Metabolism of Purine and Pyrimidine Nucleotides: Biosynthesis, Catabolism, and Disorders | Summaries Biochemistry

(CMT)

METABOLISM OF PURINE & PYRIMIDINE NUCLEOTIDES

BIOCHEMICAL IMPORTANCE

❖ Humans synthesize nucleic acids and their derivatives ATP,

NAD+, coenzyme A, etc, from amphibolic intermediates

(e.g. Oxaloacetate, Alpha-ketoglutarate, etc.).

❖ Dietary purines and pyrimidines -not incorporated directly

into tissue nucleic acids.

❖ Injected purine or pyrimidine analogs (e.g. potential

anticancer drugs)-incorporated directly into DNA.

PURINE

❖ low solubility end product of catabolism:

➢ uric acid

❖ Diseases involved:

➢ Gout

➢ Lesch-Nyhan syndrome

➢ adenosine deaminase deficiency

➢ purine nucleoside phosphorylase deficiency

PYRIMIDINE

❖ Highly water-soluble end product of catabolism:

➢ carbon dioxide

➢ ammonia

➢ β-alanine

➢ γ-amino isobutyrate

❖ Diseases of pyrimidine biosynthesis: RARE

➢ Orotic acidurias

➢ β-hydroxybutyric aciduria- due to total or partial

deficiency of the enzyme dihydropyrimidine

dehydrogenase.

➢ Uraciluria-thyminuria- also known as combined, is also

a disorder of β-amino acid biosynthesis.

PURINES & PYRIMIDINES ARE DIETARILY NONESSENTIAL

❖ Normal human tissues can synthesize purines and

pyrimidines from amphibolic intermediates in quantities

and at times appropriate to meet variable physiologic

demands.

➢ Ingested nucleic acids and nucleotides

▪ Dietarily nonessential

▪ Little or none is incorporated into tissue nucleic

acids

▪ Intestinal degradation -> mononucleotides ->

absorbed or converted to purine base (oxidized to

uric acid->absorbed and excreted in the urine) and

pyrimidine bases.

➢ Injected nucleic acids and nucleotides are

incorporated into tissue nucleic acids.

BIOSYNTHESIS OF PURINE NUCLEOTIDES

❖ All forms of life synthesize purine and pyrimidine

nucleotides, except parasitic protozoa.

❖ Propose of biosynthesis:

➢ To achieve homeostasis,

➢ intracellular mechanisms sense

➢ regulate the pool sizes of NTPs, which rise during

growth or tissue regeneration when cells are rapidly

dividing.

❖ Early investigations of nucleotide biosynthesis first

employed birds (Pigeons), and later Escherichia coli.

❖ Avian tissues also serve as a source of cloned genes that

encode enzymes of purine biosynthesis and the regulatory

proteins that control the rate of purine biosynthesis.

❖ The three processes that contribute to purine nucleotide

biosynthesis are, in order of decreasing importance:

➢ Synthesis from amphibolic intermediates (synthesis de

novo)

➢ Phosphoribosylation of purines

➢ Phosphorylation of purine nucleosides

INOSINE MONOPHOSPHATE (IMP) IS SYNTHESIZED FROM

AMPHIBOLIC INTERMEDIATES

Alpha-D-Ribose 5-phosphate -> -> -> Inosine monophosphate

(IMP)

❖ The initial reaction of purine biosynthesis:

➢ Alpha-D-Ribose 5-phosphate + ATP -> Phosphoribosyl

pyrophosphate (PRPP)

▪ Enzyme: PRPP Synthase

❖ Following IMP (Two Branches):

➢ IMP->Guanosine monophosphate (GMP)

➢ IMP-> Adenosine monophosphate (AMP)

❖ Subsequent phosphoryl transfer from ATP converts AMP

and GMP to ADP and GDP, respectively.

❖ Conversion of GDP to GTP involves a second phosphoryl

transfer from ATP, whereas conversion of ADP to ATP is

achieved primarily by oxidative phosphorylation.

