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Informational macromolecules (2)

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Nucleotides are the basic building blocks of nucleic acids DNA and RNA. They consist of a nitrogenous base, a pentose sugar and phosphate groups. Nucleotides combine to form nucleic acids and act as carriers of genetic information and cellular energy. Some synthetic nucleotide analogs are used as chemotherapy drugs by incorporating into DNA and disrupting nucleic acid synthesis. Nucleic acids DNA and RNA store and transmit genetic information through their linear polymers of nucleotides joined by phosphodiester bonds into strands with distinct primary, secondary and tertiary structures.

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INFORMATIONAL MACROMOLECULES Storage and Expression of Genetic Information
NUCLEOTIDES Nucleotides are the building blocks of Nucleic Acids. Without them, neither DNA nor RNA can be produced and therefore proteins cannot be synthesized and cells cannot proliferate. They have multiple additional functions:- They serve as carriers of activated intermediates in the synthesis of some carbohydrates, lipids and proteins.
Nucleotides are structural components of several essential coenzymes, for example, coenzyme A, FAD,NAD and NADP. Nucleotides such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP),serve as second messengers in signal transduction pathways. They also play an important role as source of energy in the cell. They act as regulatory compounds for many pathways of metabolism and inhibit or activate key enzymes.
STRUCTURE OF NUCLEOTIDES Nucleotides are composed of -a nitrogenous base -a pentose monosaccharide and -one, two or three phosphate groups. The Nitrogen containing bases are aromatic, heterocyclic bases and belong to two families of compounds: The Purines and The Pyrimidines. Their six-atom rings are numbered in opposite direction from each other.They can exist in two forms --- keto form and enol form depending on the positioning of the oxo and amino groups.
Purine and Pyrimidine Structures Purines include Adenine and Guanine abbreviated as A and G respectively. Adenine is 6-aminopurine while guanine is 2-amino,6-hydroxypurine. Hypoxanthine and xanthine are also purines Pyrimidines include cytosine(C)—2-oxy-4-amino-pyrimidine thymine(T)—2,4-dioxy-5-methyl-pyrimidine uracil(U)---2,4-dioxy-pyrimidine
 
Nucleosides Nucleosides are derivatives of purines and pyrimidines formed by the combination of a nitrogenous base (purine or pyrimidine) with a pentose sugar. If the sugar is Ribose,a Ribonucleoside is formed and if the sugar is 2-Deoxyribose,a Deoxyribonucleoside is produced. The sugar is linked at position 1´ to the heterocyclic base via a β -N-glycosidic bond,at position N-1 of a pyrimidine or to N-9 of a purine. Numerals with a prime e.g 2´ or 3´ distinguish atoms of the sugar from those of the heterocyclic base.
 
Structure of nucleosides
Mononucleotides If a phosphate group is added to a nucleoside, forming an ester linkage with the hydroxyl group of the pentose sugar, a mononucleotide is formed. In case of ribose there are three such places at carbon atoms number 2,3 and 5 where an ester linkage can be formed which are written as nucleoside 2´-monophosphate,nucleoside 3´-monophosphate and nucleoside 5´-monophosphate respectively. The first phosphate group is attached to the 5´-OH of the pentose and is called nucleoside 5´-monophosphate (NMP) or 5´-nucleotide e.g Adenosine monophosphate (AMP), also called adenylate.
In contrast to ribose which has three OH groups and thus three points for phosphate group attachment, deoxyribose has two such places(3´ and 5´) as C2 lacks an oxygen atom and thus named as deoxyribose. Thus a nucleotide can be RIBONUCLEOTIDE or DEOXYRIBONUCLEOTIDE.The letter d is placed before the name of the nucleotide containing deoxyribose e.g dAMP.
AMP
Nucleoside Diphosphate and Triphosphate If a second or third phosphate is added to the nucleoside, a nucleoside diphosphate or nucleoside triphosphate is formed. Adenosine diphosphate(ADP) Adenosine triphosphate(ATP) The second and third phosphates are each connected to the nucleotide by a “high energy bond”. These bonds are “acid anhydride bonds”.
AMP,ADP,ATP
Nitrogenous bases,their nucleoside and nucleotide derivatives base ribonucleoside ribonucleotide deoxyribonucleoside deoxyribonucleotide adenine adenosine Adenosine monophosphate Deoxy-adenosine Deoxy-adenosine –mono-phosphate guanine guanosine Guanosine monophosphate Deoxy-guanosine Deoxy-guanosine-mono-phosphate cytosine cytidine Cytidine monophosphate Deoxy-cytidine Deoxy-cytidine-mono-phosphate uracil uridine Uridine monophosphate ----- ----- thymine --- ----- Deoxy-thymidine Deoxy-thymidine-mono-phosphate
Different nucleotides and their composition A-PRESENT IN RNA:- AMP or adenylate --- adenine+ribose+phosphoric acid GMP or guanylate ---- guanine+ribose +phosphoric acid CMP or cytidylate ---- cytosine+ribose+phosphoric acid UMP or uridylate ----- uracil+ribose+phosphoric acid B-PRESENT IN DNA:- d-AMP or deoxyadenylate --- adenine+deoxyribose+phosphoric acid dGMP or deoxyguanylate ----guanine+deoxyribose+phosphoric acid dCMP or deoxycytidylate ---- cytosine+deoxyribose+phosphoric acid TMP or thymidylate ---- thymine+deoxyribose+phosphoric acid
Physiologic functions of biologically important nucleotides and nucleosides In addition to being the precursors of DNA and RNA, the serve a number of physiologic functions. Derivatives of Adenine:- a –ATP:- It is the store house of energy for cells. It has two high energy bonds and on hydrolysis, each releases energy (7.6 Kcal ) and this energy is then used for -muscle contraction -transmission of nerve impulse -phosphorylations -active transport -synthetic processes -formation of active methionine and active sulfate
b- ADP :- -acts as phosphate acceptor in oxidative phosphorylation - role in cellular respiration -muscle contraction -enzyme activation c-AMP:- - activator of several enzymes - inhibition of certain enzymes
Derivatives of guanosine:- GTP:- -role in citric acid cycle -role in protein synthesis -necessary for the formation of cyclic AMP -role in purine synthesis -role in gluconeogenesis
Derivatives of uridine:- - UDP Glucose:- formed by the rea-ction of UTP with glucose-1 phosphate------ provides glucose units to glycogen during glycogen synthesis. - UDP Glucuronic acid:- used for conjugation of bilirubin and many drugs.
Derivatives of cytosine:- CTP takes part in synthesis of phosphoglycerides, sphingomyelin, sphingosines.
Cyclic nucleotides CYCLIC AMP:- it is a cyclic nucleotide and chemically it is 3´-5´ adenosine monopohosphate. It is synthesized in tissues from ATP. FUNCTIONS OF AMP:- -second messenger in the cell -role in glycogen metabolism -triglyceride metabolism -cholesterol synthesis -activation of protein kinases -protein biosynthesis -cell differentiation -regulation of cell membrane permiability
CYCLIC GMP:- Made from GTP. FUNCTIONS OF c-GMP:- -protein phosphorylation -role in smooth muscle relaxation and vasodilatation -role in neurotransmission -role in insulin action -regulation of sodium channels
Synthetic Analogs of Nucleosides and Nucleotides Chemically synthesized analogs of purines,pyrimidines and their nucleosides and nucleotides have numerous applications in clinical medicine especially for cancer chemotherapy. BASIS OF CHEMOTHERAPY:- In these analogs,the heterocyclic ring or the sugar moiety is altered in such a way that toxic effects are produced when this analog gets incorporated into the cellular constituents of the body. The toxic effects of chemotherapeutic drugs include ---- inhibition of enzymes essential for nucleic acid synthesis OR they get incorporated into nucleic acids which results in disruption of base pairing.
