In this article we will discuss about the formation of purine and pyrimidine deoxyribonucleosies.
Formation of Purine Deoxyribonucleosides-5′- Triphosphates:
The deoxyribonucleotides are simply formed by reduction of the corresponding ribonucleotides. Thus reduction of carbon 2′ of ribose, takes place in E.coli and animal tissues at the ribonucleosides-5′-diphosphates stage, at the cost of a protein of low molecular weight, thioredoxin, comprising 2 thiol groups; thioredoxin will thus be oxidized, but it can be again reduced at the cost of NADPH.
A mutant of E.coli, without thioredoxin, but yet capable of forming deoxyribonucleotides, enabled the characterization of a protein similar to thioredoxin, glutaredoxin, which, after oxidation during the reduction of ribonucleotides, is reduced by glutaredoxin reductase, an enzyme whose prosthetic group is glutathione. Oxidized glutathione (disulphide form) will be finally reduced by NADPH.
All these reactions, schematically represented in figure 6-24, concern the reduction of purine as well as pyrimidine ribonucleosides-diphosphates. In the case of purine derivatives, one thus obtains dADP and dGDP which will be phosphorylated, by a nucleoside-diphosphate kinase, to dATP and dGTP, the purine precursors required for the synthesis of deoxyribonucleic acids.
Ribonucleoside diphosphate reductase comprises two sub-units: B1, a dimer of 160 kd which carries the binding sites of substrates (ZDP) and allosteric effectors, and which comprises the SH groups, electron donors for the reduction of ribose, and B2, a dimer of 78 kd, which participates in the catalytic activity by forming a free organic radical: the tyrosyl cation.
Sub-Unit B1 Comprises 2 Types of Allosteric Sites:
i. One, permits the control of the general activity of the enzyme; the binding of dATP results in feedback inhibition of the catalytic activity, while ATP lifts this feedback inhibition effect;
ii. The other site provides a specific control of the substrate:
The reduction of pyrimidine nucleotides, UDP and CDP, is activated by the binding of dATP on this site.
Reduction of GDP is favoured by the binding of dTTP, which also inhibits the reduction of pyrimidine nucleotides.
The resulting formation of dGTP stimulates the reduction of ADP;
It appears that ribonucleotide reductase can exist in different conformations, each having specific reducing properties, and that this system of allosteric regulation provides a balanced production of the 4 deoxy- ribonucleotides required for the synthesis of DNA.
Formation of Pyrimidine Deoxyribonucleosides-5′- Triphosphates:
UDP and CDP can be reduced ((just like ADP and GDP) to dUDP and dCDP. The latter will be phosphorylated into dCTP, one of the pyrimidine precursors required for the synthesis of deoxyribonucleic acids. The other precursor is not dUTP but dTTP, and we will now see the reactions involved in the biosynthesis of thymidylic deoxyribonucleotides.
1. Formation of dUMP:
A. From Uridylic Deoxyribonucleotides:
dUDP can
i. Either lose a phosphate group and thus give JUMP
ii. Or be phosphorylated into dUTP, but the latter, is not a precursor of deoxyribonucleic acids, and it is degraded into dUMP + PP.
B. From Cytidylic Deoxyribonucleotides:
dCMP can be deaminated into dUMP by deoxycytidylate aminohydrolase (or dCMP deaminase) which catalyzes the following reaction:
dCMP + H2O → dUMP + NH3
This is the reverse reaction of the amination reaction of UTP into CTP which we mentioned in the foregoing, with the difference that here the base is linked to a deoxyribose-5′-monophosphate and not to a ribose-5′-triphosphate. The enzyme has a very narrow specificity and does not deaminate deoxycytidine, dCDP, dCTP, cytosine, cytidine, CMP, although the structures of these compounds are closely related to that of dCMP.
2. Methylation of dUMP into dTMP:
This reaction catalyzed by (deoxy) thymidylate synthase takes place by the action of N5 — N10 methylene tetrahydrofolic acid which on the one hand, yields its one carbon atom group and on the other hand acts as a reductant and is thus finally found in the form of dihydrofolic acid.
Tetrahydrofolic acid will be regenerated by dihydrofolate reductase which catalyzes the following reaction:
Generally, cancerous cells multiply rapidly; they are therefore subjected to a significant synthesis of DNA which requires especially dTMP in large quantities. This is why thymidylate synthase and dihydrofolate reductase are choice targets in the treatment of cancer by chemotherapy.
Methotrexate and aminopterin, which are analogues of dihydrofolate, are powerful competitive inhibitors of dihydrofolate reductase, and methotrexate is used in the treatment of various rapid growth tumours; but this compound kills the cells in rapid division, malignant or otherwise; it therefore has secondary toxic effects (in bone marrow, capillary follicles, epithelial cells of the intestine, etc.).
On the other hand, it was observed that mammalian cells cultured in presence of methotrexate become resistant to this substance; mutated cells appear which are affected either in the uptake of methotrexate or in the affinity of dihydrofolate reductase for methotrexate (which is decreased), or because of an excess production of dihydrofolate reductase (1000 times more enzymes) due to an amplification of the corresponding gene (some cells can have up to 100 copies of the gene coding for this enzyme). The treatment must therefore use rather high doses of methotrexate to prevent the appearance of mutant cells increasingly resistant, due to genie amplification.
Fluorouracil is used as anti-cancerous agent because it is converted in vivo into fluorodeoxyuridylate (F-dUMP) which irreversibly inhibits thymidylate synthase. At the beginning of the reaction F-dUMP behaves as the normal substrate (dUMP) and methylene tetrahydrofolate can fix itself on the C-5 of the analogue.
But the enzyme cannot detach F+ from the C-5 of F-dUMP (as it detaches H+ from the C-5 of dUMP), with the result that the reaction cannot proceed. It is a suicide-inhibition because the enzyme transforms a substance (which first behaves as a substrate) into an inhibitor which blocks the reaction.
3. Utilization of Thymine and Deoxythymidine:
Lastly, it should be mentioned that preformed purines and pyrimidines can be incorporated in the corresponding deoxyribonucleotides, but only through deoxyribonucleosides (there is no equivalent of PRPP for deoxyribose).
One can have for example:
Figure 6-25 gives a general diagram recapitulating the main steps of the biosynthesis of precursors of ribonucleic acids (purine and pyrimidine ribonucleosides-5′-triphosphates) and deoxyribonucleic acids (purine and pyrimidine deoxyribonucleosides-5′ – triphosphates).