(NOTE: See Figure 33-2 and 33-3, pages 329-330)

MULTIFUNCTIONAL CATALYSTS PARTICIPATE IN PURINE

NUCLEOTIDE BIOSYNTHESIS (Figure 33-2)

❖ Enzymes of eukaryotes are polypeptides that possess

multiple catalytic activities whose adjacent catalytic sites

facilitate the channeling of intermediates between sites.

❖ Three distinct multifunctional enzymes catalyze reactions:

➢ Formyl transferase

➢ VII carboxylase

➢ Formyl transferase

ANTIFOLATE DRUGS & GLUTAMINE ANALOGS BLOCK PURINE

NUCLEOTIDE BIOSYNTHESIS

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(CMT) BIOCHEMICAL IMPORTANCE ❖ Humans synthesize nucleic acids and their derivatives ATP, NAD+, coenzyme A, etc, from amphibolic intermediates (e.g. Oxaloacetate, Alpha-ketoglutarate, etc.). ❖ Dietary purines and pyrimidines - not incorporated directly into tissue nucleic acids. ❖ Injected purine or pyrimidine analogs (e.g. potential anticancer drugs)-incorporated directly into DNA. PURINE ❖ low solubility end product of catabolism: ➢ uric acid ❖ Diseases involved: ➢ Gout ➢ Lesch-Nyhan syndrome ➢ adenosine deaminase deficiency ➢ purine nucleoside phosphorylase deficiency PYRIMIDINE ❖ Highly water-soluble end product of catabolism: ➢ carbon dioxide ➢ ammonia ➢ β-alanine ➢ γ-amino isobutyrate ❖ Diseases of pyrimidine biosynthesis: RARE ➢ Orotic acidurias ➢ β-hydroxybutyric aciduria- due to total or partial deficiency of the enzyme dihydropyrimidine dehydrogenase. ➢ Uraciluria-thyminuria- also known as combined, is also a disorder of β-amino acid biosynthesis. PURINES & PYRIMIDINES ARE DIETARILY NONESSENTIAL ❖ Normal human tissues can synthesize purines and pyrimidines from amphibolic intermediates in quantities and at times appropriate to meet variable physiologic demands. ➢ Ingested nucleic acids and nucleotides ▪ Dietarily nonessential ▪ Little or none is incorporated into tissue nucleic acids ▪ Intestinal degradation - > mononucleotides - > absorbed or converted to purine base (oxidized to uric acid->absorbed and excreted in the urine) and pyrimidine bases. ➢ Injected nucleic acids and nucleotides are incorporated into tissue nucleic acids. BIOSYNTHESIS OF PURINE NUCLEOTIDES ❖ All forms of life synthesize purine and pyrimidine nucleotides, except parasitic protozoa. ❖ Propose of biosynthesis: ➢ To achieve homeostasis, ➢ intracellular mechanisms sense ➢ regulate the pool sizes of NTPs, which rise during growth or tissue regeneration when cells are rapidly dividing. ❖ Early investigations of nucleotide biosynthesis first employed birds (Pigeons), and later Escherichia coli. ❖ Avian tissues also serve as a source of cloned genes that encode enzymes of purine biosynthesis and the regulatory proteins that control the rate of purine biosynthesis. ❖ The three processes that contribute to purine nucleotide biosynthesis are, in order of decreasing importance: ➢ Synthesis from amphibolic intermediates (synthesis de novo) ➢ Phosphoribosylation of purines ➢ Phosphorylation of purine nucleosides INOSINE MONOPHOSPHATE (IMP) IS SYNTHESIZED FROM AMPHIBOLIC INTERMEDIATES Alpha-D-Ribose 5-phosphate - > - > - > Inosine monophosphate (IMP) ❖ The initial reaction of purine biosynthesis: ➢ Alpha-D-Ribose 5-phosphate + ATP - > Phosphoribosyl pyrophosphate (PRPP) ▪ Enzyme: PRPP Synthase ❖ Following IMP (Two Branches): ➢ IMP->Guanosine monophosphate (GMP) ➢ IMP-> Adenosine monophosphate (AMP) ❖ Subsequent phosphoryl transfer from ATP converts AMP and GMP to ADP and GDP, respectively. ❖ Conversion of GDP to GTP involves a second phosphoryl transfer from ATP, whereas conversion of ADP to ATP is achieved primarily by oxidative phosphorylation. (NOTE: See Figure 33-2 and 33-3, pages 329-330) MULTIFUNCTIONAL CATALYSTS PARTICIPATE IN PURINE NUCLEOTIDE BIOSYNTHESIS (Figure 33-2) ❖ Enzymes of eukaryotes are polypeptides that possess multiple catalytic activities whose adjacent catalytic sites facilitate the channeling of intermediates between sites. ❖ Three distinct multifunctional enzymes catalyze reactions: ➢ Formyl transferase ➢ VII carboxylase ➢ Formyl transferase ANTIFOLATE DRUGS & GLUTAMINE ANALOGS BLOCK PURINE NUCLEOTIDE BIOSYNTHESIS