Some synthetic analogs:- -5-FLUOROURACIL, 3-DEOXYURIDINE, 6-MERCAPTOPURINE, 6-AZAURIDINE are widely used by oncologists. These drugs get incorporated into DNA before cell division. ALLOPURINOL is a purine analog, used in the treatment of hyperuricemia and gout. It acts by inhibiting purine biosynthesis and it inhibits the enzyme xanthine oxidase which acts for uric acid synthesis. CYTARABINE used in cancer chemotherapy and in viral infections. AZATHIOPRINE ,catabolized to 6-MARCAPTOPURINE, used in organ transplantation to suppress immunological rejection. -
Polynucleotides:- When two or more nucleotides are joined together by internucleotide bonds, polynucleotides are formed. The bond between the nucleotides is the “phosphodiester bond”. This bond is formed mainly between the 3´OH group of sugar of one nucleotide and 5´OH group of sugar of another nucleotide. This forms a Dinucleotide. This 3´-5´ linkage is the backbone of DNA and RNA.
dinucleotude
NUCLEIC ACIDS Nucleic acids are required for the storage and expression of genetic information. There are two chemically distinct types of nucleic acids. These are DNA and RNA. The monomeric unit of DNA is deoxyribonucleotide and that of RNA is ribonucleotide.
DNA DNA is a repository of genetic information. Present in the chromosomes in the nucleus of every eukaryotic cells. The nuclear DNA is bound to basic proteins called HISTONES. Prokaryotic cells, lack nuclei , have a single chromosome, also contain non-chromosomal DNA in the form of Plasmids. The genetic information found in DNA is copied and transferred to daughter cells through a process called DNA REPLICATION. This information is then used for RNA synthesis which finally results in protein synthesis.
Structure of DNA DNA is a polymer of deoxyribonucleoside monophosphates which are covalently linked by 3´ ->5´ phosphodiester bonds. DNA exists as a double stranded molecule in which the two strands wind around each other and form a double helix. The deoxynucleotide units of DNA include--- DEOXYADENYLATE, DEOXYGUANYLATE, DEOXYCYTIDYLATE, THYMIDYLATE.
Primary structure of DNA The primary structure of DNA is the number and sequence of different deoxyribonucleotides in the DNA strand. The back bone of the primary structure is the linear strand made by sugar phosphate residues, linked together, while the bases project laterally. The 3´OH group of the deoxypentose of one nucleotide is joined to 5´OH group of the deoxypentose of the adjacent nucleotide through a phosphate group i.e makes a phosphodiester bond. This way a long,unbranched chain is formed which has polarity--- a 5´end and 3´ end are free ( phosphate groups are free without phosphodiester linkage not attached to other nucleotides ). The bases located along the resulting deoxyribose- phosphate backbone are always present in a sequence, from 5´ to 3´ end of the chain. This sequence is the genetic code of the DNA.
polynucleotide
Secondary structure of DNA This is made up of a double stranded helix formed by two polydeoxyribonucleotide strands around a central axis called the axis of symmetry. The chains are paired in anti-parallel manner,that is, the 5´ end of one strand is paired with 3´ end of the other strand. The deoxyribose-phosphate backbone is hydrophilic and thus in each chain it is on the outside of the molecule. The bases are hydrophobic and thus located towards the inside of the molecule. The overall structure resembles a twisted ladder. The phosphodiester bonds in the two interwoven strands run in opposite directions. Therefore the strands are called Antiparallel. The 3´-5´strand is called Coding or Template strand while 5´-3´ strand is called non-coding strand.
The spatial relationship between the two strands in the helix creates a major ( wide ) groove and a minor narrow groove. These grooves are used by the regulatory proteins to bind to their specific recognition sequences along the DNA chain. BASE PAIRING: The bases of one strand of DNA are paired with the bases of the other strand. ADENINE IS ALWAYS PAIRED WITH A THYMINE AND A CYTOSINE IS ALWAYS PAIRED WITH A GUANINE. Therefore one strand of the double stranded DNA is always the complement of the other strand….. Given the sequence of bases on one chain, the sequence of bases on the other chain can be determined. This base pairing leads to CHARGAFF RULE: in any sample of dsDNA, the amount of adenine equals the amount of thymine, the amount of guanine equals amount of cytosine, and the total number of purines is equal to total number of pyrimidines. The base pairs are held together by hydrogen bonds--- two between A and T and three between G and C. They stabilize the structure of the helix.
 
 
 
Structural forms of DNA double helix There are three major structural forms;A,B and Z. B-DNA – the most common type. Described by Watson and Crick. Usually found under physiologic conditions( low salt, well hydrated ). A right handed helix. Contains ten residues per 360 degree turn of the helix. The planes of the bases is perpendicular to the helical axis. A-DNA – It is formed by moderately dehydrating the B form. It is also a right handed helix but contains eleven base pairs per turn. The planes of the bases are tilted 20 degrees away from the perpendicular to the axis of helix. Z-DNA- left handed helix. twelve base pairs per turn found in regions of DNA which has alternating purines and pyrimidines.
 
The denaturation of DNA Hydrogen bond disruption– Separation of the two strands of DNA. In lab– disruption caused by alteration in pH of the solution of DNA so that nucleotide bases are ionized OR by heating the solution. Tm--- when DNA is heated, the temperature at which one half of the helical structure is lost is defined as the melting temperature. The loss of helical structure is called Denaturation. The Tm of DNA is determined by its base composition. As there are three hydrogen bonds between G and C , and two hydrogen bonds between A and T, the DNA with higher amount of G and C, has a higher Tm.
RIBONUCLEIC ACID (RNA) RNA is a polymer of ribonucleotides of Adenine, Uracil, Guanine and Cytosine, joined together by 3´-5´phosphodiester bonds. RNA does not contain thymine except in rare cases. The pentose sugar of RNA is D-ribose. RNA is found in the nucleolus, ribosomes, mitochondria, and cytoplasm.
Structure of RNA PRIMARY STRUCTURE:- It is defined as the number and sequence of ribonucleotides in the RNA chain. The sequence is complementary to the template strand of the gene from which it was transcribed. The ribonucleotides are held together by 3´-5´ phosphodiester bonds. 3´-OH group of one nucleotide is bound to 5´-OH of the other nucleotide and form a linear strand.
SECONDARY STRUCTURE OF RNA:- This involves coil formation of the polyribonucleotide chain. The coiled structures are stabilized by hydrophobic interactions between purine and pyrimidine bases. There are intra-chain hydrogen bonds between G-C and A-U.
TERTIARY STRUCTURE OF RNA;- This involves the folding of the molecule into three dimensional structure. There is cross-linking at various sites stabilized by hydrophobic and hydrogen bonds producing a compactly coiled globular structure.
