1.10.4 Nucleotide metabolism

tube defects [the cause of a variety of severe birth defects including spina bifida (defects in the spinal column that often result in paralysis) and anencephaly (the invariably fatal failure of the brain to develop, which is the leading cause of infant death due to congenital anomalies)]. Hyperhomocysteinemia is readily controlled by ingesting the vitamin precursors of the coenzymes that participate in homocysteine breakdown, namely, B6 (pyridoxine, the PLP precursor; and folate

1.10.4 Nucleotide metabolism

Snapshot

1. The purine ring system is built up step-by-step beginning with 5-phosphoribosylamine. The amino acids glutamine, glycine, and aspartate furnish all the nitrogen atoms of purines. Two ring-closure steps form the purine nucleus. 2. Pyrimidines are synthesized from carbamoyl phosphate and aspartate, and ribose 5-phosphate is then attached to yield the pyrimidine ribonucleotides. 3. Nucleoside monophosphates are converted to their triphosphates by enzymatic phosphorylation reactions.

Ribonucleotides are converted to deoxyribonucleotides by ribonucleotide reductase, an enzyme with novel mechanistic and regulatory characteristics. The thymine nucleotides are derived from dCDP and dUMP. 4. Uric acid and urea are the end products of purine and pyrimidine degradation. ■ Free purines can be salvaged and rebuilt into nucleotides. Genetic deficiencies in certain salvage enzymes cause serious disorders suchas Lesch-Nyhan syndrome and ADA deficiency. 5. Accumulation of uric acid crystals in the joints, possibly caused by another genetic deficiency,results in gout. 6. Enzymes of the nucleotide biosynthetic pathways are targets for an array of chemotherapeutic 7. agents used to treat cancer and other diseases.

Introduction (Figure 1.10.50).

Figure 1.10.50. Chemical structure of Nucleotides

Uses of Nucleotides

1. Nucleotides are precursors of DNA and RNA and are essential carriers of chemical Energy—a role primarily of ATP and to some extent GTP. 2. Components of the cofactors NAD, FAD, S-adenosylmethionine, and coenzyme A, well as of activated biosynthetic intermediates such as UDP-glucose and CDP-diacylglycerol. 3. cAMP and cGMP, are also cellular second messengers.

Nucleotide Biosynthesis (Figure 1.10.51). 1. ribonucleotides are formed first which is then utilized in the formation of corresponding deoxyribonucleotide. 2. Purine ring structure is built up of one or a few atoms at a time, attached to ribose throughout the process.

Salvage pathway

Nucleotide Biosynthesis Recycle the free bases and nucleosides released from nucleic acid breakdown

Synthesis of nucleotides begins with their metabolic precursors: amino acids, ribose 5-phosphate, CO2, and NH3

De novo synthesis

Figure 1.10.51. Two ways of nucleotide synthesis 3. The pyrimidine ring is synthesized as orotate, attached to ribose phosphate, and then converted to the common pyrimidine nucleotides required in nucleic acid synthesis. 4. Phosphoribosyl pyrophosphate (PRPP) is important in both. 5. PRPP is synthesized from ribose 5-phosphate derived from pentose phosphate pathway and is the reaction is catalysed by ribose phosphate pyrophosphokinase.

DE NOVO PURINE SYNTHESIS

 In the first committed step of the pathway, an amino group donated by glutamine is attached at

C-1 of PRPP and is unique to purine synthesis (Figure 1.10.51).  ATP is required to activate the glycine carbonyl group in the second step of addition of three carbons of glycine to 5-Phospho –β-Dribosylamine.  Step 6 and 7 in figure 4 is present onle in bacteria and 6a is seen in higher eukaryotes including human  Carboxylation of 5-Aminoinidazole ribonucleotide (AIR) donot require biotin, instead bicarbonate from aqueous solution is used.  Inositate monophosphate(IMP) is the precursor of adenine and guanine.  Enzymes of IMP synthesis are present as large multi enzyme complexes (single polypeptide with multiple function)  In humans, a multifunctional enzyme combines the activities of AIR carboxylase and SAICAR synthetase.  Conversion of inosinate to adenylate requires the insertion of an amino group derived from aspartate (Figure 1.10.52, Figure 1.10.53)  A crucial difference is that GTP rather than ATP is the source of the high-energy phosphate in synthesizing adenylosuccinate.  Guanylate is formed by the NAD+-requiring oxidation of inosinate at C-2, followed by addition of an amino group derived from glutamine

