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1 number 34 Done by Abdulrahman Alhanbali Corrected by Mohammad Mahmoud Tarabeih Doctor Diala Nucleotide metabolism 1 P age

2 In this lecture we will talk about nucleotides; their structures, the synthesis of some of them and some clinical aspects related to them. The structure of nucleotides: Any nucleotide is composed of: A pentose sugar; Either ribose or deoxyribose. One or more phosphate group(s). A nitrogenous base; there are two types of N-bases: 1- Purines: composed of two rings; a 5-membered ring and a 6-membered ring adenine & guanine. 2- Pyrimidines: cytosine, uraciland thymine (CUT). Are composed of only one 6- membered ring. In metabolism, in some pathways the sugar and the phosphate(s) must be present to proceed the metabolism, while they aren t as important in other pathways. The importance of nucleotides: They are the monomers for nucleic acids either DNA or RNA. They act as energy molecules (ATP, GTP etc.). They are the carriers of many activated intermediates in different metabolism pathways (UDP-glucose, CDP-choline etc.). They are components in some electron carriers (NADH, FADH2) They act as second messengers (camp). Synthesis of nucleotides: Nucleotides can be obtained by: 1- Diet:most of the nucleotide intake is degraded and reused in salvage pathways. Yet, little amount is used as whole nucleotides without degrading and recycling. 2- De novo synthesis: this mean the synthesis of the nucleotides from scratch; atom by atom. 3- Salvage synthesis: building nucleotides from components that are already present, like when we add a N-base to a ribose-phosphate molecule. Modifications that can occur on the nitrogenous bases: 1- Methylation: adding a methyl group, usually for gene silencing. Remember: SAM is one of the sources of methyl group in methylation reactions. 2- Acetylation: adding an acetyl group, usually for gene activating. 2 P age

3 3- Glycosylation: adding a sugar molecule. 4- Reduction.. If we have a pentose and a nitrogenous base only without any phosphate groups we call this structure a nucleoside. However, sometimes we need to know the number of phosphate groups in a certain nucleotide so we name it as nucleoside (mono-/di-/tri-)phosphate. (e.g.:adenosine monophosphate). It is very important to note that it is a nucleotide not a nucleoside. Synthesis of purines: The synthesis of purines is expected to be more complicated than the synthesis of pyrimidines; because purines have more atoms, so more steps are required. 1- De novo synthesis of purines: a- Sources of atoms used in purine synthesis: Amino acids: aspartate, glycine and glutamate. Molecular CO2. Tetrahydrofolate (N10-formyl tetrahydrofolate) also contribute with some C atoms. b- Pathway: In purine synthesis we start with a ribose sugar with a phosphate group attached to it on carbon 5. 1) A pyrophosphate is added to carbon 1 By an enzyme called PRPP synthase making a 5-phosphoribosyl-1-pyropphosphate molecule(prpp). ATP is the source of the pyrophosphate. (ATP -> AMP) This gives a signal that this molecule is active, and it can start the synthesis of a purine nitrogenous base. This step is very important, and it is regulated by feedback mechanisms, high amounts of purines ribonucleotides will inhibit this step. Activated by inorganic phosphate The pyrophosphate molecule is only for activating, it won t make it to the final structure of purines. 2) The pyrophosphate is replaced by an amide group from glutamine.turning glutamine to a glutamate. This step is also highly regulated; it is activated by PRPP and inhibited by AMP/GMP. 3) In the next step a glycine molecule is added to amide group. This step needs ATP. The result intermediate is called glycinamide ribotide. 4) With the help of the enzyme formyl transferase, a carbonyl group is added to the structure. The source of the carbonyl group is the formyl form of tetrahydrofolate. (CHO is added to the structure) Now we have the 5 members of the first ring, but we can t close the ring until we add the first atom of the second ring. 3 P age

4 4 P age 5) Another amine group is added from another molecule of glutamine. The difference between this step and step 2 is that we need ATP in this step (synthetase enzyme), but in step 2 the release of pyrophosphate will supply the reaction with the energy it needs. 6) The first ring is closed with the usage of ATP. with the amine group outside the ring 7) A CO2 is added by a carboxylase enzyme. 8) In this step the last amino acid is used which is aspartate. Aspartate is integrated as a whole to the structure (attachment point is through its amine group) by a synthetase enzyme;however, we only need the amino group; 9) So, in this step adenylosuccinatelyase will cleave the rest of aspartate as a fumarate molecule. Remember there was a similar step in urea cycle. 10) Again, we need formyltetrahydrofolate to add a carbonyl group with the help of formyl transferase. 11) The second ring (6 atoms) is closed by a dehydration reaction. Now we have the first purine which is called Inosine monophosphate(imp). This is a branching point, it can proceed in guanine synthesis or adenine synthesis.

