⚗️Biological Chemistry II Unit 5 – Nucleotide Metabolism

Key Concepts and Definitions

• Nucleotides consist of a nitrogenous base, a pentose sugar, and one or more phosphate groups
• Purines (adenine and guanine) have a double-ring structure derived from a purine base
• Pyrimidines (cytosine, thymine, and uracil) have a single-ring structure derived from a pyrimidine base
• Nucleosides are formed when a nitrogenous base is attached to a pentose sugar (ribose or deoxyribose) via a glycosidic bond
• Nucleotides serve as building blocks for nucleic acids (DNA and RNA), energy carriers (ATP and GTP), and signaling molecules (cAMP and cGMP)
• Nucleotide metabolism encompasses the synthesis, degradation, and recycling of nucleotides in the body
• Purine and pyrimidine nucleotides are synthesized through de novo and salvage pathways

Nucleotide Structure and Function

• Nucleotides are composed of three main components: a nitrogenous base, a pentose sugar, and one or more phosphate groups
• The nitrogenous base can be a purine (adenine or guanine) or a pyrimidine (cytosine, thymine, or uracil)
• The pentose sugar is either ribose (in RNA) or deoxyribose (in DNA)
• Phosphate groups are attached to the 5' carbon of the pentose sugar, forming mono-, di-, or triphosphates
• Nucleotides serve as the monomeric units of nucleic acids, with DNA containing deoxyribonucleotides and RNA containing ribonucleotides
• ATP (adenosine triphosphate) and GTP (guanosine triphosphate) function as energy carriers in cellular processes such as metabolism and signal transduction
  • ATP is the primary energy currency of the cell, providing energy for various biochemical reactions
  • GTP is involved in protein synthesis, signal transduction, and microtubule assembly
• Cyclic nucleotides (cAMP and cGMP) act as second messengers in intracellular signaling pathways
• Nucleotide derivatives (NAD+, NADP+, FAD, and Coenzyme A) serve as coenzymes in metabolic reactions

Purine Metabolism

• Purine nucleotides (adenine and guanine) are synthesized through de novo and salvage pathways
• The de novo pathway begins with the synthesis of phosphoribosyl pyrophosphate (PRPP) from ribose-5-phosphate and ATP
• PRPP is converted to inosine monophosphate (IMP) through a series of enzymatic reactions, with the purine ring assembled step by step
• IMP serves as a branch point for the synthesis of AMP and GMP
  • AMP is formed from IMP by the action of adenylosuccinate synthetase and adenylosuccinate lyase
  • GMP is formed from IMP by the action of IMP dehydrogenase and GMP synthetase
• The salvage pathway recycles preformed purine bases (adenine, guanine, and hypoxanthine) by attaching them to PRPP
  • Adenine phosphoribosyltransferase (APRT) catalyzes the formation of AMP from adenine and PRPP
  • Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyzes the formation of IMP and GMP from hypoxanthine and guanine, respectively
• Purine nucleotides are degraded to uric acid, which is excreted in urine
  • Adenosine is deaminated to inosine by adenosine deaminase
  • Guanosine is deaminated to xanthosine by guanine deaminase
  • Inosine and xanthosine are further degraded to hypoxanthine and xanthine, respectively, and finally to uric acid by xanthine oxidase

Pyrimidine Metabolism

• Pyrimidine nucleotides (cytosine, thymine, and uracil) are synthesized through de novo and salvage pathways
• The de novo pathway begins with the formation of carbamoyl phosphate from glutamine, bicarbonate, and ATP by carbamoyl phosphate synthetase II (CPS II)
• Carbamoyl phosphate is combined with aspartate to form N-carbamoylaspartate, which is then converted to dihydroorotate by dihydroorotase
• Dihydroorotate is oxidized to orotate by dihydroorotate dehydrogenase, and orotate is combined with PRPP to form orotidine monophosphate (OMP)
• OMP is decarboxylated to form uridine monophosphate (UMP), which serves as a precursor for the synthesis of other pyrimidine nucleotides
  • UMP is phosphorylated to form UDP and UTP
  • CTP is formed from UTP by the action of CTP synthetase
  • dTMP is formed from dUMP by thymidylate synthase, using N5,N10-methylenetetrahydrofolate as a methyl donor
• The salvage pathway recycles preformed pyrimidine bases (uracil, thymine, and cytosine) by attaching them to ribose-1-phosphate
  • Uracil phosphoribosyltransferase (UPRT) catalyzes the formation of UMP from uracil and PRPP
  • Thymidine kinase catalyzes the phosphorylation of thymidine to form dTMP
  • Deoxycytidine kinase catalyzes the phosphorylation of deoxycytidine to form dCMP
• Pyrimidine nucleotides are degraded to β-alanine (from uracil and thymine) and β-aminoisobutyrate (from cytosine), which are excreted in urine

Nucleotide Synthesis: De Novo vs. Salvage Pathways

• Nucleotides are synthesized through two main pathways: de novo synthesis and salvage pathways
• The de novo pathway involves the synthesis of nucleotides from simple precursor molecules, such as amino acids and ribose-5-phosphate
  • Purine nucleotides are synthesized from phosphoribosyl pyrophosphate (PRPP) and amino acids (glycine, glutamine, and aspartate)
  • Pyrimidine nucleotides are synthesized from carbamoyl phosphate and aspartate
• The salvage pathway recycles preformed nucleobases and nucleosides by attaching them to ribose-1-phosphate or ribose-5-phosphate
  • Purine bases (adenine, guanine, and hypoxanthine) are salvaged by phosphoribosyltransferases (APRT and HGPRT)
  • Pyrimidine bases (uracil, thymine, and cytosine) are salvaged by phosphoribosyltransferases (UPRT) or kinases (thymidine kinase and deoxycytidine kinase)
• The de novo pathway is energy-intensive, requiring ATP for the synthesis of nucleotides
• The salvage pathway is more energy-efficient, as it recycles preformed bases and nucleosides, reducing the need for de novo synthesis
• The balance between de novo and salvage pathways is regulated by the availability of preformed bases and nucleosides, as well as the energy status of the cell

