In this article we will discuss about the process of biosynthesis of nucleic acids.
Biosynthesis of Nucleosides-5′-Triphosphates:
Among animals, some amino acids cannot be synthesized and must therefore be supplied through diet (essential amino acids). On the contrary, purine and pyrimidine nucleotides can be synthesized “de novo”, from precursors like CO2, NH3, formiate, glycine etc., and this is true of animals and plants as well as bacteria (except some mutants lacking an enzyme catalyzing one of the reactions leading to nucleotides).
This does not prevent the cells from using, for the biosynthesis of their nucleic acids, the already formed purines and pyrimidines which may be provided to them (e.g., through food).
Purine and pyrimidine nucleotides have a common precursor, ribose-5- phosphate, the formation of which will not be discussed here again since we already mentioned while studying the pentose-phosphates cycle that the passage from glucose-6-phosphate to ribose-5-phosphate is possible, either by the oxidative pathway (necessitating NADP+), or by the pathway of interconversions; these reactions are schematically represented in figure 4-43).
Ribose-5-phosphate binds on its carbon 1 a pyrophosphate group from ATP and thus transforms (see fig. 6-18) into 5-phosphoribosyl-1-pyrophosphate or PRPP, a precursor which will provide the “ribose-5-phosphate” part in the de novo biosynthesis of purine and pyrimidine nucleotides, as well as in the synthesis of nucleotides from preformed purines or pyrimidines.
Biosynthesis of Purine Ribonucleosides-5′- Triphosphates:
1. De Novo Biosynthesis of IMP:
Figure 6-19 shows the series of 11 reactions leading from ribose-5-phosphate to IMP (or inosine-5′-monophosphate, or inosinic acid), the base of which, we may repeat, is called hypoxanthine. Names of intermediate compounds and enzymes catalyzing the various reactions are not important here; we will confine ourselves to some remarks on some of the steps.
Reaction 2 is subjected to feedback inhibition; in other words, the enzyme is inhibited by purine nucleotides which are the final products of this metabolic series; such a mechanism, inhibiting the first reaction of the metabolic pathway when the terminal products are in excess, is obviously very economical for the cell because it prevents a useless energy expenditure.
It is indeed the first reaction of the metabolic pathway leading specifically to purine nucleotides which is subjected to feedback inhibition, because the previous reaction, leading from ribose-5-phosphate to PRPP, permits the formation of a compound (PRPP) which is a precursor of the biosynthesis of purine and pyrimidine nucleotides and it would be undesirable that it should be inhibited by an excess of purine nucleotides only.
Besides, it must be noted that at this stage there is a change of configuration at carbon 1 of ribose, since there is passage from configuration α (PRPP) to configuration β (phospho-ribosylamine) which is that of nucleotides.
Reaction 4 and reaction 10 consist of a transfer of formyl group, from N5 —N10 methenyl-tetrahydrofolic acid (reaction 4) and N10-formyl- tetrahydrofolic acid (reaction 10). But we have seen that sulphamides are structural analogues of para-aminobenzoic acid, the compound used by bacteria to synthesize folic acid. Sulphamides therefore prevent the formation of the tetrahydrofolic acid required for the biosynthesis of purine nucleotides which is blocked and this explains the bacteriostatic effect of sulphamides.
Reactions 8 and 9 consist of the input of a nitrogen atom provided by aspartic acid which is thus transformed into fumaric acid. A similar mechanism is involved in the transformation of IMP into AMP which we will study in the next paragraph (see fig. 6-21) and in the transformation of citrulline into arginine in the ureo-genesis cycle.
This series of reactions can be summarized as follows:
ribose-5-phosphate + glycine + aspartate + 2glutamine + 2 formiate + CO2 → IMP + 2glutamate + fumarate.
The diagram of figure 6-20 points out the origin of the 5 carbon atoms and 4 nitrogen atoms of the purine ring.
Lastly, it must be noted that the biosynthesis of the purine ring consumes a great deal of ATP. It is seen that 5 ATP molecules are needed for the series of reactions leading to IMP (see fig. 6-19), but one must add to it 2 ATP molecules needed for the formation of the 2 glutamine molecules and 2 ATP required for the formation of the formylated derivatives of tetrahydrofolic acid. Therefore, 9 molecules of ATP are required to synthesize one molecule of IMP.
2. Formation of AMP and GMP from IMP:
IMP, whose de novo biosynthesis we have just seen, is not a normal constituent of nucleic acids; it will be converted into adenylic and guanylic nucleotides, which are the major purine nucleotides found in ribonucleic and deoxyribonucleic acids.