 
 
Differences between DNA and RNA RNA exists as a single strand, however it is capable of folding back on itself like a hairpin and thus acquire double-stranded characteristics. Pentose sugar is ribose. It has different pyrimidine components i.e it has uracil in place of thymine. Since RNA is a single strand comlementary to only one of the two strands of the gene, its guanine content is not necessarily equal to cytosine, nor its adenine necessarily equals its uracil content. In addition to nucleus,RNA is found in cytoplasm. DNA forms RNA by transcription but RNA cannot form DNA. In experiments, reverse transcriptase can be used for this purpose. RNA can be hydrolysed to 2´,3´ cyclic mononucleotides.
Classes of RNA:- Three main classes of RNA exist. They are:- A-Messenger RNA (m-RNA) B-Transfer RNA (t-RNA) C-Ribosomal RNA (r-RNA) Another type found in eukaryotic cells is small nuclear RNA (snRNA)
A-MESSENGER RNA The most heterogeneous class regarding size and stability. FUNCTION: The members of this class function as messengers to convey the information in a gene to the protein synthesizing machinery. The mRNA carries genetic information from the nuclear DNA to the cytosol, where it is used as a template for protein synthesis. If the mRNA carries information from more than one gene, it is called polycistronic which is characteristic of prokaryotes. If the mRNA carries information from just one gene, it is called monocistronic, characteristic of eukaryotes.
Messenger RNA contains mainly adenine, guanine, cytosine and uracil as the major bases. mRNA molecules are formed with the help of DNA template strand (3´-5´) during the process called transcription. In addition to the protein coding regions that can be translated, there are untranslated regions at its 5´ and 3´ ends.
Special structural characteristics of eukaryotic messenger RNA:- It contains a long sequence of adenine nucleotides (20-250) on the 3´ end of the RNA chain and is called “poly-A tail”. It is thought to play role in stability of mRNA against 3´ exonucleases. There is a “cap” on the 5´ end consisting of a molecule of 7-methylguanosine. The cap is involved in the recognition of mRNA by the translation machinery and also helps to stabilize mRNA against 5´ exonucleases.
In mammalian cells, the messenger RNA that comes to cytoplasm is the product of processing of a precursor called, HATEROGENOUS NUCLEAR RNA (hnRNA). It is synthesized in the nucleus and is much bigger than mRNA. Most of it is degraded in the nucleus and only 25% of it forms a precursor of mRNA called pre-mRNA which is then converted to mRNA.
TRANSFER RNA (tRNA) tRNA is the smallest of the three major species of RNA. Single stranded globular molecules. Remain largely in cytoplasm. Functions; The t RNA molecules serve as ADAPTERS for the translation of information in the sequence of nucleotides of the mRNA into specific amino acids. T here is at least one specific type of tRNA molecule for each of the amino acids commonly found in proteins. Each tRNA carries its specific amino acid to the site of protein synthesis. There it recognizes the genetic code word on mRNA and this specifies the addition of its amino acids to the growing peptide chain.
Structure of tRNA:- Primary structure; tRNA molecules consist of 74-95 nucleotides in a particular sequence. The tRNA molecules contain not only the usual bases like adenine, guanine, cytosine, uracil but also contain unusual bases like dihydrouracil , pseudouridine, thymine. Secondary structure: each single stranded tRNA is folded extensively and extensive intrachain base pairing which leads to a characteristic CLOVER-LEAF structure. These folds are stabilized by hydrogen bonds between complementary bases of the same strand. This leads to the formation of double stranded structures.
Arms or loops of tRNA: All tRNA molecules contain 4 main arms or loops. 1-Acceptor arm; This is made up of unpaired sequences of cytosine-cytosine-adenine at the 3´end. The 3´OH terminal of adenine binds with the carboxylic group of a specific amino acid and carries it to ribosomes for protein synthesis. 2-Anticodon arm: it is in the form of a loop and carries specific sequences of three bases which constitute the anticodon. The bases of anticodon are bonded with three comlementary bases of codon of mRNA. 3-D arm: it contains the base dihydrouridine. 4-T Ψ C arm: contains thymine, pseudouridine and cytosine. The extra arm and the T Ψ C arms help define a specific tRNA.
 
Ribosomal RNA ( rRNA) rRNA is found as a component of ribososmes--- a cytoplasmic nucleoprotein structure that acts as the machinery for the synthesis of proteins from the mRNA template. The rRNA forms 80% of the total cellular RNA. The ribosomal subunits are defined according to their sedimentation velocity in Svedberg units. The mammalian ribosomes has a sedimentation velocity of 80S . It contains two major subunits- a larger one with 60S and a smaller one with 40S. The 60S subunit carries 60% of rRNA . There are four distinct species of rRNA in eukaryotic cells--- 28S, 18S, 5.8S and 5S. The bases in rRNA are mainly adenine, guanine, cytosine and uracil and a few pseudouridine.
Function of rRNA: - The rRNA are necessary for ribosomal assembly and play a key role in the binding of mRNA to ribosomes and its translation. Some rRNA function as catalysts in protein synthesis and are termed as “ribozymes”.
Small RNA Small nuclear RNA (SnRNA) are large number of small stable RNA species found in eukaryotic cells. Most of them are complexed with proteins to form ribonucleoproteins. They are distributed in the nucleus, in the cytoplasm or in both. They are significantly involved in mRNA processing and gene regulation.
METABOLISM OF NUCLEOTIDES
Biological significance of nucleotide metabolism Nucleotides make up DNA and RNA. Nucleotide Triphosphates are energy carriers e.g ATP. Regulate many metabolic pathways. GTP used in protein synthesis, UTP activates glucose and galactose, CTP has role in lipid metabolism AMP is a component of coenzymes e.g NAD, NADP, FAD etc.
De-Novo SYNTHESIS OF PURINE NUCLEOTIDES A complex process Not synthesized as free base Synthesized as ribonucleotides Site is cytosol of liver cells There is no dietary requirement of purines as they are synthesized sufficiently in body.
Materials required for Purine synthesis PRPP--- starting material, formed from ribose-5-phosphate obtained from HMP shunt. Amino acids and derivatives ---- glycine, aspartic acid, glutamine. Energy --- ATP CO 2 Formylated tetrahydrofolate and Mg
Steps of Biosynthesis 1 – Synthesis of PRPP:- begins with Ribose-5´-P ---- from HMP- shunt pathway. ATP is used for energy. -- Enzyme is PRPP Synthetase. Coenzyme is Mg. --activated by inorganic phosphate and inhibited by Purine Nucleotides. 2 – Synthesis of 5´-phosphoribosylamine :- Formed by the transfer of amide grp of Glutamine to PRPP. Gives N-9 of purine ring. -- Enzyme is Glutamine-PRPP amidotransferase. --Inhibitors --- AMP, GMP, IMP. -- Activators --- PRPP The committed step in purine synthesis.
3- PRA + glycine , ATP is used, formation of 5-phosphoribosyl-glycinamide. This gives C-4, C-5 and N-7 of the purine ring. 4- Formylation of amino nitrogen of glycinamide by formyl-THF forming 5-phosphoribosyl-formyl glycinamide. Formyl C becomes C-8 of purine ring.
5- Transfer of another amide group forms 5-phosphoribosyl-N formyl glycinamidine. This forms N3 of purine ring. 6- Formation of 5-phosphoribosyl-5-aminoimidazole, formed by removal of water and closure of the ring.
