Biochemistry Education BIOC 423 03.24.1 Concept 24: Nucleotide Metabolism Concept Overview The metabolism of nucleotides is complex because the molecules are large and composed of several different parts. The regulation of nucleotide metabolism; however, is very logical and fits the general scheme that we have been discussing throughout this course. Nucleotide metabolism is also important because it serves as a target for the development of several important pharmaceuticals. In this lecture we will look at general schemes for the synthesis of purines and pyrimidines, and not detailed pathways. In your studies do not waste effort memorizing de novo synthesis, rather concentrate on the important regulated steps, the general scheme of synthesis and similarities and differences between purine and pyrimidine synthesis. Also focus your attention to the steps that are the targets of drug therapies. Objectives When you have finished this module you should be able to: ? Review the structures of major purine and pyrimidine bases ? Tell the difference between a nucleosides and a nucleotides. ? Compare and contrast pyrimidine catabolism with purine catabolism ? Outline how purines are degraded and the health related problems associated with purine catabolism ? Identify the enzyme defects associated with Lesch?Nyhan syndrome and be able to discuss the significance of this association. ? Describe the role of purine catabolism in re?perfusion injury ? Describe the main features of the pathways for de novo synthesis of nucleotides, and to understand the roles of amino acids, one?carbon units and phosphoribosylpyrophosphate (PRPP). You do not need to have the entire pathway memorized. You should be able to compare and contrast pyrimidine synthesis with purine synthesis ? Describe the differences between de novo and salvage synthesis of nucleotides ? Describe the overall regulation of these pathways. ? Draw the reactions by which deoxyribonucleotides are made from ribonucleotides and describe the regulation of the enzyme ribonucleotide diphosphate reductase as well as common therapeutic targets in this pathway ? Draw the reactions associated with thymidylate synthase and describe the therapeutic potential of inhibiting this enzyme Biochemistry Education BIOC 423 03.24.2 ? Contents at a Glance Nucleotide Review Pyrimidine catabolism Purine catabolic pathways Problems related to uric acid Xanthine Oxidase and Re?perfusion injury Lesch?Nyhan syndrome ? what it is and why it is important Salvage pathway ? synthesis or disposal? DeNovo Pyrimidine Synthesis Synthetic pathway Regulation DeNovo Purine Synthesis Synthetic pathway Regulation Ribonucleotide reductase Dihydrofolate reductase inhibitors Synthesis of dTMP Thymidylate synthase inhibitors Biochemistry Education BIOC 423 03.24.3 Nucleotide Review At this point we need to refresh some of the nomenclature related to the purines and pyrimidines. The important purines and pyrimidines are illustrated below. Purines Pyrimidines Biochemistry Education BIOC 423 03.24.4 Recall that the nucleoside and nucleotide of thymine is associated with a deoxy sugar. In this course we will abbreviate thymidine as dTMP so we are consistent with the nomenclature used for the other purines and pyrimidines. You should be aware the deoxy nucleotide status is implied in the abbreviation TMP and some books use that symbol without adding the extra ?d?. Pyrimidine catabolism By this point in the course you should be able to make some fairly accurate guesses about a metabolic strategy for disposing of pyrimidine. With nucleotides in general the catabolic pathway is: Nucleotide ? Nucleoside ? Sugar + Base The above pathway is followed for both purines and pyrimidines. The entire pyrimidine pathway is shown for TMP. Only a couple of steps need extra comment. The second step in the pathway, nucleoside phosphorylase, (remember glycogen phosphorylase) is cellular. In the gut this enzyme is Biochemistry Education BIOC 423 03.24.5 replaced by a nucleoside hydrolase giving rise to a ribose sugar instead of the sugar phosphate. For your own practice, AND REVIEW, you should be able to propose a pathway to convert the end product (??methyl, ??aminopropionic acid = ?? aminoisobutyric acid) into succinyl?CoA. Most of the intermediates in this pathway are relatively soluble and deficiencies in the enzymes tend not to give rise to common catabolic problems. You are not responsible for memorizing the entire catabolic pathway of the pyrimidines. You should know the first two steps and the fact that pyrimidines can be catabolized to energy producing products (glycolytic and CAC intermediates) Purine catabolic pathways Unlike the pyrimidine catabolic pathway, purine catabolism is rich with medical problems and health issues. The pathway starts out the same way as pyrimidine catabolism with nucleosidases and nucleotidases. In addition for AMP or adenoside the amino group on the base must be removed. A similar step is necessary for guanosine. Below