According to the World Health Organization, cancer is a leading cause of death worldwide: it accounted for 7. 9 million deaths (around 13% of all deaths) in 2007. As such, most researches nowadays are focused on drug development for cancer. These researches are often centered on blocking steps in nucleotide biosynthesis, particularly in the synthesis of DNA precursors (Berg, 2002). Aside from the importance of nucleotides and nucleic acids as essential intermediates in virtually all aspects of cellular metabolism, the recent applications calls for critical understanding of the nucleotide biosynthetic pathways (Berg, 2006).
The pathways for the biosynthesis of nucleotides fall into two classes: de novo pathways and salvage pathways. In de novo (from scratch) pathways, the nucleotide bases are assembled from simpler compounds. The framework for the pyrimidine base of Cytosine, Uracil and Thymine nucleotides is assembled first and then attached to ribose. In contrast, the framework for the purine base of Adenine and Guanine is synthesized piece by piece directly onto a ribose-based structure.
These pathways comprise a small number of elementary reactions that are repeated with variation to generate different nucleotides, as might be expected for pathways that appeared very early in evolution. In salvage pathways, preformed bases are recovered and reconnected to a ribose unit (Berg, 2002). The first step in the de novo pyrimidine biosynthesis is carbamoyl phosphate synthesis pathway where bicarbonate is phosphorylated by ATP to form carboxyphosphate and ADP. This reaction is catalyzed by carbamoyl phosphate synthetase (CPS).
Ammonia then reacts with carboxyphosphate to form carbamic acid and inorganic phosphate. The active site for this reaction lies in the domain formed by the aminoterminal third of CPS which can form a structure called an ATP-grasp fold, which surrounds ATP and holds it in an orientation suitable for nucleophilic attack at the ? phosphoryl group. These ATP-grasp folds catalyze the formation of carbon-nitrogen bonds through acyl-phosphate intermediates. The final step catalyzed by carbamoyl phosphate synthetase is the phophorylation of carbamic acid with another molecule of ATP to form carbamoyl phosphate (Berg, 2002).
This takes place in a second ATP-grasp domain within CPS, which has three different active sites. The ammonia came from the hydrolization of glutamine by carbamoyl phosphate synthetase through its second polypeptide component where glutamate is also a by-product. The active site of the glutamine-hydrolyzing component of carbamoyl phosphate synthetase contains a catalytic dyad comprising a cysteine and a histidine residue (Berg, 2002). The second step is the conversion of carbamoyl phosphate into uridylate. Initially, carbamoyl phosphate reacts with aspartate to form carbamoylaspartate.
This is catalyzed by aspartate transcarbamoylase. Carbamoylaspartate then cyclizes to form dihydroorotate which is then oxidized by NAD+ to form orotate (Berg, 2002). Orotate then couples with ribose 5-phosphoribosyl-1-pyrophosphate (PRPP) to form orotidylate, a pyrimidine nucleotide. The PRPP, a form of ribose activated to accept nucleotide bases, is produced by the pentose phosphate pathway, wherein ribose-5-phosphate is added with pyrophosphate from ATP. This reaction is driven by the hydrolysis of pyrophosphate and catalyzed by pyrimidine phosphoribosyltransferase (Berg, 2002).
Orotidylate is then decarboxylated to form uridylate (UMP), a major pyrimidine nucleotide that is a precursor to RNA. This reaction is catalyzed by orotidylate decarboxylase, one of the most proficient enzymes known which makes the decarboxylation reaction occur at high speed (Berg, 2002). Finally, uridylate (UMP) is converted to uridine triphosphate. First, UMP is phosphorylated to UDP by UMP kinase-one of the specific nucleoside monophosphate kinases that utilize ATP as the phosphoryl-group donor. UDP is then interconverted by nucleoside diphosphate kinase.
This is an enzyme that has broad specificity in contrast with the monophosphate kinases (Berg, 2002). Another pyrimidine, cytidine triphosphate (CTP), is formed when uridine triphosphate (UTP) is transformed by replacement of a carbonyl group with an amino group from glutamine. This reaction requires ATP and proceeds through an analogous mechanism in which the O–4 atom is phosphorylated to form a reactive intermediate, and then the phosphate is displaced by ammonia, freed from glutamine by hydrolysis (Berg, 2002).
For the de novo purine biosynthesis, purine bases already assembled attached to ribose ring are formed from simple starting materials such as amino acids and bicarbonate-unlike the case for pyrimidines (Berg, 2002). The salvage purine biosynthesis is notable for the energy that they save compared to other pathways. The first step is the conversion of free purine bases into precursors. The free purine bases derived from the turnover of nucleotides or from the diet, are attached to PRPP to form purine nucleoside monophosphates (Berg, 2002).
Two salvage enzymes with different specificities recover purine bases. Adenine phosphoribosyltransferase catalyzes the formation of adenylate while hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyzes the formation of guanylate as well as inosinate (inosine monophosphate, IMP), a precursor of guanylate and adenylate (Berg, 2002). The second step is an initial committed step where pyrophosphate is displaced by ammonia to produce 5-phosphoribosyl-1-amine, with the amine in the ? configuration.
This reaction is catalyzed by glutamine phosphoribosyl amidotransferase which is comprised of two domains: the first is homologous to the phosphoribosyltransferases in salvage pathways, whereas the second produces ammonia from glutamine by hydrolysis. However, the glutamine-hydrolysis domain is distinct from the domain that performs the same function in carbamoyl phosphate synthetase since the cysteine residue located at the amino terminus of this enzyme facilitates the glutamine hydrolysis (Berg, 2002).