(CMT) ❖ Purine deficiency states, while rare in humans, generally reflect a deficiency of folic acid. ➢ Since the carbons added in reactions ④ and ⑩ of Purine Biosynthesis (Figure 33-2) are contributed by derivatives of tetrahydrofolate. Compounds that inhibit the formation of tetrahydrofolates and therefore block purine synthesis have been used in cancer chemotherapy. ❖ Inhibitory compounds: ➢ azaserine (inhibit reaction ⑤, Figure 33–2), ➢ diazanorleucine (inhibit reaction ②, Figure 33-2) ➢ 6 - mercaptopurine (inhibit reactions ⑬ and ⑭, Figure 33–3) ➢ mycophenolic acid (inhibit reactions and ⑭, Figure 33 – 3) “SALVAGE REACTIONS” CONVERT PURINES & THEIR NUCLEOSIDES TO MONONUCLEOTIDES Salvage reactions ❖ Involve in the conversion of purines, their ribonucleosides, and their deoxyribonucleosides to mononucleotides. ❖ Require far less energy than de novo synthesis. ❖ The more important (1st) mechanism involves phosphoribosylation by PRPP of a free purine (Pu) to form a purine 5’-mononucleotide (Pu-RP). ➢ Pu+PR-PP - > Pu-RP +PP ➢ Phosphoryl transfer from PRPP catalyzed by adenosine- and hypoxanthine-phosphoribosyl transferases. ➢ Converts adenine, hypoxanthine, and guanine to their mononucleotides. ➢ (See figure 33-4, page 331) ❖ A 2 nd^ salvage mechanism involves phosphoryl transfer from ATP to a purine ribonucleoside (Pu-R): ➢ Pu-R + ATP - > PuR-P + ADP ➢ Phosphorylation of the purine nucleotides, catalyzed by adenosine kinase converts adenosine and deoxyadenosine to AMP and dAMP. ➢ Similarly, deoxycytidine kinase phosphorylates deoxycytidine and 2’-deoxyguanosine, forming dCMP and dGMP, respectively. ❖ Liver ➢ major site of purine nucleotide biosynthesis ➢ Provides purines and purine nucleosides for salvage and for utilization by tissues incapable of their biosynthesis. ❖ Human brain tissue ➢ low level of PRPP glutamyl amidotransferase ➢ depends in part on exogenous purines. ❖ Erythrocytes and polymorphonuclear leukocytes ➢ Cannot synthesize 5-phosphoribosylamine. ➢ utilize exogenous purines to form nucleotides. HEPATIC PURINE BIOSYNTHESIS IS STRINGENTLY REGULATED AMP & GMP FEEDBACK REGULATE PRPP GLUTAMYL AMIDOTRANSFERASE ❖ Biosynthesis of IMP ➢ energetically expensive ➢ ATP, glycine, glutamine, aspartate, and reduced tetrahydrofolate derivatives all are consumed in the process. ➢ It is of survival advantage to closely regulate purine biosynthesis in response to varying physiologic needs. ❖ De novo purine nucleotide biosynthesis (Figure 33-5) ➢ The concentration of PRPP controls the overall determinant of the rate of de novo purine nucleotide biosynthesis. ➢ The rate of PRPP synthesis depends on the availability of ribose 5 - phosphate and the activity of PRPP synthetase. ➢ PRPP synthetase- an enzyme responsible for feedback inhibited by AMP, ADP, GMP, and GDP. AMP & GMP FEEDBACK REGULATE THEIR FORMATION FROM IMP (Figure 33-6) ❖ AMP ➢ AMP feedback inhibits adenylosuccinate Synthetase ➢ Conversion of IMP to adenylosuccinate to AMP requires GTP. ❖ GMP ➢ GMP inhibits IMP dehydrogenase. ➢ Conversion of xanthinylate (XMP) to GMP requires ATP. ➢ GMP feedback inhibits PRPP glutamyl amidotransferase. ❖ The cross-regulation between the pathways of IMP metabolism ➢ Balance the biosynthesis of purine nucleoside triphosphates by decreasing the synthesis of one purine nucleotide when there is a deficiency of the other nucleotide. ❖ AMP and GMP also inhibit hypoxanthine-guanine phosphoribosyltransferase, which converts hypoxanthine and guanine to IMP and GMP. REDUCTION OF RIBONUCLEOSIDE DIPHOSPHATES FORMS DEOXYRIBONUCLEOSIDE DIPHOSPHATES (Figure 33 - 7, page

