Nucleotidemetabolism-2552

Metabolism of Nucleic Acids Phanchana Sanguansermsri

Nucleic acids • Nucleic acids are linear polymers of nucleotides – Deoxyribonucleic acid (DNA) – Ribonucleic acid (RNA)

Nucleotides • Nucleotides are phosphate esters of a pentose (ribose or deoxyribose) in which a purine or pyrimidine base is linked to C1’ o f the sugar

Function of nucleotides • Monomeric unit of nucleic acid • Energy currency: ATP • Component of some of the cofactor of metabolism: FAD, NAD+, coenzyme A

Nucleotide metabolism • • • •

Synthesis of purine ribonucleotides Synthesis of pyrimidine ribonucleotides Formation of deoxyribonucleotides Nucleotide degradation

Synthesis of purine ribonucleotides

Isotonic experiment • 1948, Buchanan – feed a variety of isotopically labeled compounds to pigeons – determine the positions of the labeled atoms in their excreted uric acid (a purine)

Overview of de novo synthesis of purine ribonucleotides • Purines are initially formed as ribonucleotides rather than as free bases 1. The initial synthesized purine derivative is inosine monophosphate (IMP) 2. IMP is the precursor of both AMP and GMP

Synthesis of inosine monophosphate (IMP) • IMP is synthesized in a pathway composed of 11 reactions • Starting material is α-D-ribose-5phosphate, a product of the pentose phospha te pathway

(1) activation of ribose-5-phosphate

(2) acquisition of N9

(3) acquisition of C4, C5, and N7

(4) acquisition of C8

(5) acquisition of N3

(6) formation of imidazole ring

(7) acquisition of C6

(8) acquisition of N1

(9) elimination of fumarate

(AICAR)

(10) acquisition of C2

(AICAR)

(11) cyclization to form IMP

• In bacteria, the enzymes of IMP biosynthesis occur as independent proteins. • In animals, single polypeptides catalyze – – – –

Reaction 3, 4, and 6 Reaction 7, 8 Reaction 10, 11 The intermediate products of these multifunctional enzymes are not readily release d to the medium but are channeled to the succee ding enzymatic activities of the pahtway.

Synthesis of guanine and adenine ribonucleotides • Inosine monophosphate (IMP) does not accumulate in the cell but rapidly converted to adenosine monophosphate (AMP) and gu anosine monophosphate (GMP) in different two-reaction pathways

1st reaction to convert IMP to AMP

2nd reaction to convert IMP to AMP

1st reaction to convert IMP to GMP

2nd reaction to convert IMP to GMP

• Nucleoside diphosphates and triphosphates are synthesized by the phosphorylation of nucleoside monophosphates.

• Nucleoside diphosphates are synthesized from the corresponding monophosphates by base-specific nucleoside monophosphate ki nases. • The enzymes do not discriminate between ribose and deoxyribose. AMP + ATP

GMP + ATP

Adenylate kinase

ADP

GDP + ADP

• Nucleoside diphosphates are converted to the corresponding triphosphates by nucleosi de diphosphate kinase. • The enzyme exhibits no preference for the bases of its substrate or for ribose over deoxyribose.

GDP + ATP

GTP + ADP

Regulation of purine ribonucleotide biosynthesis • The pathways synthesizing IMP, ATP, and GTP are individually regulated in most cells so as to control the total amounts of purine nucleotides for nucleic acid synthesis, as we ll as the relative amount of ATP and GTP.

---> feed back inhibition ---> feedforward activation

Salvage of purines • In most cells, the turnover of nucleic acids releases adenine, guanine, and hypoxanthin e. These free purines are reconverted to thei r corresponding nucleotides through salvage pathways. • Salvage pathways are diverse in character and distribution. In mammals, purines are mostly salvage by the different enzymes.