These transformations are diagrammatically depicted in figure 6-21. It is seen that the passage from IMP to AMP consists in replacing the hydroxyl group of carbon 6 by an amino group. Aspartic acid yields its nitrogen by a mechanism similar to the one we have just seen in the de novo biosynthesis of IMP for the introduction of nitrogen 1 (reactions 8 and 9 of figure 6-19).
GMP differs from IMP by an amino group on carbon 2. In a first step, oxidation takes place at this carbon (coupled with the reduction of NAD+) and one obtains xanthosine-5′-monophosphate or XMP (the corresponding base, oxidized in 2 and 6, is called xanthine). Then in a second step an amination reaction takes place, at the cost of NH3 in bacteria, and glutamine (amidic N) in animal cells.
It is interesting to note that the synthesis of AMP requires GTP and that of GMP requires ATP. This permits a regulation of the synthesis of the two purine nucleotides, which are both required for the biosynthesis of nucleic acids, since an excess of one stimulates the formation of the other.
De novo biosynthesis of one AMP or GMP molecule therefore requires 10 (9 +1) ATP molecules.
On the other hand, AMP controls its own synthesis by feedback inhibiting the conversion of IMP into adenylosuccinate, and at the same time GMP feedback inhibits the conversion of IMP into XMP.
3. Utilization of Preformed Purines:
If adenine or guanine is supplied to a bacterium or higher organism, the base can be converted into nucleoside-5′-monophosphate by two processes (we are indicating them for adenine but they are identical for guanine):
(i) Either in 2 steps:
Adenine + ribose-1-phosphate → Adenosine + Pi (reaction catalyzed by a nucleoside-phosphorylase)
Adenosine + ATP→ Adenosine-5′-monophosphate (AMP) + ADP (reaction catalyzed by a nucleoside-kinase).
It is observed that nucleosides can also be utilized by the cells;
(ii) Or in one single step:
Adenine + 5′ phosphoribosyl-1′-pyrophosphate (PRPP) ←→ AMP + PP (reaction catalyzed by a nucleotide-pyrophosphorylase).
It is obvious that the formation of AMP or GMP from adenine or guanine is — from the energy point of view — much more economical than their de novo synthesis, because it requires 1 molecule of ATP instead of 10 (even in the formation of AMP or GMP in a single step, there is utilization of one molecule of ATP, required for the synthesis of PRPP, see figure 6-19).
It is therefore in the interest of living organisms to utilize the purine bases or nucleosides, which are either present in their food, or recovered during the degradation of nucleic acids and nucleotides (hence the name “salvage pathway” given to these reactions).
4. Phosphorylation of Nucleosides-5′-Monophosphates into Nucleosides-5′-Triphosphates:
We have just seen that a nucleoside like adenosine can be phosphorylated by ATP to nucleoside-5′-monophosphate. Let us note that the reaction has not been written as a reversible one, and in fact, it is not reversible, because there is rupture of a phosphoanhydride bond and formation of an ester linkage, so that the equilibrium is very strongly in favour of the ester formation.
On the contrary, the transphosphorylation reactions that we will see now are reversible because there is rupture and formation of the same type of bond (phosphoanhydride bond).
A. Reactions Catalyzed by Nucieosides-5’-Monophosphates-Kinases:
The best known enzyme of this group is AMP kinase (or adenylate kinase or myokinase) which catalyzes the reaction:
AMP + ATP
ADP + ADP (2 ADP)
i.e. phosphorylation of a nucleoside-5′-monophosphate into diphosphate.
B. Reactions Catalyzed by Nucleosides-5′-Diphosphates-Kinases:
As indicated by their name, these enzymes catalyze the phosphorylation of a nucleoside-5′-diphosphate into triphosphate.
For example, the following reaction may take place:
GDP + ATP 
GTP + ADP
This shows how, from AMP and GMP, formed either by de novo biosynthesis, or from preformed purines, can be synthesized the corresponding ribonucleosides-5′-triphosphates which are the precursors required for the synthesis of ribonucleic acids.
It should be noted that the 3 classes of transphophorylations we have just considered exist not only in the “ribo” series but also in the “deoxyribo” series and that they also exist for the pyrimidine compounds.