Figure 1.10.51. Origin of the ring atoms of purines. This information was obtained f rom isotopic experiments with 14C- or 15N-labeled precursors.Formate is supplied in the f orm of N10-f ormyltetrahydrofolate

Figure 1.10.52. De novo synthesis of purine nucleotides: construction of the purine ring of inosinate (IMP). Each addition to the purine ring is shaded to match Figure 22–32. Af ter step 2 , R symbolizes the 5-phospho-D-ribosyl group on which the purine ring is built. Formation of 5phosphoribosylamine (step 1 ) is the f irst committed step in purine synthesis. Note that the product of step 9 , AICAR, is the remnant of ATP released during histidine biosynthesis. Abbreviations are given for most intermediates to simplify the naming of the pathway enzymes. Step 6a is the alternative path f rom AIR to CAIR occurring in higher eukaryotes.

Mechanisms of purine biosynthesis regulation.

 3 mechanism work in cooperation.  Controls the relative rate of formation of adenylate and guanylate

Figure 1.10.53. Biosynthesis of AMP and GMP f rom IMP.

Mechanism 1 Enzyme is inhibited by IMP, AMP, and GMP

Mechanism 2 Enzyme is inhibited by increased concentration of GMP

Enzyme is inhibited by Increased concentration of AMP

Mechanism 3 Enzyme is inhibited by Increased concentration of AMP and GMP Allosteric regulation of enzyme glutaminePRPP

Amidotransferase which transfer of an amino group

to PRPP to form 5phosphoribosylamine

Inhibits an enzyme IMP dehydrogenase which catalyze the conversion of inosinate to xanthylate .

Does not affect synthesis of AMP

Inhibits adenylosuccinate synthetase which catalyses conversion of inosinate to adenylosuccinate.

Does not affect the synthesis of GMP

Allosteric regulation of PRPP synthesis by the by inhibition of ribose

phosphate pyrophosphokinase

Unit 1  Phosphorylation of AMP to ADP is promoted by adenylate kinase.  The ADP so formed is phosphorylated to ATP by the glycolytic enzymes or through oxidative phosphorylation.  ATP also brings about the formation of other nucleoside diphosphates by the action of a class of enzymes called

nucleoside monophosphate kinases.

 These enzymes are specific for a particular base but nonspecific for the sugar.  Nucleoside diphosphates are converted to triphosphates by the action of a ubiquitous enzyme,

nucleoside diphosphate kinase and

this enzyme is neither specific for sugar nor bases.

DE NOVO PYRIMIDINE SYNTHESIS

 Common pyrimidine nucleotides are uridylate and cytidylate (Figure

1.10.53)

 Thymidylate is derived from dCDP and dUMP  The pyrimidine ring is synthesized first and then attached to ribose 5phospahte.  Aspartate, carbamoyl phosphate and

PRPP are the precursor of pyrimidine synthesis.  Carbamoyl phosphate reacts with aspartate to yield Ncarbamoylaspartate in the first committed step of pyrimidine biosynthesis.  N-carbamoylaspartate is seen as an intermediate in urea cycle is made in mitochondria by carbamoyl phosphate synthetase I  N-carbamoylaspartate required for the pyrimidine biosynthesis is made in the cytosol by an enzyme called carbamoyl phosphate synthetase II in mammals while in bacteria single enzyme contribute carbamoyl phosphate to both arginine and pyrimidines.  The first three enzymes in pyrimidine pathway—carbamoyl phosphate synthetase II, aspartate transcarbamoylase, and dihydroorotase— are part of a single trifunctional protein. This protein is called by and acronym CAD  regulation of pyrimidine synthesis :

Figure 1.10.54. De novo synthesis of pyrimidine nucleotides: biosynthesis of UTP and CTP via orotidylate. The pyrimidine is constructed f rom carbamoyl phosphate and aspartate. The ribose 5-phosphate is then added to the completed pyrimidine ring by orotate phosphoribosyltransf erase. The f irst step in this pathway (not shown here; see is the synthesis of carbamoyl phosphate f rom CO2 and NH4 , catalyzed in eukaryotes by carbamoyl phosphate synthetase II.

o In bacteria, regulation occurs through aspartate carbamoyl transferase (ACTase) and is inhibited by CTP. o The bacterial ACTase molecule consists of six catalytic subunits and six regulatory subunits. o The bacterial ACTase exists in two conformations: active and inactive. o When CTP is not bound the the regulatory subunits, the ACTase is active in maximum.

