number Done by Corrected by Doctor

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1 number 35 Done by حسام ا بوعوض Corrected by عبدالرحمن الحنبلي Doctor Diala 1 P age

2 We mentioned at the end of the last lecture that ribonucleotide reductase enzyme can be inhibited preventing the synthesis of deoxyribonucleotides (the building blocks of DNA) and so preventing DNA synthesis. This can be used as an anti-cancer treatment (no DNA present for cancer cells to replicate). One of these ribonucleotide reductase inhibitors is hydroxyurea which is used in some types of cancer including chronic myelogenous leukaemia (CML). At the beginning of the last lecture, we said that the sources of nucleotides are the diet (minor) and the synthesis processes (major). We discussed some of the synthesis processes last time and now we ll be discussing the degradation processes (i.e. the source is the diet) and some other synthesis processes. Degradation of Purine Nucleotides: At the end of the day, the food which we eat is made up of cells which contain DNA and RNA. These DNA and RNA(just like other macromolecules) need to be degraded in preparation for the absorption process (in the intestines). The nucleic acids are hydrolysed by phospho-nucleases (do not produce monomers) and phospho-diesterases (produce monomers). Then, when the (deoxyribo)nucleotides enter the intestinal mucosalcells, they are further degraded into (deoxyribo)nucleosidesand are further degraded to free bases which are then released to the circulation[according to the body s need, the free bases may be further degraded (purines to uric acid )major pathway for dietary purines) and pyrimidines to other metabolites] in the intestinal mucosal cells or may join other metabolic pathways in the intestinal mucosal cells or other cells. (Purines that come from de novo synthesis are degraded in the liver and then the free bases are sent to the circulation so that other cells can use them in the salvage pathway). In both GMP and AMP (it s purine degradation) degradation pathways there are some steps that are exactly the same and other steps that are separate (not the same enzyme), but are still similar. 1. GMP I- Nucleotide to Nucleoside GMP + H₂O guanosine + Pi 2 P age

3 The reaction requires the action of the 5 - Nucleosidase enzyme (refer tothe previous sheet and to the diagram at page 4 for the structures). II- Separating the Purine Ring Guanosine + Pi Guanine + Ribose-1-phosphate The enzyme Purine Nucleoside Phosphorylase is used. III- Xanthine Production Guanine + H₂O Xanthine + NH₃ The enzyme Guanase is needed here. IV- Uric Acid Production Xanthine + H₂O + O₂ Uric Acid + H₂O₂ The enzyme Xanthine Oxidase is required. 2- AMP Degrading AMP is not as straight-forward as degrading GMP; there are different degradation pathways possible and they meet (with each other and with different pathways at several points). AI- Nucleotide to Nucleoside AMP + H₂O Adenosine + Pi The enzyme 5 Nucleotidase is used (the same enzyme in step I in GMP degradation). AII- Inosine Production Adenosine + H₂O Inosine + NH₃ The enzyme Adenosine deaminase is needed. BI- IMP production AMP + H₂O IMP + NH₃ The enzyme AMP deaminase is required. IMP can enter into the degradative pathway from the synthetic pathway at this point. This means that the cell still has the chance to stop the 3 P age

4 synthesis of the purines when it reaches the point of IMP production in the synthesis pathway. BII- Inosine Production IMP + H₂O Inosine + Pi The enzyme 5 - Nucleotidase is necessary here (the same enzyme in step I in GMP degradation). From step III onwards both AMP degradation pathways (pathway A and pathway B) meet. III- Hypoxanthine Production Inosine + Pi Hypoxanthine + Ribose-1-phosphate This step is catalysed by the enzyme Purine Nucleoside Phosphorylase, which is the same enzyme that was used to remove the pentose sugar (the pentose sugar is removed here as well) in step II in GMP degradation. IV- Xanthine Production Hypoxanthine + H₂O + O₂ Xanthine + H₂O₂ The enzyme used here is Xanthine Oxidase, the same enzyme used in step III in GMP degradation. As you can see, Xanthine is also the product of step III of GMP degradation, this means that both the pathways meet at this point. V- Uric Acid Production Xanthine + H₂O + O₂ Uric Acid + H₂O₂ Again, the enzyme is Xanthine Oxidase. (This step is the exact same step as step IV in GMP degradation). 4 P age