7- Carboxylation gives C6 of purine ring. Formation of 5-phosphoribosyl-5-aminoimidazole-4-carboxylate. 8- Condensation of Aspartic acid with the above compound forms 5-phosphoribosyl-5-aminoimidazole-4-N succino-carboxamide.
9- Cleavage enzymes act and form 5-phosphoribosyl-5-aminoimidazole-4-carboxamide. 10- Formyl tetrahydrofolate donates C2 of the purine ring with the help pf formyl transferase. This forms 5-phosphoribosyl-5-formamido –imidazole-4-carboxamide.
11- Formation of IMP–( inosine-5-monophosphate). Formed by removal of water molecule and ring closure. Inosinic acid (IMP) is also called Hypoxanthine nucleotide and is the first purine synthesized in the body.
Conversion of IMP to AMP Two step energy requiring pathway. IMP condenses with Aspartic acid and forms Adenylosuccinate. Enzyme is adenylosuccinate synthetase which is inhibited by end product(AMP). Energy comes from GTP. Adenylosuccinase cleaves adenylosuccinate to form AMP.
Conversion of IMP to GMP Two steps Energy comes from ATP IMP dehydrogenase oxidises IMP into Xanthosine monophosphate. This step is inhibited by end product GMP. GMP synthase transfers amide group of glutamine to C-2 of XMP and forms GMP.
Mycophenolic acid is a drug that acts as a reversible, uncompetitive inhibitor of IMP dehydrogenase. It is used to prevent graft rejection as it stops the rapid proliferation of T and B cells.
 
Formation of Nucleoside Diphosphates & Triphosphates Phosphorylation converts mono form to di and tri phosphate forms. AMP is converted to ADP and then ATP by action of adenylate kinase. GMP is converted to GDP and then GTP by action of guanylate kinase.
Regulation/control of purine synthesis At the level of PRPP synthase. Allosteric feed back inhibition by PRPP, AMP, GMP, ADP, GDP etc. At the level of Glutamine-PRPP amidotransferase. Rate limiting. Inhibited by AMP and GMP. Balance between ATP and GTP. Each one stimulates the synthesis of the other.
Inhibitors of purine synthesis PABA analogs ---- Sulfonamides are structural analogs of PABA and thus competitively inhibit folic acid synthesis in bacteria. Human beings get folic acid from external sources and thus are not affected by the drug. Folic acid analogs---- methotrexate is one such drug. It inhibits the reduction of dihydrofolate into tetrahydrofolate and thus no purines synthesis. This slows down DNA replication . Used for the treatment of cancer. Harmful to all the cells.
Salvage Pathway for Purines:- This is the synthesis of nucleotides and nucleosides from the free purines already present in the body. These purines may be derived from -diet -or come from the normal turnover of cellular nucleic acids.
Salvage pathway needs much less energy as compared to the de-novo synthesis of purine nucleotides. It is a re-cycling pathway. There are two mechanisms for salvage pathway
1-conversion of free purines to their mononucleotide form:- Conversion of Guanine to GMP:- enzyme--HGPRT. Conversion of Hypoxanthine to IMP:- enzyme---HGPRT. Conversion of Adenine to AMP:- enzyme--- APRTase PRPP is the source of ribose-5P in all these reactions. Irreversible reactions
2- Conversion of nucleosides to nucleotides:- Nucleosides can be converted into their respective nucleotides by the action of the particular nucleoside kinase. ATP is utilized during the process. e.g adenosine +ATP AMP + ADP
Lesch-Nyhan Syndrome X-linked recessive disorder Complete absence of HGPRT Hypoxanthine and Guanine cannot be salvaged. PRPP increases, IMP and GMP lower, more de novo purine synthesis excessive uric acid formation--- gout and neurological features.
Synthesis of Deoxyribonucleotides:- Ribonucleotides are converted to deoxyribonucleotides by the enzyme Ribonucleotide Reductase. Its co-enzyme is Thioredoxin which requires NADPH for its regeneration. This enzyme is strongly inhibited by dATP .
DEGRADATION OF PURINE NUCLEOTIDES/Uric acid formation Pancreatic enzymes hydrolyze dietary nucleotides into nucleosides and free bases in the intestines. Sequential break down takes place inside cells and end product Uric acid is formed. Uric acid is the end-product of purine catabolism in human beings and other primates.
Degradation of adenine nucleotides AMP is converted to Adenosine by the action of enzyme 5-nucleotidase. Adenosine deaminase takes out ammonia and converts Adenosine to Inosine. Phosphorylation of inosine forms Hypoxanthine and Ribose phosphate. Enzyme is purine nucleoside phosphorylase.
Oxidation of hypoxanthine forms xanthine. Enzyme is xanthine oxidase. Xanthine oxidase again oxidises xanthine into uric acid.
Degradation of Guanine Nucleotides GMP releases Guanosine by the action of 5-nucleotidase. Guanosine gives Guanine and Ribose P by the action of phosphorylase. Guanine deaminase converts Guaninine to xanthine. Xanthine oxidase forms uric acid.
Uric acid End product of purine metabolism in humans and primates. Normally 500-600 mg is synthesized in body daily and most of it is excreted in urine. Serum contains 3-7mg/100 ml of uric acid.
Clinical disorders of purine metabolism 1- Primary hyperuricemia/gout 2- Secondary Hyperuricemia 3- Lesch Nyhan syndrome 4- Xanthinuria 5-Adenosine Deaminase deficiency
Gout Chronic disorder characterized by - Hyperuricemia - deposition of urate crystals in joints and tissues ---- Tophi - attacks of acute arthritis - urate stones in kidneys
Primary Gout -Excessive production of uric acid -Decreased excretion of uric acid from kidney Excessive production --- excessive purine synthesis due to enzyme defects, and thus more uric acid synthesis.
Resistance to feed back inhibition of PRPP synthetase. Defective HGPRT leading to decreased utilization of PRPP in salvage pathway. High level of PRPP in cells enhances de-novo purine synthesis.
High purine foods --- all meats, organ meats, alcoholic beverages, beans, peas, lentils, spinach, cauliflowers and mushrooms.
Secondary Hyperuricemia Increased break down of purines during diseases like leukemia, prolonged fasting, polycethemia and psoriasis. Cytotoxic drugs. Renal failure leading to decreased uric acid excretion. Von Gierke’s disease --- deficiency of glucose 6 phosphatase, increased pentose, increased purine synthesis.
Treatment of Gout Anti-inflammatory drugs– Colchicine and NSAIDs. Uricosuric drugs---- increased renal excretion of uric acid. Probenecid is one such drug. Given to patients with normal renal function. Enzyme inhibitor--- Allopurinol--- competitive inhibitor of Xanthine Oxidase.
Xanthinuria Inherited disorder Deficiency of xanthine oxidase Hypoxanthine and xanthine cannot be converted to uric acid Low blood levels of uric acid Excessive excretion of xanthine, xanthine stones in kidneys
Adenosine Deaminase Deficiency Genetic deficiency of adenosine deaminase. B and T lymphocytes are deficient--- immunodeficiency. Accumulation of deoxyribonucleotides, inhibition of further production of precursors of DNA. Hypouricemia due defective break down of purine nucleotides.
METABOLISM OF PYRIMIDINES

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Informational macromolecules (2)

  • 1. INFORMATIONAL MACROMOLECULES Storage and Expression of Genetic Information
  • 2. NUCLEOTIDES Nucleotides are the building blocks of Nucleic Acids. Without them, neither DNA nor RNA can be produced and therefore proteins cannot be synthesized and cells cannot proliferate. They have multiple additional functions:- They serve as carriers of activated intermediates in the synthesis of some carbohydrates, lipids and proteins.
