Chapter 32

Exam Information

Date and Time: Monday, April 20, from 3:30 PM to 4:20 PM
Location: W290 Chemistry Building, same as the lecture (BMB 3110)
Requirements: Show University ID when handing in the exam; bring it with you.
Prohibited Items: Calculators are not allowed during the exam.

Lecture Overview

Course: BMB 3110, taught by William Hacker, PhD
Lecture Number: 32
Chapter: 32 - Nucleotide Metabolism
  - Outline Topics:
    - Overview
    - Pyrimidines
    - Purines
    - Making deoxynucleotides
    - Regulation (and dysregulation)
  - Practice Problems: 1-4, 7-8, and 11-15 at the end of Chapter 32
  - Source: Lane and Fan, 2015. Nucleic Acids Res. 43: 2467
  - Copyright: Notes for personal, educational, and noncommercial use only; cannot be reproduced or distributed without permission from the instructor.

Learning Goals

At the end of this lecture, students should be able to:

Recognize: The five common bases (adenine, guanine, cytosine, thymine, uracil)
Distinguish: Differences between bases, nucleosides, and nucleotides
Understand: Key steps in the de novo synthesis of pyrimidines and purines
Generate: Deoxyribonucleotides from ribonucleotides
Familiarize: Mechanisms for regulating nucleic acid synthesis

Importance of Amino Acids

Biosynthetic Precursors: Amino acids serve as key precursors for the synthesis of nucleotides, including:
  - Serine
  - Histidine
  - Tyrosine
  - Tryptophan

Nucleotide Components

Nucleoside vs. Nucleotide:
- Nucleoside: Includes a base (adenine, guanine, cytosine, thymine, or uracil) and a sugar (ribose or deoxyribose).
- Nucleotide: Includes a base, sugar, and at least one phosphate group (can be mono-, di-, or triphosphate; e.g., cAMP as a cyclic nucleotide).

Nucleotide Biosynthesis Overview

Pathways: Nucleotide biosynthesis is divided into two main groups:
  - De novo pathways:
    - Bases are assembled from simpler compounds (energetically expensive).
  - Salvage pathways:
    - Utilize already-complete bases; energetically cheaper than de novo synthesis.
Outcome: Both pathways typically lead to ribonucleotides, with deoxy forms generated through reduction of ribonucleotides.

Synthesis of Pyrimidines (De Novo)

Raw Materials:
  - Bicarbonate
  - Aspartate
  - Ammonia
Process Overview:
  - Pyrimidine ring synthesized independently before linking to ribose; ring attached to activated ribose (PRPP).
  - Final product is Uridine Monophosphate (UMP) through decarboxylation.
  - Cytidine Triphosphate (CTP) formation from Uridine Triphosphate (UTP) via amination.

Formation of Carbamoyl Phosphate

Catalyzing Reaction:
  - Enzyme: Carbamoyl phosphate synthetase II (CPS II)
    - Requires two ATP molecules.
    - Nitrogen donated from glutamine.

Pyrimidine Ring Formation Steps

Step 1: Combine carbamoyl phosphate and aspartate through aspartate transcarbamoylase.
Step 2: Cyclization of the product occurs through dihydroorotase, resulting in orotate.

Attachment of Pyrimidine to Ribose

Mechanism:
- Use of activated ribose form PRPP (5-phosphoribosyl-1-pyrophosphate).
- Links to form orotidylate, which is then decarboxylated to uridylate (UMP).

Modifications to Nucleotides

Conversion to Triphosphates:
- Nucleoside monophosphate kinases convert monophosphates to diphosphates (e.g., UMP kinase).
- Nucleoside diphosphate kinases convert diphosphates to triphosphates (single enzyme with broad specificity).
Amination: Converts UTP to CTP.

Pyrimidine Salvage Pathway

Recycling Mechanism:
  - Pyrimidine bases from degraded DNA/RNA can be reused.
  - Example: Formation of Thymidylate from Thymine:
    - Steps: Thymine to Thymidine (nucleoside; catalyzed by thymidine phosphorylase), and then to Thymidylate (nucleotide; catalyzed by thymidine kinase).
    - Thymidine kinase is critical for making TMP and is a target for viral kinase drugs.

Synthesis of Purines (De Novo)

Assembly Process: Purine rings are synthesized directly on ribose phosphate.
  - Replacement of pyrophosphate on PRPP with amine (5-phosphoribosyl-1-amine produced).
  - Nine total steps lead to Inosine Monophosphate (IMP); required precursors include:
    - Glycine
    - Glutamine
    - Aspartate
    - Tetrahydrofolate (carrier of activated one-carbon units).

Modification of PRPP in Purine Synthesis

Key Reaction:
- Amine from Glutamine replaces pyrophosphate on PRPP, catalyzed by glutamine phosphoribosyl amidotransferase.

Synthesis of Inosine Monophosphate (IMP)

Reactions Required: Total of nine reactions including:
  - Addition of glycine.
  - Requires:
    - Four ATP
    - Two one-carbon units
    - Bicarbonate
    - Glycine
    - Aspartate
  - Nomenclature note: Inosinate is the nucleotide, Inosine is the nucleoside, and Hypoxanthine is the base.

