member ring, which becomes a 6-member ring through the separation of H2O. The C-1 fragment is obtained from the N10-formyltetrahydrofolate.
The resulting product is called inosine monophosphate (IMP). It serves as a pre-amplifier to adenosine monophosphate (AMP) and guanosine monophosphate (GMP) synthesis.
The synthesis to AMP is also GTP-dependent and aspartate-dependent. Within this reaction, the keto group is replaced with an NH2 group in position C6.
The synthesis of GTP from IMP is, on the contrary, composed of two steps. In the first step, IMP is oxidized to xanthine monophosphate in an NAD+-dependent reaction. Xanthine monophosphate, however, is later aminated, and the amine group (NH2) is obtained from the glutamine. The second step is both glutamine and ATP-dependent.
Note: Glutamine and aspartate belong to the amino acids involved in the purine nucleotide synthesis, where they serve as donors of N atoms and thus become glutamate or fumarate.
Role of Folic Acids
Folic acid is a very important component of an efficient purine nucleotide synthesis. A lack of folic acids leads to a strongly reduced purine nucleotide synthesis.
This reduction becomes particularly clear in processes with high cell turnover, e.g., erythropoiesis.
A consequence of a folic acid deficiency in this context is megaloblastic anemia, where a malfunction is present, both in DNA synthesis, as well as in the nuclear maturation of the myelopoiesis, which leads to the appearance of megaloblasts.
Besides a folic acid deficiency, megaloblastic anemia can also occur due to a lack of B-12 vitamin, which, overall, is a more frequent cause of megaloblastic anemia than a folic acid deficiency.
Further symptoms of folic acid deficiency are, among others, gastritis or dermatitis. The lack of folic acid during pregnancy can somewhat increase the risk of the baby being born with spina bifida.
Folic Acid Metabolism Process
Folic acids are composed of a p-aminobenzoic acid rest, a glutamine rest and a pteridine rest. Folic acid is available in its biologically active form as tetrahydrofolic acid (TH-4), in which it is involved, among other things, in the construction of the purine nucleotide (see above).
The active form of folic acid serves all in all as a coenzyme in the C-1 transmission, in which the rest of the C-1 position is bound to the N atoms of position 5 and 10 of the pteridine or 4-aminobenzoic acid rest. Possible rests that could be transferred in this context are methyl, hydroxyl, formic, and formyl rests.
The conversion to the active form takes place, among others, in a reaction which is dependent on NADP+ and vitamin C. In its first step 7, 8-dihydrofolic acid is created from folic acid – with the investment of NADPH + H+ and is mediated by folate reductase.
In an NADPH + H+-dependent reaction, 5, 6, 7, 8-tetrahydrofolate is also created by this process. Tetrahydrofolate synthesis, in its active form, takes place thanks to the dihydrofolate reductase enzyme. This enzyme can be inhibited through a series of substances like e.g., trimethoprim.
Pyrimidine Nucleotide De Novo Synthesis Process
The temporary product of the pyrimidine synthesis is initially a ribonucleotide. During the process, the ribose is reduced to a 2’-deoxyribose, so it can be incorporated in the DNA as such.
The entire pyrimidine nucleotide biosynthesis takes place in multiple individual steps which involve different enzymes. Here, the pyrimidine ring is synthesized first, and ribose is subsequently added to it.
Within the first step of pyrimidine synthesis, carbamoyl phosphate and aspartate react and produce carbamoyl aspartate through a phosphate release. The involved enzyme is called aspartate transcarbamoylase.
Note: The pyrimidine synthesis key reaction represents the reaction between carbamoyl phosphate and aspartate to carbamoyl-aspartate.
Dihydroorotic acid develops from carbamoyl aspartate through a water (H2O) separation. This second step of the pyrimidine synthesis includes a carbamoyl aspartate cyclization and involves the dihydroorotase enzyme.
The following step is the oxidation of dihydroorotic acid to orotic acid through the orotic-acid-dehydrogenase enzyme. This enzyme has NAD+ as a coenzyme, which comes out as NADH + H+ at the end of this reaction.
Orotic acids form orotidine-5-phosphate with phosphoribosyl pyrophosphate (PRPP) through the separation of pyrophosphate (PP). This reaction is mediated by the orotate-phosphoribosyltransferase.
From orotidine-5-phosphate, uridine-5-phosphate (UMP) develops within a decarboxylation process (through a separation of CO2) through the orotidine-5-phosphate-decarboxylase enzyme.
The uridine-5-phosphate constitutes the primary product of a series of further reactions. Firstly, UDP results from UMP via phosphorylation, which can be converted to a UTP through further phosphorylation. The phosphate group required for this is obtained through an ATP-to ADP-reaction.
In turn, through an ATP and glutamine-dependent reaction, CTP (cytidine triphosphate) can be obtained from UTP. This reaction is mediated by the CTP-synthetase enzyme.
On the other hand, uridine-5-phosphate can be reduced to d-UMP through d-TMP-synthetase (thymidylate-synthase). This reduction is NADPH + H+-mediated. The following step of the d-TMP-synthetase consists of methylation of d-UMP to d-TMP. The necessary methyl group is obtained through the N5-N10-Methylene-H4-Folate, which subsequently emerges as H2-Folate.
Deoxy-forms Synthesis of Purine and Pyrimidine Nucleotides
The final product of both the purine and the pyrimidine nucleotide synthesis (see above) is the ribonucleotide, which must be reduced further into the 2’-deoxy-form in order to be incorporated in the DNA.