  • 3. Nucleotides are structural components of several essential coenzymes, for example, coenzyme A, FAD,NAD and NADP. Nucleotides such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP),serve as second messengers in signal transduction pathways. They also play an important role as source of energy in the cell. They act as regulatory compounds for many pathways of metabolism and inhibit or activate key enzymes.
  • 4. STRUCTURE OF NUCLEOTIDES Nucleotides are composed of -a nitrogenous base -a pentose monosaccharide and -one, two or three phosphate groups. The Nitrogen containing bases are aromatic, heterocyclic bases and belong to two families of compounds: The Purines and The Pyrimidines. Their six-atom rings are numbered in opposite direction from each other.They can exist in two forms --- keto form and enol form depending on the positioning of the oxo and amino groups.
  • 5. Purine and Pyrimidine Structures Purines include Adenine and Guanine abbreviated as A and G respectively. Adenine is 6-aminopurine while guanine is 2-amino,6-hydroxypurine. Hypoxanthine and xanthine are also purines Pyrimidines include cytosine(C)—2-oxy-4-amino-pyrimidine thymine(T)—2,4-dioxy-5-methyl-pyrimidine uracil(U)---2,4-dioxy-pyrimidine
  • 6.  
  • 7. Nucleosides Nucleosides are derivatives of purines and pyrimidines formed by the combination of a nitrogenous base (purine or pyrimidine) with a pentose sugar. If the sugar is Ribose,a Ribonucleoside is formed and if the sugar is 2-Deoxyribose,a Deoxyribonucleoside is produced. The sugar is linked at position 1´ to the heterocyclic base via a β -N-glycosidic bond,at position N-1 of a pyrimidine or to N-9 of a purine. Numerals with a prime e.g 2´ or 3´ distinguish atoms of the sugar from those of the heterocyclic base.
  • 8.  
  • 10. Mononucleotides If a phosphate group is added to a nucleoside, forming an ester linkage with the hydroxyl group of the pentose sugar, a mononucleotide is formed. In case of ribose there are three such places at carbon atoms number 2,3 and 5 where an ester linkage can be formed which are written as nucleoside 2´-monophosphate,nucleoside 3´-monophosphate and nucleoside 5´-monophosphate respectively. The first phosphate group is attached to the 5´-OH of the pentose and is called nucleoside 5´-monophosphate (NMP) or 5´-nucleotide e.g Adenosine monophosphate (AMP), also called adenylate.
  • 11. In contrast to ribose which has three OH groups and thus three points for phosphate group attachment, deoxyribose has two such places(3´ and 5´) as C2 lacks an oxygen atom and thus named as deoxyribose. Thus a nucleotide can be RIBONUCLEOTIDE or DEOXYRIBONUCLEOTIDE.The letter d is placed before the name of the nucleotide containing deoxyribose e.g dAMP.
  • 12. AMP
  • 13. Nucleoside Diphosphate and Triphosphate If a second or third phosphate is added to the nucleoside, a nucleoside diphosphate or nucleoside triphosphate is formed. Adenosine diphosphate(ADP) Adenosine triphosphate(ATP) The second and third phosphates are each connected to the nucleotide by a “high energy bond”. These bonds are “acid anhydride bonds”.
  • 15. Nitrogenous bases,their nucleoside and nucleotide derivatives base ribonucleoside ribonucleotide deoxyribonucleoside deoxyribonucleotide adenine adenosine Adenosine monophosphate Deoxy-adenosine Deoxy-adenosine –mono-phosphate guanine guanosine Guanosine monophosphate Deoxy-guanosine Deoxy-guanosine-mono-phosphate cytosine cytidine Cytidine monophosphate Deoxy-cytidine Deoxy-cytidine-mono-phosphate uracil uridine Uridine monophosphate ----- ----- thymine --- ----- Deoxy-thymidine Deoxy-thymidine-mono-phosphate
  • 16. Different nucleotides and their composition A-PRESENT IN RNA:- AMP or adenylate --- adenine+ribose+phosphoric acid GMP or guanylate ---- guanine+ribose +phosphoric acid CMP or cytidylate ---- cytosine+ribose+phosphoric acid UMP or uridylate ----- uracil+ribose+phosphoric acid B-PRESENT IN DNA:- d-AMP or deoxyadenylate --- adenine+deoxyribose+phosphoric acid dGMP or deoxyguanylate ----guanine+deoxyribose+phosphoric acid dCMP or deoxycytidylate ---- cytosine+deoxyribose+phosphoric acid TMP or thymidylate ---- thymine+deoxyribose+phosphoric acid
  • 17. Physiologic functions of biologically important nucleotides and nucleosides In addition to being the precursors of DNA and RNA, the serve a number of physiologic functions. Derivatives of Adenine:- a –ATP:- It is the store house of energy for cells. It has two high energy bonds and on hydrolysis, each releases energy (7.6 Kcal ) and this energy is then used for -muscle contraction -transmission of nerve impulse -phosphorylations -active transport -synthetic processes -formation of active methionine and active sulfate
  • 18. b- ADP :- -acts as phosphate acceptor in oxidative phosphorylation - role in cellular respiration -muscle contraction -enzyme activation c-AMP:- - activator of several enzymes - inhibition of certain enzymes
  • 19. Derivatives of guanosine:- GTP:- -role in citric acid cycle -role in protein synthesis -necessary for the formation of cyclic AMP -role in purine synthesis -role in gluconeogenesis
  • 20. Derivatives of uridine:- - UDP Glucose:- formed by the rea-ction of UTP with glucose-1 phosphate------ provides glucose units to glycogen during glycogen synthesis. - UDP Glucuronic acid:- used for conjugation of bilirubin and many drugs.
  • 21. Derivatives of cytosine:- CTP takes part in synthesis of phosphoglycerides, sphingomyelin, sphingosines.
  • 22. Cyclic nucleotides CYCLIC AMP:- it is a cyclic nucleotide and chemically it is 3´-5´ adenosine monopohosphate. It is synthesized in tissues from ATP. FUNCTIONS OF AMP:- -second messenger in the cell -role in glycogen metabolism -triglyceride metabolism -cholesterol synthesis -activation of protein kinases -protein biosynthesis -cell differentiation -regulation of cell membrane permiability
  • 23. CYCLIC GMP:- Made from GTP. FUNCTIONS OF c-GMP:- -protein phosphorylation -role in smooth muscle relaxation and vasodilatation -role in neurotransmission -role in insulin action -regulation of sodium channels
  • 24. Synthetic Analogs of Nucleosides and Nucleotides Chemically synthesized analogs of purines,pyrimidines and their nucleosides and nucleotides have numerous applications in clinical medicine especially for cancer chemotherapy. BASIS OF CHEMOTHERAPY:- In these analogs,the heterocyclic ring or the sugar moiety is altered in such a way that toxic effects are produced when this analog gets incorporated into the cellular constituents of the body. The toxic effects of chemotherapeutic drugs include ---- inhibition of enzymes essential for nucleic acid synthesis OR they get incorporated into nucleic acids which results in disruption of base pairing.