is an outline of purine catabolism. Biochemistry Education BIOC 423 03.24.6 There is only one enzyme responsible for the last two steps in purine catabolism and that enzyme is xanthine oxidase. The enzyme is unusual in that it uses Mo IV ion as a cofactor and generates hydrogen peroxide as a product. Uric acid is the end product of metabolism in humans. Other species can continue breaking down uric acid. Note that purine catabolism in humans does not produce glycolytic or CAC intermediates. Consequently, the purine bases are not a source of catabolic energy. Moreover, the end product, uric acid, is a new waste product. What can go wrong? Unfortunately the answer to the ?what can go wrong?? question is a rather large and important list. The problems usually relate to the chemical properties of uric acid. Uric acid is very insoluble and under normal conditions we function close to the limits of solubility. Any event that causes increased purine catabolism (anti? neoplastic chemotherapy, radiation injury, increased tissue death, etc.) or decreased total body water, or decreased kidney function or dehydration will allow the uric acid levels to increase to the saturation point and ureate crystals start precipitating leading to ureate crystals depositing in the tissues and joints. The disease gout is the result of an inflammatory response to these crystals. Reperfusion injury: A second issue related to the end products of purine catabolism is reperfusion injury. In this situation when a tissue is anoxic and all other anaerobic energy sources are consumed, the cell begins using RNA as a fuel source. The sugar found in RNA is useful and you should be able to describe how ribose enters glycolysis. But the purines require oxygen for their continued catabolism. In the absence of oxygen elevated levels of hypoxanthine build up from AMP and xanthine from GMP and the catabolism of purines stops at this stage. When oxygen is restored to the cell, given the high levels of hypoxanthine and xanthine there is a rapid production of uric acid with the accompanying production of hydrogen peroxide. The elevated hydrogen peroxide rapidly leads to tissue death. Drugs that inhibit xanthine oxidase, such as allopurinol, are used to slow down the xanthine oxidase activity and the associated production of hydrogen peroxide. You should notice the similarity between allopurinol and hypoxanthine. Severe Combined ImmunoDeficiency (SCID): Everyone is familiar with the ?Bubble Boy? who had no B?cell or T?cell immune response and needed to be isolated from all infectious agents. His condition was caused by the lack of the enzyme adenosine deaminase. This is one of the first condition for which in vivo enzyme replacement therapy has been attempted. We will wait until the next lecture to discuss how an ADA deficiency can give rise to a malfunctioning immune system. Biochemistry Education BIOC 423 03.24.7 Salvage Pathway If we were dependent only on the above pathway for purine catabolism we would be continuously depositing uric acid crystals in our tissues and inducing inflammation. There is a second pathway to supplement to disposal of purines. This second pathway, the salvage pathway, is a method to recycle or reuse the purine bases. Consequently, the salvage pathway is both a catabolic and anabolic pathway. The key enzyme in the salvage pathway is Hypoxanthine, Guanine?PhosphoRibosyl Transferase (HGPRT). There are analogous transferases for each of the purines and pyrimidines. In order to use this enzyme we need to make a ribose phosphate that is activated at position 1 of the ribose. The reaction is catalyzed by the enzyme ribose 5? phosphate pyrophosphokinase. The salvage enzyme HGPRT will transfer either hypoxanthine or guanine onto a PRPP molecule to generate IMP or GMP, respectively. The salvage of a guanine base is shown in the reaction below. You should be able to also write out the reaction salvaging the hypoxanthine base. What can go wrong? Again this is a relevant question. Although not common, the deficiency of HGPRT leads to a severe clinical syndrome called Lesch?Nyhan syndrome. This is an X?linked disease in which there is crippling gouty arthritis and severe neurological problems that lead to symptoms of self?mutilation. Historically this disease is one of the first ?mental diseases? in which a biochemical basis for the disease was identified. Applications of biotechnology Biochemistry Education BIOC 423 03.24.8 The biotechnology field has taken advantage of the fact that there are two independent pathways for the synthesis of nucleotides. By manipulating these two pathways it is possible to fuse two different cells and then select specific cells that have desired properties. Manipulation of these pathways represent the basic science behind hybridoma technology. SYNTHESIS of the NUCLEOTIDES There are two different pathways for the synthesis of both purine and pyrimidine nucleotides. One pathway is based on the synthesis of new purines or pyrimidines (de novo synthesis). The second pathway is a method of salvaging preformed purine and pyrimidine bases, the Salvage Pathway, that we have already discussed. Pyrimidine synthesis One of the very first metabolic reactions that we studied in the course is the first step in pyrimidine synthesis (look back to your notes on enzyme regulation). The enzyme that catalyzes that regulatory step in pyrimidine synthesis is aspartate transcarbamoylase. For this reaction we need aspartate (no problem); however, we also need carbamoyl phosphate. Synthesis is in the cytoplasm and the carbamoyl phosphate we made in the urea cycle is in the matrix of the mitochondria (problem). In this case we do not invest a new transport system. Instead, we will synthesize carbomoyl phosphate in the cytoplasm. The reaction is: Gln + 2 ATP + CO 2 ? carbamoyl phosphate + 2 ADP + Pi Note that this is not the same reaction we studied in the urea cycle. The nitrogen source is Gln instead of ammonium and the enzyme that catalyzes this cytoplasmic reaction is carbamoyl phosphate synthetase ?2. Once we have synthesized carbamoyl aspartate, the scheme for making pyrimidines is to first make the pyrimidine base, and then add the ribose phosphate, in a salvage type of reaction, to make the pyrimidine nucleotide (OMP). Biochemistry Education BIOC 423 03.24.9 OMP is then decarboxylated to form UMP. UMP is converted to UTP and the CTP molecule is synthesized by using an amido transferase enzyme. Remember our nitrogen donor for synthetic reactions is usually Gln. You should not memorize this entire pathway but rather have a general idea of what is happening in pyrimidine synthesis. You already know the details of the first and regulated step in pyrimidine synthesis. The regulation of pyrimidine synthesis is slightly more complex in that it varies with different species. In humans the regulation is in the production of cytoplasmic carbamoyl phosphate rather than the production of carbamoyl aspartate, which is the regulatory enzyme in E. coli. \ Biochemistry Education BIOC 423 03.24.10 Purine Synthesis The pathway for purine synthesis is very different than that of the pyrimidines. This pathway starts with PRPP and slowly builds the purine base on the ribose phosphate molecule by essentially adding one atom at a time until the molecule inosine monophosphate (IMP) is constructed. The pathway is reproduced below. Biochemistry Education BIOC 423 03.24.11 You are not responsible for the details of this pathway other than a general overall approach to synthesis that is outlined in the figure above. Go through the above pathway once to try to convince yourself that we can synthesize purines. In addition it is important to note that some of the carbon atoms in the purine are donated by THF derivatives, so that THF analogues can often serve as medically important inhibitors of the purine synthetic pathway. Also, a deficiency of folic acid in the growing individual can have major consequences in proper development. The AMP and GMP molecules are then synthesized from IMP. The following figure shows the details of these modifications of reactions. You are not expected to be able to draw from memory the details of this figure. You should however note the similarity of the AMP synthetic route with the urea cycle that we have already discussed. Biochemistry Education BIOC 423 03.24.12 In this pathway you will again see Gln and ATP used by an amido transferase to replace a carbonyl group with a primary amine. In the synthesis of ATP however, the amino group is added using Aspartate just like in the urea cycle. Regulation Regulation of purine synthesis fits the model we have been talking about all semester with the end product inhibiting the first committed step in the reaction. In this case however there is a problem in that the first committed step varies with different species. For this class we will not differentiate between species. There is also regulation at branch points. IMP will feedback to inhibit the first committed step and the end products AMP and GMP will feed back inhibiting the reactions catalyzed at the branch points. The result of this set of allosteric inhibitors is a balanced production of both ATP and GTP. Biochemistry Education BIOC 423 03.24.13 Reactions necessary for DNA synthesis The reactions discussed above provide the precursors need for the synthesis of RNA or the nucleic acid component of the cofactors. To uses these materials in DNA synthesis required two additional reactions. First, the deoxyribose needs to be synthesized and secondly it is necessary to convert the dUMP to Thymidylate (dTMP) Ribonucleotide Diphosphate Reductase The enzyme that reduces ribose to 2??deoxyribose is ribonucleotide diphosphate reductase. This enzyme will accept any of the purine or pyrimidine diphosphate nucleotides (NDP) and reduce them to the 2??deoxyribose derivative (dNDP). One complication in this reaction is that the reducing agents for this reaction are the thiol groups on the