• Adenine phosphoribosyltransferase (APRT) mediates AMP formation using PRPP. Adenine + PRPP

AMP + PPi

• Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyz es the analogous reaction for both hypoxant hine and guanine. Hypoxanthine + PRPP Guanine + PRPP

IMP + PPi GMP + PPi

Synthesis of pyrimidine ribonucleotides

Isotopic experiment

Overview of synthesis of pyrimidine ribonucleotides 1. Synthesis of uridine monophosphate (UMP) 2. Synthesis of uridine triphosphate (UTP) and cytidine triphosphate (CTP)

Synthesis of uridine monophosphate • Uridine monophosphate (UMP) is synthesized in a six-reaction pathway • In contrast to purine nucleotide synthesis, pyrimidine ring is coupled to the ribose-5-p hosphate moiety after the ring has been synt hesized

(1) synthesis of carbamoyl phosphate

(2) synthesis of carbamoyl aspartate

(3) ring closure to form dihydroorotate

(4) oxidation of dihydroorotate

(5) acquisition of ribose phosphate moiety

(6) decarboxylation to form UMP

• In bacteria, the six enzymes of UMP biosynthesis occur as independent proteins. • In animals – Enzymatic activity of the reaction 1, 2, and 3 occur in a single 210 kD polypeptide chain. – Reaction 5 and 6 are catalyzed by a single polypeptide. – The intermediate products are channeled to the succeeding enzymatic activities of the pathway.

Synthesis of UTP and CTP • The synthesis of UTP from UMP occurs by the sequential actions of a nucleoside mono phosphate kinase and nucleoside diphosphat e kinase: UMP + ATP

UDP + ADP

UDP + ATP

UTP + ADP

• CTP is form by amination of UTP by CTP synthethase.

• In animals, the amino group is donated by glutamine, whereas in bacteria it is supplied by ammonia.

Regulation of pyrimidine ribonucleotide biosynthesis

---> feed back inhibition ---> feedforward activation

Formation of deoxyribonucleotides

Ribonucleotide & deoxyribonucleotide • DNA differs from RNA in two respects: 1. Its nucleotides contain 2’-deoxyribose residue rather than ribose residues, and 2. It contains the base thymine (5methyluracil) rather than uracil.

Production of deoxyribose residues • Deoxyribonucleotides are synthesized from their corresponding ribonucleotides by the r eduction of their C2’ position using the enz yme ribonucleotide reductase rather than by their de novo synthesis from deoxyribose-co ntaining precursors.

• The final step in the production of all dNTPs is the phosphorylation of the corresp onding dNDPs. • The reaction is catalyzed by nucleoside diphosphate kinase (as in phosphorylation o f NDPs).

dADP + ATP

dATP + ADP

dGDP + ATP

dGTP + ADP

dCDP + ATP

dCTP + ADP

dUDP + ATP

dUTP + ADP

NTP or dNTP can function as the phosphoryl donor

Origin of thymine • dUMP is generated through the hydrolysis of dUTP by dUTP diphosphohydrolase (dU TPase): dUTP + H2O

dUMP + PPi

• Thymidylate (dTMP) is synthesized from dUMP by thymidylate synthase with N5, N10 -methylenetetrahydrofolate as methyl donor .

• dTMP, once it is formed, is phosphorylated to form dTTP.

dTMP + ATP

dTDP + ADP

dTDP + ATP

dTTP + ADP

• The apparent reason for the energetically wasteful process of dephosphorylating dUT P and rephosphorylating dTMP is that cells must minimize their concentration of dUTP in order to prevent incorporation of uracil in to their DNA.

regeneration of tetrahydrofolate

• A deficiency of any dNTPs is lethal, whereas an excess is mutagenesis because t he probability that a given dNTPs will be er rornously incorporated into a growing DNA strand increase with its concentration relativ e to those of the other dNTPs.

Nucleotide degradation

Overview of nucleotide degradation • Dietary nucleic acids are degraded to nucleotides mainly in intestine by pancreati c nucleases and intestinal phosphodiesterase s. • Nucleotides are hydrolyzed to nucleosides by a variety of group-specific nucleotidases and nonspecific phosphatases.

• Nucleosides may be directly absorbed by the intestinal mucosa or further degraded to free bases and ribose or ribose-1-phosphate through the action of nucleosidases and nuc leoside phosphorylases Nucleoside + H2O Nucleoside + Pi

base + ribose base + ribose-1-P

• Radioactive labeling experiments have demonstrated that only a small fraction of the bases of ingested nucleic acid are incorp orated into tissue nucleic acids. • Ingested bases are mostly degraded and excreted. • Cellular nucleic acids are also subjected to degradation as part of the continual turnover of nearly all cellular component.

