I. NUCLEOTIDES. Nitrogenous bases are frequently represented by single letter abbreviations. The abbreviations for several bases are shown below:

Transcription

1 Biochemistry II Nucleotide Metabolism I. NUCLEOTIDES Nitrogenous bases are frequently represented by single letter abbreviations. The abbreviations for several bases are shown below: Common Nitrogenous Bases Infrequent Nitrogenous Bases Adenine A Hypoxanthine I Guanine G Xanthine X Uracil U Dihydrouracil D Cytosine C Pseudouridine Thymine T You will also see bases referred to using single letter abbreviations for the type of base: Any Base Purine Pyrimidine N R Y The nucleoside associated with a particular base, will carry the same abbreviation as the base. Hence, the nucleoside of adenine is symbolized as A. A nucleotide, on the other hand, is symbolized by the letter for the base, plus the type of phosphate that is attached: Monophosphate Diphosphate Triphosphate MP DP TP So, adenosine monophosphate is A + MP = AMP cytidine diphosphate is C + DP = CDP xanthine triphosphate is X + TP = XTP purine monophosphate is R + MP = RMP any base diphosphate is N + DP = NDP II. NUCLEOTIDE BIOSYNTHESIS Nucleotides must be present or produced whenever nucleic acids have to be produced. There are two fundamental types of nucleic acid: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The only time DNA must be produced is when cells are dividing. For most adult organisms, this means for the growth of tissue like hair, nails, and/or sex cells. Everything else stops growing and that signals the passage into adulthood. However, RNA is almost constantly needed; so there is a heavy demand for RNA. DNA is used to produce RNA. DNA is the primary copy of all information in an organism. It is not used in secondary processes and only a few types of tissue will produce it consistently. RNA, on the other hand, is used for producing proteins and for regulating certain processes. Whenever you need to make new protein, RNA is used. But the RNA that s used comes from DNA: The system requests a product, like hexokinase. The response is to use DNA to make an RNA copy (transcript) of the DNA instruction (gene) for the production of hexokinase. The transcript is carried to the production site and used to make some amount of the hexokinases. Once the required amount of hexokinase is made, the transcript is destroyed. The next time the product is required, a new transcript is produced from the hexokinase gene. This is analogous to a construction firm. The firm is housed in a building. Inside the building are file cabinets containing the blueprints for making each type of building the firm can produce. These blueprints represent DNA. When the firm receives a request for a particular building, they open the cabinet containing the required blueprint, and make a copy of it. The copy represents RNA. They package up the copy and send it to the construction site. At the site they use the copy to make the building. In the process, the copy gets written on, coffee spilled on it, torn in different places, smudged, and otherwise poorly treated. Bottom line, it ll probably be almost useless once they re done at the construction site. Hence, the good sense in not sending the main copy to the site. So we just discard the damaged copy and create a new one when the time comes. The enzymes that destroy old nucleic acids are called nucleases (nucleases hydrolyze nucleic acid to nucleotides). So, how is nucleic acid produced when required? You have to string together nucleotides. The enzymes that do this are called polymerases and their substrates include nucleotide triphosphates. Where do the nucleotides come from? Well, we don t get em from eating. They must either be made from scratch or recycled from old nucleotides.

2 III. PURINE NUCLEOTIDE BIOSYNTHESIS Purine Nucleotide Purine nucleotides consists of a 6-membered ring fused to a 5-membered ring. The approximate molar mass of the purine system is 120 daltons. There are two main purine bases: adenine and guanine. Other purine bases include hypoxanthine, xanthine, and uric acid. Position 9 of the molecule is the attachment point for the ribose of a nucleotide. Purines may be made by recycling older molecules or by making completely new ones from scratch. When a purine is made from scratch, it is constructed from amino acids, folate derivatives, water CO 2 and ATP (ingredients). FROM SCRATCH Making purine nucleotides from scratch is called de novo synthesis. When purines are produced from scratch the base is built on a ribose scaffold; ribose-5- phosphate (R5P). R5P is activated by phosphorylating carbon-1 with ATP to make 5- phosphoribosyl-1-pyrophosphate, PRPP. The base is then built upon PRPP by adding the ingredients stepwise to carbon-1 (the building point). There are 11 steps in the synthesis and the end product is a nucleotide that has a hypoxanthine base; the nucleotide is called inosine 5'-monophosphate, or IMP. IMP is the precursor of AMP and GMP. R5P -> PRPP ->->->->->->->->->->->->IMP ->->->AMP or GMP Di- and triphosphate nucleotides are made by phosphorylating monophosphate nucleotides with kinase enzymes and ATP or inorganic phosphate. GMP + ATP -> GDP + ADP Once produced, nucleotides are used and reused to make and/or repair nucleic acid until they are too damaged (worn out) to be useful. Bases from nucleic acid degradation or that have hung around too long can be targeted for removal or used to repair nucleic acid or used to make new nucleotides. SALVAGE REACTIONS Nucleotides are also synthesized by addition of purine bases from nucleic acid degradation products to PRPP. This is called salvage because the base is essentially saved from destruction. When nucleic acids have outlived their purpose, they are degraded by enzymes called nucleases. Nucleases hydrolyze nucleic acid: DNA ribose + phosphate + purine bases The products of hydrolysis are riboses (which can be converted into R5P or PRPP), phosphates (which may be used to phosphorylate), and purine bases, which can be used to regenerate nucleotides. The regeneration process involves transferase enzymes that reattach free purine bases to PRPP. transferases purine bases + PRPP RMP (A, G, or I) + PPi