In reality that following reactions can take place:
Purine or pyrimidine ribonucleoside
(Z) → ZMP ←→ ZDP ←→ ZTP
Purine or pyrimidine deoxyribonucleoside
(dZ) → dZMP ←→ dZDP ←→ dZTP
These reactions are therefore also involved in the formation of pyrimidine ribonucleotides and purine and pyrimidine deoxyribonucleotides, but we will not revert to this question, and while studying the synthesis of these nucleotides we will assume the reactions known.
Biosynthesis of Pyrimidine Ribonucleosides-5 – Triphosphates:
1. De Novo Biosynthesis of UMP:
The series of reactions leading to UMP is shown in figure 6-22. It is observed that it is much shorter than the one leading to the biosynthesis of purine nucleotides. It also differs in that the “ribose-5′-phosphate” part is introduced in the molecule only at the last but one step, when the pyrimidine ring is already formed (whereas in the biosynthesis of purine nucleotides, ribose-5′- phosphate is the starting point on which are grafted the constitutive atoms of the purine ring and therefore, an aliphatic ribonucleotide is obtained right at the start).
Another difference lies in the fact that the de novo biosynthesis of pyrimidine nucleotides leads to UMP, one of the four major nucleotides of ribonucleic acids, whereas IMP is not a nucleotide frequently found in nucleic acids. However, it may be noted that in both cases, de novo biosynthesis leads to a nucleotide deprived of any amino group, and that the amination reactions must take place subsequently.
In microorganisms, carbamyl-phosphate can be formed from CO2 and NH3, with formation of carbamic acid which is phosphorylated by a kinase to car- bamyl-phosphate. Carbamyl-aspartate is synthesized by aspartate-transcar- bamylase, an allosteric enzyme which was extensively studied (especially the one extracted and purified from E. coli).
This enzyme is inhibited by CTP, one of the terminal products of this pathway of biosynthesis of pyrimidine ribonucleotides, which represents a standard example of feedback inhibition. But it may be already indicated that it has been possible, in the case of this enzyme, to separate the sub-units possessing catalytic activity and the sub-units responsible for the regulating activity.
In animals, from the reactions point of view, the steps of this biosynthesis are essentially identical to those in bacteria, but the regulation is entirely different; the first 3 enzymatic steps are catalyzed by a single multifunctional polypeptide chain present in the cytosol, of a mass of 240 kd, called CAD because it has the activities of Carbamylphosphate synthetase, Aspartate transcarbamylase and Dihydrorotase.
The synthesis of carbamyl phosphate uses glutamine as the donor of the amino group and represents the step subjected to allosteric regulation (activation by PRPP, inhibition by UTP). Steps 5 and 6 are also catalyzed by a multifunctional polypeptide, which has the activities of orotate phosphoribosyl transferase and OMP decarboxylase.
As just mentioned in connection with the first three enzymes of the biosynthesis pathway of pyrimidines, such a grouping offers several advantages to the multienzymatic complex itself (whose formation is simpler and more effective than if it would require joining by non-covalent bonds, different enzymes which were synthesized separately), as well as to the metabolic pathway because the substrates can thus be effectively transferred from one catalytic site to the other.
Some believe that the genes corresponding to thete plurifunctional polypeptides result from rearrangements of various exons. This is, in animals, a striking example of compartmentation or metabolic channeling for two pathways having a common precursor. The carbamyl-phosphate required for the synthesis of urea (see fig. 7-30) is indeed produced by a mitochondrial enzyme characterized by different substrate (NH3) and regulation.
2. Utilization of Preformed Pyrimidines:
Uracil can be converted into UMP by reactions identical to those we have seen in connection with the utilization of preformed purines
(i) Either in 2 steps:
Uracil + ribose-1-P ←→ Uridine + Pi
(reaction catalyzed by a nucleoside-phosphorylase)
Uridine + ATP → UMP +ADP
(reaction catalyzed by a nucleoside-kinase)
(ii) Or in 1 step:
Uracil + PRPP ←→ UMP + PP
(reaction catalyzed by a nucleotide pyrophosphorylase)
Cytosine on the contrary, is not incorporated as such; it is generally deaminated into uracil, which is transformed into UMP as we have just seen. However cytidine can be incorporated after phosphorylation into CMP.
3. Formation of Uridylic and Cytidylic Ribonucleotides:
With the help of the kinases studied in connection with the formation of purine nucleotides, UMP (formed de novo, or from uracil) can be phosphorylated into UDP and then UTP. The latter will be aminated into CTP, as shown by figure 6-23, thanks to NH3 (in microorganisms) or amidic nitrogen of glutamine (in higher organisms).