5 The following diagram summarises all the reactions above: You can notice that the diagram also mentions three enzyme deficiencies related to these pathways, we ll discuss two of them (I don t know if the third is required for exam purposes). 1- Gout ( )ﺍﻟﻨﻘﺮﺱ This disease occurs when the uric acid levels become high in the blood (hyperuricemia). It could be either due to the overproduction or underexcretion of uric acid(underexcretion is more common). Uric acid (in gout) would accumulate in the synovial joints and form monosodium urate crystals which are detected by the body s immune system as antigens and so an inflammatory response begins (begins as an acute inflammation and later becomes chronic gouty arthritis). Nodular masses of monosodium urate crystals (also known as tophi) may get deposited in these synovial joints leading to what is known as Chronic Tophaceous Gout. Also, the increased levels of uric acid result in higher amounts of uric acid in the urine. The accumulation of uric acid in the kidneys may result in the building up of nucleic acids there forming kidney stones (urolithiasis). No symptoms are associated with hyperuricemia (without gout), but it is noted that gout is always preceded by hyperuricemia. To diagnose gout, you need to aspire and examine the synovial fluid of an affected joint (or the material of any tophus noted in the body) under a light microscopeusing polarised light microscopy, needle-shaped urate crystals confirm the presence of the disease (the yellow things in the second diagram). The underexcretion of uric acid (mentioned as a cause of uric acid accumulation) could be due to an inherent defect (primary), due to a known disease that affects the kidneys ability to excrete uric acid (e.g. lactic acidosis), due to an 5 Page

6 environmental factor (e.g. drugs) or due to exposure to lead (saturnine gout) (the three latter reasons are referred to as secondary causes). The overproduction of uric acid, on the other hand, is linked with several known mutations like that of the X-linked PRPP synthetase gene (increases PRPP production). To treat gout, you need to first determine the cause (overproduction or underexcretion) and, accordingly, you are supposed to prescribe drugs. The drugs offer a symptomatic treatment or might even limit the disease, but we are yet to find a cure to this disease (refer to slide #28 for some more details). (Don t mix between gout and LeischNyhan disease which was mentioned in the last lecture). 2- Adenosine Deaminase Deficiency (ADA) This is an autosomal recessive disease that is expressed in many tissues, but the lymphocytes are the ones to be affected the most. When adenosine deaminase enzyme is deficient, the levels of AMP rise (see the degradation of AMP pathway) and this increased AMP level serves as an activator to the ribonucleotide reductase enzyme, which means that more deoxyribo-nucleotides will be produced. When deoxyribo-nucleotides levels increase, they inhibit the action of ribonucleotide reductase which means that cells can no longer make DNA and so, they stop division earlier than usual and apoptosis occurs. This is seen the most in lymphocytes. To treat this disease, the patient must get a bone marrow transplant (BMT) or undergo an enzyme replacement therapy (ERT) and unless an appropriate treatment was given, the children affected usually die by the age of two. Pyrimidine Synthesis: There are two pathways: De novo (atoms added one by one) and Salvage (already built up parts are joined together). De Novo Pyrimidine Synthesis: First the pyrimidine ring is built up then it gets attached to the ribose-5- phosphate (in purines, the sugar was first added then the rings were built). 6 P age

7 A. Synthesis of Carbamoyl Phosphate: 2ATP + CO₂ + glutamine Carbamoyl phosphate + 2ADP + Pi + glutamate Glutamine acts as a source of NH₃. This reaction is catalysed by carbamoyl phosphate synthetase II (CPS II). Remember that CPS I was used in the urea cycle also to make carbamoyl phosphate. This step is the rate determining step and the one regulated the most. CPS II is inhibited by UTP (the end product of the pathway, feedback inhibition) and is activated by PRPP. B. Synthesis of Carbamoyl Aspartate: Carbamoyl Phosphate + Aspartate Carbamoyl Aspartate + Pi The enzyme used here is Aspartate transcarbamoylase. Just like usual, aspartate gets added to other molecules by adding its entire structure to the other molecule and a small molecule is lost in the process (here it is Pi). The carbamoyl aspartate is a 6 membered molecule which means that the build up reactions are over and from now on only modifications will occur (pyrimidine rings are 6 membered). C. Synthesis of Dihydro-orotate: Carbamoyl Aspartate + H+ Dihydro-orotate + H₂O The enzyme used is Dihydroorotase. The Dihydro-orotate is the first ring-structured molecule in this pathway. D. Synthesis of Orotate: Dihydro-orotate + FAD Orotate + FADH₂ The enzyme used is Dihydroorotate Dehydrogenase. The only difference between dihydro-orotate and orotate is the introduction of a double bond. E. Synthesis of Orotidine 5 -monophosphate (OMP): Orotate + PRPP OMP + PPi The enzyme used is Orotate Phosphoribosyl-transferase. The ribose-5- phosphate is transferred from PRPP to orotate and in the process PPi is lost. OMP is the first nucleotide produced in this pathway. The release of PPi (pyrophosphate) makes the reaction irreversible. 7 P age