  • 25. Some synthetic analogs:- -5-FLUOROURACIL, 3-DEOXYURIDINE, 6-MERCAPTOPURINE, 6-AZAURIDINE are widely used by oncologists. These drugs get incorporated into DNA before cell division. ALLOPURINOL is a purine analog, used in the treatment of hyperuricemia and gout. It acts by inhibiting purine biosynthesis and it inhibits the enzyme xanthine oxidase which acts for uric acid synthesis. CYTARABINE used in cancer chemotherapy and in viral infections. AZATHIOPRINE ,catabolized to 6-MARCAPTOPURINE, used in organ transplantation to suppress immunological rejection. -
  • 26. Polynucleotides:- When two or more nucleotides are joined together by internucleotide bonds, polynucleotides are formed. The bond between the nucleotides is the “phosphodiester bond”. This bond is formed mainly between the 3´OH group of sugar of one nucleotide and 5´OH group of sugar of another nucleotide. This forms a Dinucleotide. This 3´-5´ linkage is the backbone of DNA and RNA.
  • 28. NUCLEIC ACIDS Nucleic acids are required for the storage and expression of genetic information. There are two chemically distinct types of nucleic acids. These are DNA and RNA. The monomeric unit of DNA is deoxyribonucleotide and that of RNA is ribonucleotide.
  • 29. DNA DNA is a repository of genetic information. Present in the chromosomes in the nucleus of every eukaryotic cells. The nuclear DNA is bound to basic proteins called HISTONES. Prokaryotic cells, lack nuclei , have a single chromosome, also contain non-chromosomal DNA in the form of Plasmids. The genetic information found in DNA is copied and transferred to daughter cells through a process called DNA REPLICATION. This information is then used for RNA synthesis which finally results in protein synthesis.
  • 30. Structure of DNA DNA is a polymer of deoxyribonucleoside monophosphates which are covalently linked by 3´ ->5´ phosphodiester bonds. DNA exists as a double stranded molecule in which the two strands wind around each other and form a double helix. The deoxynucleotide units of DNA include--- DEOXYADENYLATE, DEOXYGUANYLATE, DEOXYCYTIDYLATE, THYMIDYLATE.
  • 31. Primary structure of DNA The primary structure of DNA is the number and sequence of different deoxyribonucleotides in the DNA strand. The back bone of the primary structure is the linear strand made by sugar phosphate residues, linked together, while the bases project laterally. The 3´OH group of the deoxypentose of one nucleotide is joined to 5´OH group of the deoxypentose of the adjacent nucleotide through a phosphate group i.e makes a phosphodiester bond. This way a long,unbranched chain is formed which has polarity--- a 5´end and 3´ end are free ( phosphate groups are free without phosphodiester linkage not attached to other nucleotides ). The bases located along the resulting deoxyribose- phosphate backbone are always present in a sequence, from 5´ to 3´ end of the chain. This sequence is the genetic code of the DNA.
  • 33. Secondary structure of DNA This is made up of a double stranded helix formed by two polydeoxyribonucleotide strands around a central axis called the axis of symmetry. The chains are paired in anti-parallel manner,that is, the 5´ end of one strand is paired with 3´ end of the other strand. The deoxyribose-phosphate backbone is hydrophilic and thus in each chain it is on the outside of the molecule. The bases are hydrophobic and thus located towards the inside of the molecule. The overall structure resembles a twisted ladder. The phosphodiester bonds in the two interwoven strands run in opposite directions. Therefore the strands are called Antiparallel. The 3´-5´strand is called Coding or Template strand while 5´-3´ strand is called non-coding strand.
  • 34. The spatial relationship between the two strands in the helix creates a major ( wide ) groove and a minor narrow groove. These grooves are used by the regulatory proteins to bind to their specific recognition sequences along the DNA chain. BASE PAIRING: The bases of one strand of DNA are paired with the bases of the other strand. ADENINE IS ALWAYS PAIRED WITH A THYMINE AND A CYTOSINE IS ALWAYS PAIRED WITH A GUANINE. Therefore one strand of the double stranded DNA is always the complement of the other strand….. Given the sequence of bases on one chain, the sequence of bases on the other chain can be determined. This base pairing leads to CHARGAFF RULE: in any sample of dsDNA, the amount of adenine equals the amount of thymine, the amount of guanine equals amount of cytosine, and the total number of purines is equal to total number of pyrimidines. The base pairs are held together by hydrogen bonds--- two between A and T and three between G and C. They stabilize the structure of the helix.
  • 35.  
  • 36.  
  • 37.  
  • 38. Structural forms of DNA double helix There are three major structural forms;A,B and Z. B-DNA – the most common type. Described by Watson and Crick. Usually found under physiologic conditions( low salt, well hydrated ). A right handed helix. Contains ten residues per 360 degree turn of the helix. The planes of the bases is perpendicular to the helical axis. A-DNA – It is formed by moderately dehydrating the B form. It is also a right handed helix but contains eleven base pairs per turn. The planes of the bases are tilted 20 degrees away from the perpendicular to the axis of helix. Z-DNA- left handed helix. twelve base pairs per turn found in regions of DNA which has alternating purines and pyrimidines.
  • 39.  
  • 40. The denaturation of DNA Hydrogen bond disruption– Separation of the two strands of DNA. In lab– disruption caused by alteration in pH of the solution of DNA so that nucleotide bases are ionized OR by heating the solution. Tm--- when DNA is heated, the temperature at which one half of the helical structure is lost is defined as the melting temperature. The loss of helical structure is called Denaturation. The Tm of DNA is determined by its base composition. As there are three hydrogen bonds between G and C , and two hydrogen bonds between A and T, the DNA with higher amount of G and C, has a higher Tm.
  • 41. RIBONUCLEIC ACID (RNA) RNA is a polymer of ribonucleotides of Adenine, Uracil, Guanine and Cytosine, joined together by 3´-5´phosphodiester bonds. RNA does not contain thymine except in rare cases. The pentose sugar of RNA is D-ribose. RNA is found in the nucleolus, ribosomes, mitochondria, and cytoplasm.
  • 42. Structure of RNA PRIMARY STRUCTURE:- It is defined as the number and sequence of ribonucleotides in the RNA chain. The sequence is complementary to the template strand of the gene from which it was transcribed. The ribonucleotides are held together by 3´-5´ phosphodiester bonds. 3´-OH group of one nucleotide is bound to 5´-OH of the other nucleotide and form a linear strand.
  • 43. SECONDARY STRUCTURE OF RNA:- This involves coil formation of the polyribonucleotide chain. The coiled structures are stabilized by hydrophobic interactions between purine and pyrimidine bases. There are intra-chain hydrogen bonds between G-C and A-U.
  • 44. TERTIARY STRUCTURE OF RNA;- This involves the folding of the molecule into three dimensional structure. There is cross-linking at various sites stabilized by hydrophobic and hydrogen bonds producing a compactly coiled globular structure.
  • 45.  
  • 46.  
  • 47. Differences between DNA and RNA RNA exists as a single strand, however it is capable of folding back on itself like a hairpin and thus acquire double-stranded characteristics. Pentose sugar is ribose. It has different pyrimidine components i.e it has uracil in place of thymine. Since RNA is a single strand comlementary to only one of the two strands of the gene, its guanine content is not necessarily equal to cytosine, nor its adenine necessarily equals its uracil content. In addition to nucleus,RNA is found in cytoplasm. DNA forms RNA by transcription but RNA cannot form DNA. In experiments, reverse transcriptase can be used for this purpose. RNA can be hydrolysed to 2´,3´ cyclic mononucleotides.