Catabolism of purines • All catabolism pathways of purine nucleotides and purine deoxynucleotides lea d to uric acid. • Pathway intermediates may be directed into purine nucleotide synthesis via salvage reac tions.

• Adenosine and deoxyadenosine are not degraded by mammalian purine nucleoside phosphorylase (PNP). • Rather adenine nucleosides and nucleotides are deaminated by adenosine deaminase (ADA) and AMP deaminase to their corresp onding inosine derivatives.

• Ribose-1-phosphate, a product of the reaction catalyzed by purine nucleoside pho sphorylase (PNP) is a precursor of PRPP.

phosphoribomutase Ribose-1-P

Ribose-5-P

• Deamination of AMP to IMP, when combined with the synthesis of AMP from I MP (purine nucleotide cycle), has the net ef fect of deaminating aspartate to yeild fumar ate.

Purine nucleotide cycle

• Muscle replenishes its citric acid cycle intermediates with fumarate generated in th e purine nucleotide cycle.

Gout (excess of uric acid) • Caused by deposition of crystals of sodium urate. • Painful arthritis, joint inflammation of sudden onset, most often in big toe. • May precipitate in the kidneys and ureters as stones, resulting in renal damage and urinary tract obstruction.

• The most prevalent cause of gout is impaired uric acid excretion. • Gout may also result from a number of metabolic insufficiencies (eg. HGPRT defic iency) • Gout can be treated by the xanthine oxidase inhibitor, allopurinol. The product, alloxanthine, remain tightly bound to the re duce form of the enzyme.

Fate of uric acid • In human and other primates, the final product of purine degradation is uric acid, w hich is excreted in the urine. • The same is true for birds, terrestrial reptiles, and many insects. – These organism also catabolize their excess amino acid nitrogen to uric acid via purine bios ynthesis.

• In all other organisms, uric acid is further processed before excretion

Catabolism of pyrimidines • Animal cells degrade pyrimidine nucleotides to their component bases. • These reactions, like those in purine nucleotides, occur through dephosphorylati on, deamination, and glycosidic bond cleav ages.

• The resulting uracil and thymine are then broken down in the liver through reduction rather than by oxidation as occurs in purine catabolism.

• The end products of pyrimidine catabolism, beta-alanine and beta-aminoisobutyrate, are amino acids and are metabolized as such. • They are converted, through transamination and activation reactions, to malonyl-CoA and methylmalonyl-CoA

• Malonyl-CoA is a precursor of fatty acid synthesis, and methylmalonyl-CoA is conve rted to the citric acid cycle intermediate suc cinyl-CoA. • Catabolism of pyrimidine nucleotides contributes to the energy metabolism of the cell.

Summary

Synthesis of purine ribonucleotides • The purine nucleotide IMP is synthesized in 11 steps from ribose-5-phosphate, aspartate, fumarate, glutamine, glycine, and HCO3-. • IMP synthesis is regulated at its first and second steps. • IMP is the precursor of AMP and GMP, which are phosphorylated to produce the co rresponding di- and triphosphates.

Synthesis of pyrimidine ribonucleotides • Pyrimidine nucleotide UMP is synthesized from 5-phosphoribosyl pyrophosphate, aspartate, glutamine, and HCO3- in six reactions. • UMP is converted to UTP and CTP by phosphorylation and amination. • Biosynthesis is regulated in bacteria at the ATCase step and in animals at the step catalyzed by carbamoyl phosphate synthetase II

Formation of deoxyribonucleotides • Deoxyribonucleoside diphosphates are synthesized from corresponding NDP by th e action of ribonucleotide reductase. • dTMP is synthesized from dUMP by thymidine synthase.

Nucleotide degradation • Purine nucleotides are degraded by nucleosidases and purine nucleoside phosph orylase (PNP). • Adenine nucleotides are deaminated by adenosine deaminase and AMP deaminase. • The synthesis and degradation of AMP in the purine nucleotide cycle yield the citric a cid cycle intermediate fumarate in muscles.

• Xanthine oxidase catalyzes the oxidation of hypoxantine to xanthine and of xanthine to uric acid. • In humans, the ultimate product of purine degradation is uric acid, which is excreted. Other organisms degrade urate further. • Pyrimidines are broken down to intermediates of fatty acid metabolism.

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