Formation of Adenylate and Guanylate from IMP

Standard Purine Nucleotides Produced:
  - Adenosine Monophosphate (AMP)
  - Guanosine Monophosphate (GMP)
  - Synthesis connections: AMP requires GTP and GMP requires ATP, also requiring NAD+.

Purine Salvage Pathways

Recycling Purine Bases: From DNA/RNA breakdown.
  - Enzymatic Examples:
    - Adenine to AMP via adenine phosphoribosyl transferase.
    - Guanine to GMP via hypoxanthine-guanine phosphoribosyl transferase.
    - Hypoxanthine to IMP via the same enzyme.

Generation of Deoxyribonucleotides

Process: Formed by reduction of ribonucleotides:
  - Diphosphate nucleotides are reduced (rNDP → dNDP).
  - Enzyme: Ribonucleotide reductase; requires NADPH.
  - Acts on all four ribonucleoside diphosphates (rNDPs).

Mechanism of Ribonucleoside Reductase

Electron Transfer Process: NADPH's reducing power does not act directly on ribonucleotides but moves through a chain that includes thioredoxin.

Production of Thymidylate

Key Conversion Steps:
  - Conversion of deoxyuridine phosphates (dU) to thymidylate (T), which are components of DNA.
  - Requires:
    - Conversion of dUDP to dUMP
    - Conversion of dUMP to TMP (catalyzed by thymidylate synthase, needing tetrahydrofolate for the methyl group).
  - Tetrahydrofolate is regenerated by dihydrofolate reductase.

Clinical Insight: Anticancer Drugs

Targeting Rapidly-Dividing Cells: These cells require abundant thymidylate.
- Drug Action: Fluorouracil is converted to fluorodeoxyuridylate (F-dUMP) in cells and acts as a suicide inhibitor on thymidylate synthase.
- Other Drug Actions: Methotrexate and aminopterin inhibit dihydrofolate reductase, preventing tetrahydrofolate regeneration.

Regulation of Nucleotide Production in Pyrimidines

Key Regulators:
- Aspartate transcarbamoylase is crucial for pyrimidine biosynthesis in bacteria. Activated by ATP and inhibited by CTP to balance purine/pyrimidine levels.
- Carbamoyl phosphate synthetase II (CPS II) also regulated by feedback inhibition in bacteria and eukaryotes.

Regulation of Nucleotide Production in Purines

Committed Step: Synthesis of phosphoribosylamine is inhibited by purine monophosphates IMP, AMP, and GMP.
- Feedback Mechanism: Synthesis of AMP and GMP is coordinated; each feedback-inhibited by their products, balancing synthesis through ATP and GTP dependency.

Regulation of Production of Deoxyribonucleotides

Ribonucleotide Reductase Activity Regulation: Each subunit has two allosteric sites regulating overall activity and substrate specificity:
- dATP binding reduces activity, ATP binding increases it.
- This complex pattern balances relative dNTP levels, emphasizing its significance as a cancer therapy target.

Catabolism of Nucleotides

Continual Turnover Reasons: Nucleotides continuously broken down:
  - Steps include:
    - Conversion from nucleotides to nucleosides by nucleotidases.
    - Conversion of nucleosides to free bases and (deoxy)ribo-1-phosphate by phosphorylases.
  - Example: Purine catabolism:
    - Adenosine to inosine via adenosine deaminase.
    - Guanine to xanthine.
  - Defects: High levels of dATP due to deaminase defects can inhibit ribonucleotide reductase, affecting DNA production (causes severe immunodeficiency).

Clinical Insight: Purine Catabolism and Gout

Xanthine Oxidase Role: Converts xanthine to uric acid, which at physiological pH, forms urate.
  - High urate levels (hyperuricemia) lead to gout:
    - Symptoms include severe joint pain caused by crystallization of sodium urate in the joints and can lead to kidney damage.
  - Treatment: Inhibition of xanthine oxidase with allopurinol (suicide inhibitor).

Clinical Insight: Lesch-Nyhan Syndrome

Cause: Defect in HGPRT (Hypoxanthine-guanine phosphoribosyltransferase) leads to elevated urate levels and gout symptoms.
Behavioral Symptoms: Includes self-destructive behavior and cognitive deficits, although the molecular basis of such behaviors is unclear.

Key Concepts Summary

Structures: Recognize the structures of the five common bases including U and T.
Classification/Nomenclature: Differentiate between bases, ribonucleosides, and nucleotides, and understand precursor sources for pyrimidines.
Pyrimidine Synthesis: Key questions on pyrimidine synthesis processes.
Purine Synthesis: Understanding initial purine formation; reactions catalyzed by CPS II and aspartate transcarbamoylase.
Nucleotide Salvage: Types of reactions in salvage pathways.
Deoxynucleotides: Role of ribonucleoside reductase in dNTP production; pathways converting UMP to TMP; regulation of reductase activity.
Regulation: Feedback inhibition mechanisms in AMP and GMP synthesis.
Clinical Implications: Awareness of anticancer drug pathways; causes of gout; effects of enzyme defects leading to SCID and Lesch-Nyhan syndrome.