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6 6 P age -guanine synthesis: IMP dehydrogenase will start an oxidizing reaction coupled with the reduction of NAD+ to NADH making Xanthosine monophosphate. Then GMP synthetase will replace the oxygen with an amino group from glutamine making guanosine monophosphate (GMP). An ATP molecule is hydrolyzed to AMP and pyrophosphate in the last reaction. -adenine synthesis: An aspartate molecule is added on IMP by adenylosuccinate synthetase with the usage of a GTP molecule making adenylosuccinate. Then fumarate is cleaved by adenylosuccinaselyase which will leave us with Adenosine monophosphate AMP. Note that in both AMP and GMP the last result is the addition of an amino group but the differences are: -the place of the addition. -the source of the amino group. (aspartate in adenine and glutamate in guanine). -the energy molecule used in the process. (GTP in adenine and ATP in guanine). Note:The result is AMP and GMP are both monomers of RNA. DNA monomers should be deoxyribose

7 Nucleotides synthesis is usually targeted in cancer treatment, because when I inhibit nucleotide synthesis there won t be enough nucleotides for DNA replication, which will stop the cell division. (they don t discriminate between cancer cells and normal cells. That s why we see side effects epically in highly proliferating cells) Some antibiotics (such as sulphonamides) also target the synthesis of nucleotides (purines) in microorganisms. 2- Salvage pathway: If I have the N-base, I can add a phosphosugar (in the form of PRPP) to it in order to make a nucleotide. Enzymes that catalyze these reactions are: a) Adenine phosphoribosyl transferase (APRT) in adenine. b) hypoxanthine guanine phosphoribosyl transferase (HGPRT) has 2 substrates 1) Hypoxanthine (an intermediate in adenine synthesis!)imp 2) guaninegmp Clinical application: Lesch-nyhan syndrome: Is an inherited deficiency of HGPRT. 7 P age

8 This will affect the salvage synthesis of purines (unable to use hypoxanthine and Guanine). So, the body will try to fix that by activating de novo synthesis. This will partially fix the guanine problem but remember that APRT is still active so that will result in over production of AMP. AMP level will increase a lot because it will be synthesized by de novo synthesis and by salvage pathways de novo synthesis is activated to compensate for GMP and due to excess substrate (PRPP) not utilized in salvage pathway. High amounts of AMP will activate the degradation pathways which will lead to high amount of uric acid (degradation is discussed in the next lecture). This will result in gout-like symptoms: Uric acid accumulation in joints inflammatory response (pain/fever/edema ) arthritis. Uric acid accumulates also in kidneys, it will form a nucleus for a kidney stone (urolithiasis). Psychological effects: some behavioral effects such as biting lips or nails. Modifications on AMP and GMP: AMP and GMP aren t the only forms that we need in our bodies; to make other forms a further modification is required, and this modification can be: -the addition of phosphate groups: The addition of the second phosphate group is completed by a base-specific nucleoside kinase, that means each type of nucleosides-monophosphate has its own enzyme. these kinases don t discriminate between deoxyribose and ribose. discriminate the nitrogenous base only (adenylate kinase and guanylate kinase) To add the third phosphate, we need a different type of enzymes which is not base specific. Non-base specific kinase can work on any type of di-phosphate nucleosides either purines or pyrimidines. (nucleoside diphosphate kinase) The source of phosphate groups is ATP in both reactions. Because it s the most abundant -the reduction of the sugar: An enzyme called Ribonucleotide reductase (RR) works on di-phospho nucleotides by the removal of an OH group making the sugar a deoxyribose. Ribonucleotide reductase works on any di-phospho nucleotide either purine or pyrimidine. A cofactor called thioredoxin is oxidized in this process.nucleotidesare reduced Thioredoxin contains two cysteine residues which will form a disulfide bridge when thioredoxin is oxidized. To recycle thioredoxin back to its reduced form we need the enzyme thioredoxin reductase, which will oxidize a NADPH to NADP+ in the reaction. RR is a very important enzyme, it is highly regulated and it is the target of many drugs. It is regulated because deoxy-nucleotides are only used in DNA synthesis 8 P age

9 RR is formed from four subunits; two regulatory (R1) and two catalytic (R2) subunits. The regulatory subunits havetwo allosteric sites; a) Activity sites: deoxy adenosine triphosphate (datp) binds to this site to inhibit the enzyme, while ATP activates it. b) Substrate specificity sites: an example to understand the concept: deoxy thymidine triphosphate (dttp) binds to this site to activate the reduction of GTP to dgtp. So, binding of one type of nucleotides will affect the reduction of other types of nucleotides. importance: inhibition of Ribonucleotide reductaseso no deoxy nucleotides. DNA synthesis will stop specifically while nucleotide synthesis will continue. ANTI-Cancer to treat Chronic Myeloid leukemia 9 P age

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