❖ Reduction of the 2’hydroxyl (2’-deoxyribonucleoside diphosphate)

(CMT) ❖ Since humans lack uricase, the end product of purine catabolism in humans is uric acid. DISORDERS OF PURINE METABOLISM ❖ Various genetic defects in PRPP synthetase present clinically as gout. ❖ Each defect—for example, an elevated Vmax, increased affinity for ribose 5-phosphate, or resistance to feedback inhibition—results in overproduction and overexcretion of purine catabolites. ❖ When serum urate levels exceed the solubility limit, sodium urate crystalizes in soft tissues and joints and causes an inflammatory reaction, gouty arthritis. ❖ Most cases of gout reflect abnormalities in renal handling of uric acid. ❖ Hyperuricemias may be differentiated based on whether patients excrete normal or excessive quantities of total urates. ❖ Hyperuricemias reflect specific enzyme defects. ❖ Secondary to diseases such as cancer or psoriasis that enhance tissue turnover. LESCH-NYHAN SYNDROME ❖ An overproduction hyperuricemia ❖ Frequent episodes of uric acid lithiasis and a bizarre syndrome of self-mutilation. ❖ Reflects a defect in hypoxanthine-guanine phosphoribosyl transferase, an enzyme of purine salvage. ❖ The accompanying rise in intracellular PRPP results in purine overproduction. ❖ Mutations that decrease or abolish hypoxanthine-guanine phosphoribosyltransferase activity include: ➢ Deletions ➢ Frame-shift mutations ➢ Base substitutions ➢ Aberrant mRNA splicing. VON GIERKE DISEASE ❖ Purine overproduction and hyperuricemia in von Gierke disease (glucose- 6 - phosphatase deficiency) occurs secondary to enhanced generation of the PRPP precursor ribose 5-phosphate. ❖ An associated lactic acidosis elevates the renal threshold for urate, elevating total body urates. HYPOURICEMIA ❖ Hypouricemia an increased excretion of hypoxanthine and xanthine are associated with a deficiency in xanthine oxidase. ❖ Due to a genetic defect or to severe liver damage. ❖ Patients with a severe enzyme deficiency may exhibit xanthinuria and xanthine lithiasis. ADENOSINE DEAMINASE & PURINE NUCLEOSIDE PHOSPHORYLASE DEFICIENCY ❖ Associated with an immunodeficiency disease in which both thymus-derived lymphocytes (T cells) and bone marrow–derived lymphocytes (B cells) are sparse and dysfunctional. ❖ Patients suffer from severe immunodeficiency. ❖ In the absence of enzyme replacement or bone marrow transplantation, infants often succumb to fatal infections. ❖ Defective activity of purine nucleoside phosphorylase is associated with a severe deficiency of T cells, but apparently normal B-cell function. ❖ Immune dysfunctions appear to result from accumulation of dGTP and dATP, which inhibit ribonucleotide reductase and thereby deplete cells of DNA precursors. PYRIMIDINE CATABOLITES ARE WATER SOLUBLE ❖ Catabolism of the pyrimidines forms highly water-soluble products—CO2, NH3, β-alanine, and β-aminoisobutyrate. ❖ Excretion of β-aminoisobutyrate increases in leukemia and severe x-ray radiation exposure due to increased destruction of DNA. ❖ Disorders of β-alanine and β-aminoisobutryrate metabolism arise from defects in enzymes of pyrimidine catabolism. ❖ A disorder of pyrimidine catabolism, known also as combined uraciluria-thyminuria, is also a disorder of β- amino acid metabolism, since the formation of β-alanine and of β-aminoisobutyrate is impaired. ❖ When caused by a genetic error, there are serious neurologic complications. ❖ A nongenetic form is triggered by the administration of the anticancer drug 5-fluorouracil to patients with low levels of dihydropyrimidine dehydrogenase. PSEUDOURIDINE IS EXCRETED UNCHANGED ❖ No human enzyme catalyzes hydrolysis or phosphorolysis of the pseudouridine (ψ) derived from the degradation of RNA molecules. ❖ This unusual nucleotide therefore is excreted unchanged in the urine of normal subjects. ❖ Pseudouridine was indeed first isolated from human urine. OVERPRODUCTION OF PYRIMIDINE ❖ In hyperuricemia associated with severe overproduction of PRPP, there is overproduction of pyrimidine nucleotides and increased excretion of β-alanine.