enzyme itself. Consequently, during the reaction the thiol groups are oxidized to disulfides and the enzyme is inactivated. In order to continue the reaction it is necessary to reduce the oxidized enzymes. There are two different systems that can be used to accomplish this reduction. One system is thioredoxin while the other system is a glutathione based system that we have seen previously in our discussions of reactive oxygen species. Biochemistry Education BIOC 423 03.24.14 BASE O OHOH HH HH PPO BASE O HOH HH HH PPO Ribonucleotide reducatase SH SH Ribonucleotide reducatase S S Thioredoxin SH SH Thioredoxin S S FAD Reductase NADPH NADP Glutaredoxin HS HS Glutaredoxin S S GSH GSSG Reductase ribonucleotide reductase reductase The regulation of the enzyme is remarkable. It is regulated by feedback inhibition by each one of the four dNTPs. Each of the dNTPs feeds back and specifically inhibits its own production. For example if the level of dCTP increases, dCTP is an allosteric regulator that will specifically decrease the production of more dCDP, without affecting the rate of production of the other dNDPs. The one exception to this regulatory scheme is dATP. High levels of dATP have an inhibitory activity for production of all dNDPs. The regulation of this enzyme is extremely important in maintaining a balance of dNTPs that is necessary for DNA synthesis. Consequently, the regulation of this enzyme is far more complex than we have discussed above. In our last lecture we described Severe Combined Immunodeficiency Syndrome that is caused by a deficiency in adenosine deaminase enzyme. In this deficiency ATP and importantly dATP will begin building up in the cell. The consequences of too much ATP are not very significant because ATP is usually present in excess. However, when dATP builds up because of this enzyme deficiency, the excess dATP will inhibit ribonucleotide reductase and prevent the synthesis of dTTP, dGTP and dCTP. Consequently, DNA cannot be made. For a successful B or T cell immune response, cell proliferation is essential. Without a balance in the cellular concentration of the four deoxy?nucleotides, DNA is not synthesized and cell proliferation cannot happen. Biochemistry Education BIOC 423 03.24.15 Thymidylate Synthase The second enzyme system necessary to make precursors for DNA synthesis is the method for converting dUMP to dTMP. This reaction involves both the transfer of a methyl group from N5,N10?methylene?THF (N5,N10 methylene FH4) and the transfer of reducing equivalents. As a result of both transfers in the reaction, the THF (FH4) is oxidized into the dihydrofolate derivative (DHF or FH2) from THF and the cofactor is inactivated. To support DNA synthesis it is necessary to re?reduce the DHF (FH2) back to THF (FH4) (dihydrofolate reductase). In addition, a methylene group needs to be transferred back onto the THF cofactor (serine hydroxymethyl transferase) to regeneratee the N5,N10 methylene?THF. HN O O CH 3 N O HOH HH HH OP - O O - O HN O ON O HOH HH HH OP - O O - O FH 2 FH 4 NADPH NADP dUMP dTMP N5, N10 Methylene FH4 Thymidylate synthase Fluorouracil Inhibits dihydrofolate reductase methotrexate aminopterin inhibits Serine Glycine Serine hydroxymethyl- transferase By interfering with this process and preventing the synthesis of dTMP it is possible to control or kill cells entering the S phase of the cell cycle. Consequently, several drugs have been developed for cancer therapy that either prevent the recycling of the FH 2 cofactor (examples: methotrexate, aminopterin) or directly inhibit the thymidylate synthase enzyme (5?fluorouracil). Biochemistry Education BIOC 423 03.24.16 PROBLEMS Describe the different approaches used by purine synthesis and pyrimidine synthesis The nitrogen and carbon atoms of the pyrimidine ring are derived most directly from: A. alanine, ammonia, bicarbonate B. ammonia, aspartate, bicarbonate C. ammonia, aspartate, formyl-THFA D. bicarbonate,formyl-THFA glutamine, glycine E. aspartate, carbamoyl phosphate Draw the structures of the following: A. 5-ribosyl-1-amine B. ribose-5-phosphate C. PRPP D. AMP E. IMP F. thymidylic acid (dTMP) Biochemistry Education BIOC 423 03.24.17 What is the Pentose Phosphate Pathway? Include its function and the overall reactants and products. In what cells/tissues is it active? What is the role of the PPP in nucleotide catabolism and synthesis? In many tissues, one of the earliest responses to cellular injury is a rapid increase in the levels of enzymes involved in the pentose phosphate pathway (PPP), up to 20-30 times higher than normal. Which of the following explain the need for these high levels of PPP enzymes? A. NADPH is needed for synthesis of fatty acids and cholesterol, components of cellular membranes B. Ribose-5-phosphate is necessary for synthesis of RNA and DNA C. Higher concentrations of ATP are needed D. A and B E. A, B and C William Anderson Microsoft Word - 24 Nucleotide Metabolism.docx
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