The third step is the assembly of the purine ring by successive steps of activation by phosphorylation followed by displacement to produce inositate. These consist of nine (9) additional steps where the first six steps are analogous reactions, most of which are catalyzed by enzymes with ATP-grasp domains. Each step consists of the activation of a carbon-bound oxygen atom (typically a carbonyl oxygen atom) by phosphorylation, followed by the displacement of a phosphoryl group by ammonia or an amine group acting as a nucleophile. The final product of these reactions is the formation of inositate (Berg, 2002).
The final step is the conversion of inosinate into either AMP or GMP. Adenylate is synthesized from inosinate by substitution of an amino group for the carbonyl oxygen atom at C-6. The amino group came from the addition of aspartate followed by the elimination of fumarate. GTP, rather than ATP, is the phosphoryl-group donor in the synthesis of the adenylosuccinate intermediate from inosinate and aspartate. The reaction is catalyzed by the enzyme adenylsuccinate synthase (Berg, 2002). Guanylate (GMP) is synthesized by the oxidation of inosinate to xanthylate (XMP), followed by the incorporation of an amino group at C-2.
NAD+ is the hydrogen acceptor in the oxidation of inosinate. Xanthylate is activated by the transfer of an AMP group (rather than a phosphoryl group) from ATP to the oxygen atom in the newly formed carbonyl group. Ammonia, generated by the hydrolysis of glutamine, then displaces the AMP group to form guanylate, in a reaction catalyzed by GMP synthetase. Note that the synthesis of adenylate requires GTP, whereas the synthesis of guanylate requires ATP. This reciprocal use of nucleotides by the pathways creates an important regulatory opportunity (Berg, 2002).
The precursors of DNA, the deoxyribonucleotides, are synthesized by the reduction of ribonucleotides through a radical mechanism—more specifically the replacement of the 2? -hydroxyl group on the ribose moiety by a hydrogen atom. The substrates are ribonucleoside diphosphates or triphosphates, and the ultimate reductant is NADPH. The enzyme ribonucleotide reductase is responsible for the reduction reaction for all four ribonucleotides. Since the enzyme structure may differ depending on the organism, the ribonucleotide reductase mechanism of the two subunits R1 (an 87-kd dimer) and R2 (a 43-kd dimer) studied in an E.
coli is shown: . The ribonucleotide reductase mechanism starts with the transferring of an electron from a cysteine residue on R1 to a tyrosine radical on R2, generating a highly reactive cysteine thiyl radical. This radical abstracts a hydrogen atom from C-3? of the ribose unit. The radical at C-3? causes the removal of the hydroxide ion from the C-2? carbon atom. Combined with a hydrogen atom from a second cysteine residue, the hydroxide ion is eliminated as water. A hydroxide ion is transferred from a third cysteine residue. The C-3? radical recaptures the originally abstracted hydrogen atom.
An electron is transferred from R2 to reduce the thiyl radical. The deoxyribonucleotide is free to leave R1. The disulfide formed in the active site must be reduced to begin another reaction cycle (Berg, 2002). To produce thymine, generation of thymidylate from uracil is catalyzed by thymidylate synthase through methylating deoxyuridylate (dUMP) to thymidylate (TMP). The methylation of this nucleotide facilitates the identification of DNA damage for repair and, hence, helps preserve the integrity of the genetic information stored in DNA.
The methyl donor in this reaction is N5,N10-methylenetetrahydrofolate rather than S-adenosylmethionine (Berg, 2002). Tetrahydrofolate, produced in the synthesis of thymidylate, is regenerated from the dihydrofolate by dihydrofolate reductase with the use of NADPH as the reductant (Berg, 2002). The significance of ensuring successful regulation of the nucleotide biosynthesis is indeed placed naturally through the feedback inhibition mechanism. This controls both the overall rate of purine biosynthesis and the balance between AMP and GMP production at several sites.
These sites are: (1) the committed step of conversion of PRPP into phosphoribosylamine by glutamine phosphoribosyl amidotransferase in purine nucleotide biosynthesis, (2) the reactions leading away from inositate being the branch point in synthesis of AMP and GMP and (3) the reciprocate substrate relation in synthesis of adenine and guanine. The regulation of nucleotide biosynthesis is also important in ensuring the adequate production of important biomolecules such as nicotinamide adenine dinucleotide (NAD+), flavin adenine dinucleotide (FAD) and co-enzyme A.
The importance of understanding nucleotide biosynthesis covers both the actions man can do to prevent or try to cure cancer and other related diseases. The mutations in genes that encode nucleotide biosynthetic enzymes can result to devastating results such as the absence of hypoxanthine-guanine phosphoribosyltransferase on people with Lesch-Nyhan syndrome, a disease with inborn error of metabolism. The high serum levels of urate resulting from active purine degradation can induce gout, a disease in which salts of urate crystallize and damage joints and kidneys.
As such, drugs like allopurinol are prescribed to give relief by inhibiting the xanthine oxidase. For cancer, the synthesis and abundant supply of thymidylate for DNA synthesis was seen as the key in developing drugs. Most of these drugs block the synthesis of thymidylate through the (1) inhibition of the actions of the enzymes thymidylate synthase and dihydrofolate reductase (fluorouracil drug) or (2) the inhibition of tetrahydrofolate regeneration (aminopterin and methotrexate drugs).
Some drugs involve killing of cancer cells such as methotrexate while others like trimethoprim offers potent antibacterial and antiprotozoal activity. References Berg, J. M. , Tymoczko, J. L. and Stryer L. (2002). Biochemistry 5th edition. New York: W. H. Freeman and Company. Retrieved from http://www. ncbi. nlm. nih. gov/bookshelf/br. fcgi? book=stryer&part=stryer_book_info http://www. ncbi. nlm. nih. gov/bookshelf/br. fcgi? book=stryer&part=A3472 http://www. ncbi. nlm. nih. gov/bookshelf/br. fcgi? book=stryer&part=A3477