  • 48. Classes of RNA:- Three main classes of RNA exist. They are:- A-Messenger RNA (m-RNA) B-Transfer RNA (t-RNA) C-Ribosomal RNA (r-RNA) Another type found in eukaryotic cells is small nuclear RNA (snRNA)
  • 49. A-MESSENGER RNA The most heterogeneous class regarding size and stability. FUNCTION: The members of this class function as messengers to convey the information in a gene to the protein synthesizing machinery. The mRNA carries genetic information from the nuclear DNA to the cytosol, where it is used as a template for protein synthesis. If the mRNA carries information from more than one gene, it is called polycistronic which is characteristic of prokaryotes. If the mRNA carries information from just one gene, it is called monocistronic, characteristic of eukaryotes.
  • 50. Messenger RNA contains mainly adenine, guanine, cytosine and uracil as the major bases. mRNA molecules are formed with the help of DNA template strand (3´-5´) during the process called transcription. In addition to the protein coding regions that can be translated, there are untranslated regions at its 5´ and 3´ ends.
  • 51. Special structural characteristics of eukaryotic messenger RNA:- It contains a long sequence of adenine nucleotides (20-250) on the 3´ end of the RNA chain and is called “poly-A tail”. It is thought to play role in stability of mRNA against 3´ exonucleases. There is a “cap” on the 5´ end consisting of a molecule of 7-methylguanosine. The cap is involved in the recognition of mRNA by the translation machinery and also helps to stabilize mRNA against 5´ exonucleases.
  • 52. In mammalian cells, the messenger RNA that comes to cytoplasm is the product of processing of a precursor called, HATEROGENOUS NUCLEAR RNA (hnRNA). It is synthesized in the nucleus and is much bigger than mRNA. Most of it is degraded in the nucleus and only 25% of it forms a precursor of mRNA called pre-mRNA which is then converted to mRNA.
  • 53. TRANSFER RNA (tRNA) tRNA is the smallest of the three major species of RNA. Single stranded globular molecules. Remain largely in cytoplasm. Functions; The t RNA molecules serve as ADAPTERS for the translation of information in the sequence of nucleotides of the mRNA into specific amino acids. T here is at least one specific type of tRNA molecule for each of the amino acids commonly found in proteins. Each tRNA carries its specific amino acid to the site of protein synthesis. There it recognizes the genetic code word on mRNA and this specifies the addition of its amino acids to the growing peptide chain.
  • 54. Structure of tRNA:- Primary structure; tRNA molecules consist of 74-95 nucleotides in a particular sequence. The tRNA molecules contain not only the usual bases like adenine, guanine, cytosine, uracil but also contain unusual bases like dihydrouracil , pseudouridine, thymine. Secondary structure: each single stranded tRNA is folded extensively and extensive intrachain base pairing which leads to a characteristic CLOVER-LEAF structure. These folds are stabilized by hydrogen bonds between complementary bases of the same strand. This leads to the formation of double stranded structures.
  • 55. Arms or loops of tRNA: All tRNA molecules contain 4 main arms or loops. 1-Acceptor arm; This is made up of unpaired sequences of cytosine-cytosine-adenine at the 3´end. The 3´OH terminal of adenine binds with the carboxylic group of a specific amino acid and carries it to ribosomes for protein synthesis. 2-Anticodon arm: it is in the form of a loop and carries specific sequences of three bases which constitute the anticodon. The bases of anticodon are bonded with three comlementary bases of codon of mRNA. 3-D arm: it contains the base dihydrouridine. 4-T Ψ C arm: contains thymine, pseudouridine and cytosine. The extra arm and the T Ψ C arms help define a specific tRNA.
  • 56.  
  • 57. Ribosomal RNA ( rRNA) rRNA is found as a component of ribososmes--- a cytoplasmic nucleoprotein structure that acts as the machinery for the synthesis of proteins from the mRNA template. The rRNA forms 80% of the total cellular RNA. The ribosomal subunits are defined according to their sedimentation velocity in Svedberg units. The mammalian ribosomes has a sedimentation velocity of 80S . It contains two major subunits- a larger one with 60S and a smaller one with 40S. The 60S subunit carries 60% of rRNA . There are four distinct species of rRNA in eukaryotic cells--- 28S, 18S, 5.8S and 5S. The bases in rRNA are mainly adenine, guanine, cytosine and uracil and a few pseudouridine.
  • 58. Function of rRNA: - The rRNA are necessary for ribosomal assembly and play a key role in the binding of mRNA to ribosomes and its translation. Some rRNA function as catalysts in protein synthesis and are termed as “ribozymes”.
  • 59. Small RNA Small nuclear RNA (SnRNA) are large number of small stable RNA species found in eukaryotic cells. Most of them are complexed with proteins to form ribonucleoproteins. They are distributed in the nucleus, in the cytoplasm or in both. They are significantly involved in mRNA processing and gene regulation.
  • 60. METABOLISM OF NUCLEOTIDES
  • 61. Biological significance of nucleotide metabolism Nucleotides make up DNA and RNA. Nucleotide Triphosphates are energy carriers e.g ATP. Regulate many metabolic pathways. GTP used in protein synthesis, UTP activates glucose and galactose, CTP has role in lipid metabolism AMP is a component of coenzymes e.g NAD, NADP, FAD etc.
  • 62. De-Novo SYNTHESIS OF PURINE NUCLEOTIDES A complex process Not synthesized as free base Synthesized as ribonucleotides Site is cytosol of liver cells There is no dietary requirement of purines as they are synthesized sufficiently in body.
  • 63. Materials required for Purine synthesis PRPP--- starting material, formed from ribose-5-phosphate obtained from HMP shunt. Amino acids and derivatives ---- glycine, aspartic acid, glutamine. Energy --- ATP CO 2 Formylated tetrahydrofolate and Mg
  • 64. Steps of Biosynthesis 1 – Synthesis of PRPP:- begins with Ribose-5´-P ---- from HMP- shunt pathway. ATP is used for energy. -- Enzyme is PRPP Synthetase. Coenzyme is Mg. --activated by inorganic phosphate and inhibited by Purine Nucleotides. 2 – Synthesis of 5´-phosphoribosylamine :- Formed by the transfer of amide grp of Glutamine to PRPP. Gives N-9 of purine ring. -- Enzyme is Glutamine-PRPP amidotransferase. --Inhibitors --- AMP, GMP, IMP. -- Activators --- PRPP The committed step in purine synthesis.
  • 65. 3- PRA + glycine , ATP is used, formation of 5-phosphoribosyl-glycinamide. This gives C-4, C-5 and N-7 of the purine ring. 4- Formylation of amino nitrogen of glycinamide by formyl-THF forming 5-phosphoribosyl-formyl glycinamide. Formyl C becomes C-8 of purine ring.
  • 66. 5- Transfer of another amide group forms 5-phosphoribosyl-N formyl glycinamidine. This forms N3 of purine ring. 6- Formation of 5-phosphoribosyl-5-aminoimidazole, formed by removal of water and closure of the ring.
  • 67. 7- Carboxylation gives C6 of purine ring. Formation of 5-phosphoribosyl-5-aminoimidazole-4-carboxylate. 8- Condensation of Aspartic acid with the above compound forms 5-phosphoribosyl-5-aminoimidazole-4-N succino-carboxamide.