The enzyme involved in this is ribonucleotide-reductase, which has thioredoxin as a cofactor. Thioredoxin, in turn, contains two SH groups, which are converted to a disulfide form in the reduction process. Thioredoxin in its disulfide form is reconverted to its original form through NADP+-dependent thioredoxin reductase.
Degradation of Purine Nucleotide or Purine Bases
Like the purine nucleotide construction, their degradation also takes place in multiple individual steps. In this process, each step’s differentiation from the others is partly based on their dependence from their respective purine base (adenosine or guanosine).
However, the first step of the degradation is a conversion from nucleotide to nucleoside. This takes place through a hydrolytic separation through the nucleotidase enzyme. Additionally, a phosphorylic separation in a free base (purine or pyrimidine) and in ribose-1-phosphate takes place. The nucleoside phosphorylase enzyme mediates this step. The degradation of the purine bases adenosine and guanosine takes place afterward.
In a first step, adenosine is converted into inosine through NH3 separation (deamination), involving the adenosine deaminase enzyme. The second step is identical for both inosine and guanosine. Both are converted to hypoxanthine (inosine) or guanine (guanosine) through an ATP-dependent ribose separation. The enzyme involved in this is nucleoside phosphorylase.
During the next step, xanthine is obtained from inosine and hypoxanthine. However, this step is different in both cases. Guanine is deaminated to xanthine, while hypoxanthine is oxidized to xanthine through xanthine oxidase.
Note: Xanthine oxidase is an iron-bearing flavoprotein, which contains a molybdate atom in its active centrum.
A further step which is mediated by xanthine oxidase is the conversion of xanthine to uric acid. This step also represents oxidation, where molecular oxygen serves as a means for oxidation. Uric acid will be pruned over urine.
Enzyme Defects That Can Lead to an Altered Uric Acid Level
The uric acid level plays a clinical role since, during a solubility products exceedance (ca. 7 mg/100 ml), a urate crystal deficit in the tissue can occur. Moreover, this can cause local inflammation – the corresponding clinically manifest disease is called gout.
Urate crystals fail particularly in badly capillarized tissues, probably because a low temperature promotes a urate crystal deficit. That is why e.g., the metatarsophalangeal joint (podagra), but also the cornea or the lens, are particularly affected by this.
The gout-affected joint appears flushed, overheated and swollen, besides causing a very strong pain. These clinical signs are the cardinal ones of inflammation (rubor, calor, tumor, and dolor).
Along with purine metabolism, enzyme defects can lead to either an increased or a diminished uric acid level. Among others, enzyme defects that can cause an increased uric acid level are a partial and a complete lack of hypoxanthine-guanine-phosphoribosyltransferase. A partial lack prevents the recycling of IMP to GMP, thus increasing the de novo purine synthesis. The respective disease is called Kelley Seegmiller syndrome.
A complete hypoxanthine-guanine-phosphoribosyltransferase lack is called Lesch Nyhan syndrome. Clinically, affected children present a trio of hyperuricemia, progressive kidney insufficiency, and neurologic symptoms, for example, the tendency to self-mutilate.
One enzyme defect, which, on the other hand, can lead to a diminished uric acid level, is a reduced xanthine oxidase activity (xanthinurie).
Note: Xanthine oxidase activity can be intentionally diminished through the allopurinol agent within a gout therapy.
Besides enzyme defects, other factors, like the renal function (uric acid secretion), an increased cell turnover (e.g., in diseases like leukemia) or a strongly purine-based diet (e.g., flesh), can also influence the uric acid level.
Pyrimidine Nucleotide and Bases Degradation
The first step of pyrimidine nucleotide degradation is the conversion of nucleotides to nucleosides, similar to the purine nucleotide degradation process (see above). Here, the respective steps are identical.
The degradation of cytosine and thymine, which has been obtained during the first step of the pyrimidine bases degradation, takes place in the liver. Here, the pyrimidine ring is broken down through multiple steps and pyrimidine bases are consequently degraded.
Note: The ring is degraded during the pyrimidine nucleotide degradation, while the purine ring is preserved during the pyrimidine nucleotide degradation.
The two bases go through two independent degradation ways in which the reaction steps are identical, except for the first step regarding cytosine degradation.
In the first step, cytosine is degraded to uracil through a separation of the amino group. Uracil and thymine are, in turn, reduced to dihydrouracil and dihydrothymine through a NADPH + H+-dependent reaction.
Through a separation of CO2 and NH3, β-alanine develops from dihydrouracil, while β-aminobutyrate results from dihydrothymine.
β-alanine and β-aminobutyrate are partially further degraded into respectively acetate and propionate, NH3 and CO2 via multiple intermediate steps. The nitrogen atoms that were obtained by this become a part of the urea cycle.
Recycle of Purine and Pyrimidine Nucleotides (Salvage-Pathways)
Since purine nucleotide degradation doesn’t result in any energy gain and pyrimidine nucleotide degradation only provides a small one, while the synthesis of both needs a great amount of energy, recycling is energetically more convenient.
However, the exact steps of recycling are only known for purine bases, which is why only those are covered here.
First, during the purine bases recycling, they are phosphoribosylized to nucleotides through PRPP. The transmission of each purine base to PRPP takes place for adenine through adenine-phosphoribosyltransferase and for hypoxanthine and guanine through hypoxanthine-guanine-phosphoribosyltransferase.
Through the synthesized final product, both enzymes are inhibited. The final product is AMP for adenine, while hypoxanthine results in IMP. The end product of guanine is GMP.