3 IV. PYRIMIDINE BIOSYNTHESIS Pyrimidine nucleotides consists of a 6-membered ring. The approximate molar mass of the purine system is 80 daltons. There are three main pyrimidine bases: cytosine, uracil, and thymine. Other purine bases include dihydrouracil and pseudouridine. Position 1 of the molecule is the attachment point for the ribose of a nucleotide. Pyrimidines may be made by recycling older molecules (salvage) or by making completely new ones from scratch (de novo). When a pyrimidine is made from scratch, it is constructed from amino acids, water, bicarbonate and ATP (ingredients). Pyrimidine Nucleotide FROM SCRATCH Pyrimidines are also synthesized by a de novo and salvage pathway. However, in the de novo path, the base is not built upon PRPP, but is constructed from glutamine and bicarbonate with the PRPP being added after the base is built. The end result of the de novo pathway is uridine 5'-monophosphate, UMP. Mono-, di-, and tri- nucleotides are created by the transfer of phosphate from NTPs in phosphate transfer reactions by kinases. UMP + ATP -> UDP + ADP CTP is formed from UTP by the transfer of an amine group from glutamine. The reaction is a typical amination reaction. HO 2 2H + glutamine glutamate ATP ADP + Pi UTP CTP

4 V. SYNTHESIS OF DEOXYRIBONUCLEOTIDES Deoxyribonucleotides are produced by the reduction of ribonucleotides through free radicals - and e transfer. ribonucleotide reductase NADPH + H H O + NADP + Note the enzyme: Ribonucleotide reductase (Fig ) 2 subunits 2 B1 subunit - Contains the regulatory sites and functions as a sulfhydryl (-SH) e - donors. B2 subunit - Catalytic site. Has stable free radicals on tyrosines, and also has iron 2+ (Fe ) centers. The mechanism of radical intermediate formation is: Note how various NTPs effect the activity and binding of the reductase (p. 612). instance, datp inhibits overall catalytic activity; For WHY MIGHT THIS BE EXPECTED??? How are the electrons transferred? E transferred from NADPH to the catalytic site of the reductase is carried out by way of a small peptide called thioredoxin.

5 VI. ACTION OF RIBONUCLEOTIDE REDUCTASE Basically, two other peptides take part in the transfer of electrons from NADPH to ribonucleotide reductase. These are implicated in the mechanism of electron transfer as shown below. thioredoxin thioredoxin ribonucleotide reductase reductase VII. DNA COMPONENTS Now, DNA does not contain uracil or uracil analogs; it contains thymine. The thymine is derived from UMP that has been turned into dump. The enzyme responsible for the transformation is thymidylate synthetase. DHF Reductase THF 5 10 N,N -methylene tetrahydrofolate > + DHF thymidylate synthetase dump dtmp This type of reaction makes an interesting point about evolution of nucleic acids. Note that not only the deoxy- sugar, but actual bases themselves for DNA composition are derived from ribonucleotides. The implication is that DNA is derived from RNA. This is a fairly well accepted scenario. Indeed, the further implication is that RNA came first, and in all likelyhood the earliest forms of replicating entities were probably RNA chains. Note that several aspects of the above reaction are exploited for use in cancer treatment. suicide inhibition - directly inhibits thymidylate synthetase by first acting like a substrate (resembles uridine), then an inhibitor. {Due to some unreactive portion of the drug} Flourouracil is an example. The overall result is the inhibitioon of DNA synthesis due to the lack of dtmp. Other anticancer drugs are competitive inhibitors of THF; aminopterin and methotrexate. They are effective unless a resistance is developed to the drug (from gene duplication, etc...). CAN ANYONE THINK OF THE DRAWBACK OF THESE DRUGS???

6 VIII. NUCLEIC ACID DEGRADATION PRODUCTS AND THEIR EFFECTS As stated earlier, nucleic acids are constantly being degraded. nucleotidases nucleoside phosphorylase nucleotides nucleosides phosphoriboses + bases More specifically, the purines are degraded to metabolites which must be maintained in a specific balance to maintain an organism's health. For example, consider AMP : AMP IMP hypoxanthine xanthine uric acid Uric acid is degraded further by way of an enol/keto conversion, into urate, which is excreted in primates. GOUT: uric acid (enol) urate (keto) Buildup of urate leads to the condition known as gout. The condition comes about due to a deficiency in the enzyme, hypoxanthine-guanine phosphoribosyl transferase (HGPRT). This enzyme is necessary for the salvage synthesis of IMP and GMP. PRPP + hypoxanthine IMP + PPi HGPRT + guanine GMP + PPi If HGPRT is deficient, then excess levels of hypoxanthine and guanine build up. This offset in cell equilibrium levels of these bases is corrected urate formation, as shown above. Essentially, reduction in production at the level of the salvage pathway leads to excess urate formation as shown above. PRPP + hypoxanthine/guanine IMP/GMP + PPi alloxanthine xanthine urate allopurinol The drug, allopurinol is used to relieve the gout condition. xanthine and disrupts urate buildup through suicide inhibition. This substrate resembles Note that although an excess of urate is detrimental, urate is an essential metabolite. It is an antioxidant that removes radicals, superoxide anions, singlet oxygen, and other highly reactive oxidants. Lesch-Nyhan Syndrome: There is another disease that is associated with deficiencies in base metabolism. This is the Lesch Nyhan syndrome. It is a sex-linked recessive disease. * High urate and PRPP levels (as in gout) * Extreme neurological conditions (such as retardation and self-mutilation at ages around 2-3). * HGPRT absent (no salvage pathway) * High levels of de novo synthesis The level of urate may be decreased by allopurinol, but the neurological symtoms, PRPP levels, and amount of de novo synthesis are not altered.

7 Note that in normal brain HGPRT levels are very high, and the activity amidotransferase (enzyme of committed step in de novo pathway) is low.