  • 68. 9- Cleavage enzymes act and form 5-phosphoribosyl-5-aminoimidazole-4-carboxamide. 10- Formyl tetrahydrofolate donates C2 of the purine ring with the help pf formyl transferase. This forms 5-phosphoribosyl-5-formamido –imidazole-4-carboxamide.
  • 69. 11- Formation of IMP–( inosine-5-monophosphate). Formed by removal of water molecule and ring closure. Inosinic acid (IMP) is also called Hypoxanthine nucleotide and is the first purine synthesized in the body.
  • 70. Conversion of IMP to AMP Two step energy requiring pathway. IMP condenses with Aspartic acid and forms Adenylosuccinate. Enzyme is adenylosuccinate synthetase which is inhibited by end product(AMP). Energy comes from GTP. Adenylosuccinase cleaves adenylosuccinate to form AMP.
  • 71. Conversion of IMP to GMP Two steps Energy comes from ATP IMP dehydrogenase oxidises IMP into Xanthosine monophosphate. This step is inhibited by end product GMP. GMP synthase transfers amide group of glutamine to C-2 of XMP and forms GMP.
  • 72. Mycophenolic acid is a drug that acts as a reversible, uncompetitive inhibitor of IMP dehydrogenase. It is used to prevent graft rejection as it stops the rapid proliferation of T and B cells.
  • 73.  
  • 74. Formation of Nucleoside Diphosphates & Triphosphates Phosphorylation converts mono form to di and tri phosphate forms. AMP is converted to ADP and then ATP by action of adenylate kinase. GMP is converted to GDP and then GTP by action of guanylate kinase.
  • 75. Regulation/control of purine synthesis At the level of PRPP synthase. Allosteric feed back inhibition by PRPP, AMP, GMP, ADP, GDP etc. At the level of Glutamine-PRPP amidotransferase. Rate limiting. Inhibited by AMP and GMP. Balance between ATP and GTP. Each one stimulates the synthesis of the other.
  • 76. Inhibitors of purine synthesis PABA analogs ---- Sulfonamides are structural analogs of PABA and thus competitively inhibit folic acid synthesis in bacteria. Human beings get folic acid from external sources and thus are not affected by the drug. Folic acid analogs---- methotrexate is one such drug. It inhibits the reduction of dihydrofolate into tetrahydrofolate and thus no purines synthesis. This slows down DNA replication . Used for the treatment of cancer. Harmful to all the cells.
  • 77. Salvage Pathway for Purines:- This is the synthesis of nucleotides and nucleosides from the free purines already present in the body. These purines may be derived from -diet -or come from the normal turnover of cellular nucleic acids.
  • 78. Salvage pathway needs much less energy as compared to the de-novo synthesis of purine nucleotides. It is a re-cycling pathway. There are two mechanisms for salvage pathway
  • 79. 1-conversion of free purines to their mononucleotide form:- Conversion of Guanine to GMP:- enzyme--HGPRT. Conversion of Hypoxanthine to IMP:- enzyme---HGPRT. Conversion of Adenine to AMP:- enzyme--- APRTase PRPP is the source of ribose-5P in all these reactions. Irreversible reactions
  • 80. 2- Conversion of nucleosides to nucleotides:- Nucleosides can be converted into their respective nucleotides by the action of the particular nucleoside kinase. ATP is utilized during the process. e.g adenosine +ATP AMP + ADP
  • 81. Lesch-Nyhan Syndrome X-linked recessive disorder Complete absence of HGPRT Hypoxanthine and Guanine cannot be salvaged. PRPP increases, IMP and GMP lower, more de novo purine synthesis excessive uric acid formation--- gout and neurological features.
  • 82. Synthesis of Deoxyribonucleotides:- Ribonucleotides are converted to deoxyribonucleotides by the enzyme Ribonucleotide Reductase. Its co-enzyme is Thioredoxin which requires NADPH for its regeneration. This enzyme is strongly inhibited by dATP .
  • 83. DEGRADATION OF PURINE NUCLEOTIDES/Uric acid formation Pancreatic enzymes hydrolyze dietary nucleotides into nucleosides and free bases in the intestines. Sequential break down takes place inside cells and end product Uric acid is formed. Uric acid is the end-product of purine catabolism in human beings and other primates.
  • 84. Degradation of adenine nucleotides AMP is converted to Adenosine by the action of enzyme 5-nucleotidase. Adenosine deaminase takes out ammonia and converts Adenosine to Inosine. Phosphorylation of inosine forms Hypoxanthine and Ribose phosphate. Enzyme is purine nucleoside phosphorylase.
  • 85. Oxidation of hypoxanthine forms xanthine. Enzyme is xanthine oxidase. Xanthine oxidase again oxidises xanthine into uric acid.
  • 86. Degradation of Guanine Nucleotides GMP releases Guanosine by the action of 5-nucleotidase. Guanosine gives Guanine and Ribose P by the action of phosphorylase. Guanine deaminase converts Guaninine to xanthine. Xanthine oxidase forms uric acid.
  • 87. Uric acid End product of purine metabolism in humans and primates. Normally 500-600 mg is synthesized in body daily and most of it is excreted in urine. Serum contains 3-7mg/100 ml of uric acid.
  • 88. Clinical disorders of purine metabolism 1- Primary hyperuricemia/gout 2- Secondary Hyperuricemia 3- Lesch Nyhan syndrome 4- Xanthinuria 5-Adenosine Deaminase deficiency
  • 89. Gout Chronic disorder characterized by - Hyperuricemia - deposition of urate crystals in joints and tissues ---- Tophi - attacks of acute arthritis - urate stones in kidneys
  • 90. Primary Gout -Excessive production of uric acid -Decreased excretion of uric acid from kidney Excessive production --- excessive purine synthesis due to enzyme defects, and thus more uric acid synthesis.
  • 91. Resistance to feed back inhibition of PRPP synthetase. Defective HGPRT leading to decreased utilization of PRPP in salvage pathway. High level of PRPP in cells enhances de-novo purine synthesis.
  • 92. High purine foods --- all meats, organ meats, alcoholic beverages, beans, peas, lentils, spinach, cauliflowers and mushrooms.
  • 93. Secondary Hyperuricemia Increased break down of purines during diseases like leukemia, prolonged fasting, polycethemia and psoriasis. Cytotoxic drugs. Renal failure leading to decreased uric acid excretion. Von Gierke’s disease --- deficiency of glucose 6 phosphatase, increased pentose, increased purine synthesis.
  • 94. Treatment of Gout Anti-inflammatory drugs– Colchicine and NSAIDs. Uricosuric drugs---- increased renal excretion of uric acid. Probenecid is one such drug. Given to patients with normal renal function. Enzyme inhibitor--- Allopurinol--- competitive inhibitor of Xanthine Oxidase.
  • 95. Xanthinuria Inherited disorder Deficiency of xanthine oxidase Hypoxanthine and xanthine cannot be converted to uric acid Low blood levels of uric acid Excessive excretion of xanthine, xanthine stones in kidneys
  • 96. Adenosine Deaminase Deficiency Genetic deficiency of adenosine deaminase. B and T lymphocytes are deficient--- immunodeficiency. Accumulation of deoxyribonucleotides, inhibition of further production of precursors of DNA. Hypouricemia due defective break down of purine nucleotides.