Regulation of Nucleotide Metabolism

• Nucleotide metabolism is tightly regulated to maintain balanced pools of nucleotides for DNA and RNA synthesis
• The de novo synthesis of purine nucleotides is regulated by feedback inhibition of the first committed step, catalyzed by glutamine phosphoribosylpyrophosphate amidotransferase (GPAT)
  • AMP and GMP allosterically inhibit GPAT, reducing the synthesis of purine nucleotides when their levels are high
• The de novo synthesis of pyrimidine nucleotides is regulated by the activity of carbamoyl phosphate synthetase II (CPS II)
  • CPS II is allosterically activated by PRPP and inhibited by UTP, ensuring that pyrimidine nucleotide synthesis is responsive to the availability of precursors and the levels of end products
• The salvage pathways are regulated by the availability of preformed bases and nucleosides, as well as the activity of phosphoribosyltransferases and kinases
• Ribonucleotide reductase, the enzyme responsible for the conversion of ribonucleotides to deoxyribonucleotides, is regulated by the cell cycle and DNA damage response
  • The activity of ribonucleotide reductase increases during S phase of the cell cycle to support DNA synthesis
  • DNA damage leads to the activation of ribonucleotide reductase to ensure an adequate supply of deoxyribonucleotides for DNA repair
• Imbalances in nucleotide pools can lead to increased mutation rates and genomic instability

Clinical Relevance and Disorders

• Disorders of purine metabolism include gout, Lesch-Nyhan syndrome, and adenosine deaminase deficiency
  • Gout is caused by the accumulation of uric acid crystals in joints, leading to inflammation and pain
  • Lesch-Nyhan syndrome is an X-linked disorder caused by a deficiency in hypoxanthine-guanine phosphoribosyltransferase (HGPRT), leading to hyperuricemia, neurological symptoms, and self-injurious behavior
  • Adenosine deaminase deficiency is an autosomal recessive disorder that causes severe combined immunodeficiency (SCID) due to the accumulation of deoxyadenosine and its toxic effects on lymphocytes
• Disorders of pyrimidine metabolism include orotic aciduria and pyrimidine nucleoside phosphorylase deficiency
  • Orotic aciduria is caused by a deficiency in uridine monophosphate synthase (UMPS), leading to the accumulation of orotic acid and a deficiency in pyrimidine nucleotides
  • Pyrimidine nucleoside phosphorylase deficiency is an autosomal recessive disorder that causes immunodeficiency and neurological symptoms due to the accumulation of deoxyuridine and deoxyinosine
• Chemotherapeutic agents targeting nucleotide metabolism include antimetabolites such as 5-fluorouracil, methotrexate, and 6-mercaptopurine
  • These drugs interfere with nucleotide synthesis, leading to the inhibition of DNA and RNA synthesis and cell division
  • Antimetabolites are used to treat various types of cancer, including leukemia, lymphoma, and solid tumors
• Nucleoside and nucleotide analogs are used as antiviral agents, such as acyclovir, zidovudine (AZT), and tenofovir
  • These analogs are incorporated into viral DNA or RNA, leading to chain termination and the inhibition of viral replication
  • Antiviral nucleoside and nucleotide analogs are used to treat infections caused by herpesviruses, HIV, and hepatitis B and C viruses

Lab Techniques and Applications

• Radiolabeled nucleotides (e.g., ³H-thymidine, ³²P-dCTP) are used to study DNA replication and repair, as well as to measure cell proliferation
• Polymerase chain reaction (PCR) is a widely used technique for amplifying specific DNA sequences, which relies on the use of deoxyribonucleotides (dNTPs) as substrates for DNA polymerase
• Sanger sequencing, a method for determining the nucleotide sequence of DNA, involves the use of dideoxynucleotides (ddNTPs) as chain terminators
• Next-generation sequencing (NGS) technologies, such as Illumina and Oxford Nanopore, rely on the incorporation of fluorescently labeled or modified nucleotides for the parallel sequencing of millions of DNA fragments
• Nucleotide analogs, such as bromodeoxyuridine (BrdU) and ethynyldeoxyuridine (EdU), are used to label newly synthesized DNA in cell proliferation assays
• Aptamers, short single-stranded oligonucleotides that bind specific targets, are used in various applications, including drug delivery, biosensing, and targeted therapy
  • Aptamers are selected through an in vitro process called systematic evolution of ligands by exponential enrichment (SELEX)
  • The specificity and affinity of aptamers can be enhanced by incorporating modified nucleotides, such as 2'-O-methyl or 2'-fluoro nucleotides
• Nucleotide-based probes, such as TaqMan and molecular beacons, are used in real-time PCR assays for the detection and quantification of specific DNA or RNA sequences
• Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are used to modulate gene expression by targeting specific mRNA sequences for degradation or translational inhibition
  • ASOs are single-stranded oligonucleotides that bind to complementary mRNA sequences and recruit RNase H for mRNA degradation
  • siRNAs are short double-stranded RNA molecules that are incorporated into the RNA-induced silencing complex (RISC) and guide the cleavage of complementary mRNA sequences