(CMT) ❖ N5, N10-methylenetetrahydrofolate is required for thymidylate synthesis, disorders of folate and vitamin B metabolism result in deficiencies of TMP. OROTIC ACIDURIA ❖ Accompanies the Reye syndrome probably is a consequence of the inability of severely damaged mitochondria to utilize carbamoyl phosphate, which then becomes available for cytosolic overproduction of orotic acid. ❖ Type I orotic aciduria reflects a deficiency of both orotate phosphoribosyltransferase and orotidylate decarboxylase. ❖ The rarer Type II orotic aciduria is due to a deficiency only of orotidylate decarboxylase. DEFICIENCY OF A UREA CYCLE ENZYME RESULTS IN EXCRETION OF PYRIMIDINE PRECURSORS ❖ Increased excretion of orotic acid, uracil, and uridine accompanies a ❖ deficiency in liver mitochondrial ornithine transcarbamoylase. ❖ Excess carbamoyl phosphate exits to the cytosol, where it stimulates pyrimidine nucleotide biosynthesis. ❖ The resulting mild orotic aciduria is increased by high- nitrogen foods. DRUGS MAY PRECIPITATE OROTIC ACIDURIA ❖ Allopurinol an alternative substrate for orotate Phosphoribosyltransferase competes with orotic acid. ❖ The resulting nucleotide product also inhibits orotidylate decarboxylase, resulting in orotic aciduria and orotidinuria. ❖ 6 - Azauridine, following conversion to 6-azauridylate, also competitively inhibits orotidylate decarboxylase enhancing excretion of orotic acid and orotidine. ❖ Four genes that encode urate transporters have been identified. ❖ Two of the encoded proteins are localized to the apical membrane of proximal tubular cells. CHECK PO NINYO ANG MGA FIGURE SA BOOK

Metabolism of Purine and Pyrimidine Nucleotides: Biosynthesis, Catabolism, and Disorders, Summaries of Biochemistry

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