8 F. Synthesis of Uridine 5 -monophosphate (UMP): OMP UMP + CO₂ The enzyme OMP Decarboxylase removes a CO₂ from OMP changing it to UMP. G. Moving On: ATP + UMP UDP + ADP AND UDP + ATP UTP + ADP The first reaction is catalysed by a kinase specific to UMP, while the second reaction is catalysed by a non-specific kinase. CTP can be produced from the made UTP.UTP + ATP + glutamine CTP + ADP + Pi + glutamatethe enzyme is CTP synthetase. Some CTP is dephosphorylated to CDP (a substrate for ribonucleotide reductase) to make dcdp which can be changed to dctp to be used in DNA synthesis (ammonia group is the only difference between Uracil and Cytosine). UDP can be changed to dudp by ribonucleotide reductase, dudp is then phosphorylated to dutp which is quickly changed to dump by the enzyme dutpase to prevent dutp from entering into DNA synthesis (changing UDP to dudp to dutp to dump is necessary for the production of dtmp for it can only be produced in this pathway which will be discussed shortly). H. Synthesis of Thymidine Monophosphate (TMP): dump + N⁵, N¹⁰-methylene tetrahydrofolate dtmp + Dihydrofolate The enzyme is Thymidylate Synthase. Of course dihydrofolate (DHF) has to be changed back to tetrahydrofolate (THF) for it to be able to act as a carbon carrier again, so the enzyme Dihydrofolate Reductase (uses NADPH + H+) changes it to tetrahydrofolate which can then be changed to N⁵, N¹⁰-methylene tetrahydrofolate to be used in producing more dtmp. Since this is the ONLY way for the production of dtmp (the precursor of dttp which is essential for DNA synthesis) many anti-cancer chemotherapies work by inhibiting this step (synthesis of dtmp). 5-8 P age

9 fluorouracil, for example, is a suicide inhibitor that is changed to 5- FdUMP and binds to the inactive thymidylate synthase locking it in that state and inhibiting dtmp synthesis. Methotrexate, another molecule, inhibits Dihydrofolate reductase and so locking the enzyme and preventing it from changing DHF to THF and so the carbon carrier becomes unavailable for this reaction preventing dtmp synthesis (Methotrexate also inhibits purine synthesis) which results in DNA synthesis inhibition and cell growth slow down (these examples are IMPORTANT). Notes on the Pathway: The substrates of the CPS II and CPS I enzymes are very similar, so how do they choose to go to the correct enzyme? The location of these two enzymes is different, CPS II is in the cytosol while CPS I is in the matrix of the mitochondria and their sources of their respective nitrogen atoms (i.e. one of the substrates) is slightly different (but this has a minor role in the specificity) with one using glutamine (CPS II) and the other using free ammonia (CPS I). Also, these enzymes differ by their allosteric regulators. The first three enzymes in the pathway (CPS II, aspartate transcarbamoylase and dihydroorotase) are three catalytic domains of a single polypeptide chain (CAD). The fourth enzyme in the pathway, dihydroorotate dehydrogenase, is associated with the inner mitochondrial membrane (ALL the other enzymes in the pathway are cytosolic). The last two enzymes, orotate phosphoribosyltransferase and OMP decarboxylase, are two catalytic domains of the same polypeptide chain called the UMP synthase. 9 P age

10 Both, purine and pyrimidine synthesis, require glutamine, aspartate and PRPP as essential precursors. Salvage Pyrimidine Synthesis: This is just a single step reaction (few pyrimidine bases are salvaged in human cells) Pyrimidine Nucleosides + ATP Pyrimidine Nucleotides Nucleoside Kinases are used. Pyrimidine Degradation: The pyrimidine ring is opened (unlike the purine ring which remains intact) and degraded to highly soluble products (β-alanine and β- aminoisobutyrate) (NH₃ and CO₂ are also produced). The following diagrams summarise almost everything from the last two lectures: 10 P age