Ribonucleotides Are The Precursors Of Deoxyribonucleotides

 Deoxyribonucleotides, the building blocks of DNA, are derived from the corresponding ribonucleotides by direct reduction at the 2’-carbon atom of the D-ribose to form the 2’-deoxy derivative.  The enzyme catalyzing this reaction is called ribonucleotide reductase.  Glutaredoxin and thioredoxin transfer their reducing power to ribonucleotide reductase (Figure

1.10.55)

Figure 1.10.55. Reduction of ribonucleotides to deoxyribonucleotides by ribonucleotide reductase. Electrons are transmitted (blue arrows) to the enzyme f rom NADPH by (a) glutaredoxin or (b) thioredoxin. The sulf ide groups in glutaredoxin reductase are contributed by two molecules of bound glutathione (GSH; GSSG indicates oxidized glutathione). Note that thioredoxin reductase is a f lavoenzyme, with FAD as prosthetic group.

Ribonucleotide reductase

 The enzyme in E. coli and most eukaryotes is a dimer, with subunits designated R1 and R2.  The R1 subunit contains two kinds of regulatory sites (Figure 1.10.56)  Active site is at the interface of R1 and R2 subunit.

Unit 1  At each active site, R1 contributes two sulfhydryl groups required for activity and R2 contributes a stable tyrosyl radical.  The R2 subunit also has a binuclear iron (Fe3+) cofactor that helps generate and stabilize the tyrosyl radicals.  The tyrosyl radical is too far from the active site to interact directly with the site, but it generates another radical at the active site that functions in catalysis.

THYMIDYLATE IS DERIVED

FROM dCDP AND dUMP.

 DNA contains thymine rather than uracil, and the de novo pathway to thymine involves only deoxyribonucleotides  The immediate precursor of thymidylate (dTMP) is dUMP (Figure 1.10.57;Figure

1.10.58)

Figure 1.10.56. Ribonucleotide reductase. (a) Subunit structure. The f unctions of the two regulatory sites.Each active site contains two thiols and a group (OXH) that can be converted to an activesite radical; this group is probably the OSH of Cys439, which f unctions as a thiyl radical.

Figure 1.10.57. Biosynthesis of thymidylate (dTMP). The pathways are shown beginning with the reaction catalyzed by ribonucleotide reductase.

Figure 1.10.58. Conversion of dUMP to dTMP by thymidylate synthase and dihydrofolate reductase. Serine hydroxymethyltransf erase is required for regeneration of the N5,N10methylene f orm of tetrahydrofolate. In the synthesis of dTMP, all three hydrogens of the added methyl group are derived f rom N5,N10-methylenetetrahydrof olate

Degradation Of Nucleotides Purine catabolism

Unit 1  Adenosine deaminase deficiency causes severe immunodeficiency disease in which T-lymphocyte and B-lymphocyte do not develop properly. o High concentration of dATPs causes deficiency of other dNTPs.

Figure 1.10.59. Catabolism of purine nucleotides. Note that primates excrete much more nitrogen as urea via the urea cycle (Chapter 18) than as uric acid f rom purine degradation. Similarly, fish excrete much more nitrogen as NH4 than as urea produced by the pathway shown here.

Figure 1.10.60. Catabolism of a pyrimidine. Shown here is the pathway f or thymine. The ethylmalonylsemialdehyde is f urther degraded to succinyl-CoA.

Catabolism of cytosine and uracil

SALVAGE PATHWAY

 Free adenine reacts with PRPP to yield AMP in the presence of enzyme adenosine

phosphoribosyltransferase.

Adenine + PRPP  AMP+PPi

 Guanine and hypoxanthine (deamination product of adenine) are salvaged by hypoxanthine-guanine phosphoribosyl transferase.  Lesch-Nyhan syndrome is a inherited disorder caused due to the deficiency of enzyme HGPRT, produced by gene hprt gene on X-chromosome.

 GOUT:

 Under excretion of uric acid causes gout.  Predominantly in males.

Sl.No enzyme

1. glutamine amidotransferases Enzyme role

glutamine is a nitrogen donor in at least half a dozen separate reactions drug

Azaserine and acivicin

in nucleotide biosynthesis

2. thymidylate synthase provide the only cellular pathway for Thymine synthesis. dUMP  dTMP

Fluorouracil –

converted to FdUMP by salvage pathway. FdUMP binds to enzyme and inhibit them

3. dihydrofolate reductase Catalyze conversion of dihydrofolate to tetrahydrofolate.

Methotrexate –competitive inhibitor of enzyme.