Biochemistry Lecture 6: Nitrogen Fixation and Metabolization

Course Overview

  • Course Name: Biochemistry

  • Lecture Number: 6

  • Topic: Nitrogen fixation and metabolization

  • Instructor: Prof. Dr. C. Vink

  • Institution: Erasmus University College

Learning Goals

  • Understand the following concepts:

    • Nitrogen fixation: conversion of atmospheric N₂ into bioavailable forms of nitrogen.

    • General aspects of amino acid anabolism:

    • Transaminations

    • Links with sugar metabolism

    • General aspects of amino acid catabolism:

    • The urea cycle

    • Links with sugar metabolism

    • General aspects of nucleotide biosynthesis, including the role of folate in one-carbon transfers.

Part 1: How Does Nitrogen End Up in Organic Molecules?

  • Atmospheric Composition:

    • Nitrogen is abundant in the atmosphere as inorganic molecular nitrogen (N₂): 78%

    • Other gases: O₂ (21%), Argon (0.9%), CO₂ (0.04%)

  • Incorporation of Nitrogen:

    • In its N₂ form, nitrogen cannot be incorporated into organic molecules.

  • Process of Nitrogen Fixation:

    • Through nitrogen fixation, N₂ can be converted into bioavailable forms of nitrogen including:

    • Ammonium (NH₄⁺)

    • Nitrite (NO₂⁻)

    • Nitrate (NO₃⁻)

    • Nitrous oxide (N₂O)

    • Nitric oxide (NO)

Nitrogen Cycle

  1. N₂ Fixation:

    • Conversion of N₂ into bioavailable forms (NH₄⁺ and NOₓ) by:

      • Lightning (atmospheric)

      • Bacteria (biological)

      • Humans (industrial)

  2. Ammonification (Mineralization):

    • Production of NH₄⁺ through breakdown of organic nitrogen compounds from decaying plant and animal matter by bacteria and fungi.

  3. Assimilation:

    • Absorption of NO₃⁻ or NH₄⁺ from the soil by plants.

  4. Nitrification:

    • Conversion of NH₄⁺ to NO₃⁻ primarily by soil-living bacteria and nitrifying bacteria.

  5. Denitrification:

    • Reduction of NO₃⁻ to N₂ by denitrifying bacteria.

Nitrogen Fixation Processes

  • Natural Processes:

    • Biological: Diazotrophy; by bacteria producing NH₄⁺; organisms capable of natural nitrogen fixation are diazotrophs.

    • Physical: By lightning producing NOₓ.

  • Artificial Processes:

    • By humans through burning fossil fuels or industrial (fertilizer) production.

Biological Nitrogen Fixation

  • Typical nitrogen-fixing bacteria.

  • Form symbiotic relationships with leguminous plants (e.g., beans, alfalfa).

  • Ammonium Ion (NH₄⁺):

    • The conjugate acid form of NH₃, used in the first stages of organic compound synthesis.

  • Reduction Reaction in Nitrogen Fixation:

    • Involves an eight-electron reduction, catalyzed by nitrogenase enzyme complex.

Part 2: How Does NH₄⁺ End Up in Amino Acids?

  • Key Amino Acids: Glutamate (Glu) and Glutamine (Gln) play central roles in amino acid synthesis:

    • Glutamate is produced from α-ketoglutarate (α-KG).

    • Glutamine is synthesized from glutamate.

Production of Glu from α-KG

  • Reaction Type: Reductive amination catalyzed by glutamate dehydrogenase.

    • Definition: Reductive = electrons are gained, amination = amino group transferred.

    • This reversible reaction establishes the stereochemistry of the α-carbon atom, leading to the production of L-Glu.

Production of Gln from Glu

  • Reaction Type: Amidation, catalyzed by glutamine synthetase.

  • Amidation refers to the formation of an amide bond (-NHCO-).

  • Glutamate dehydrogenase and glutamine synthetase are present in all organisms.

  • Most amino acids derive their amino group from Glu and Gln.

Part 3: General Aspects of Amino Acid Synthesis Pathways

Amino Acid Biosynthesis

  • Pathway Features:

    • Amino acid biosynthesis involves a common set of reactions.

    • Transamination reactions and one-carbon unit transfers (formyl or methyl groups) occur frequently.

    • Amino acids can be grouped into families based on common precursors.

  • Citric Acid Cycle:

    • Amphibolic with roles in both catabolism and anabolism; important for amino acid biosynthesis.

  • Amino Acid Families:

    • Glutamate Family:

    • Derived from α-Ketoglutarate.

    • Includes: Glutamate, Glutamine, Proline, Arginine.

    • Oxaloacetate Family:

    • Includes: Aspartate, Methionine, Asparagine, Threonine, Lysine, Isoleucine.

    • Serine Family:

    • Derived from 3-Phosphoglycerate; includes: Serine, Cysteine, Glycine.

    • Pyruvate Family:

    • Includes: Pyruvate, Valine, Alanine, Leucine.

    • Aromatic Family:

    • Derived from Phosphoenolpyruvate; includes: Phenylalanine, Tyrosine, Tryptophan.

Part 4: Amino Acid Catabolism: The Disposal of Nitrogen

Forms of Excess Nitrogen Excretion

  1. Ammonia (NH₄⁺):

    • Excreted by fish (ammonotelic).

  2. Uric Acid:

    • Excreted by birds (uricotelic).

  3. Urea:

    • Excreted by terrestrial animals (ureotelic).

Urea Production

  • Occurs almost exclusively in the liver.

  • Urea passes into the bloodstream to kidneys and is excreted in urine.

Transport of Amino Groups to the Liver

  • Glutamine (Gln): Acts as a non-toxic transporter of amino groups to the liver.

  • Gln loses its amino group during the urea cycle.

  • Alanine (Ala): Another important NH₄⁺ transporter through transaminations.

  • **Urea Cycle Mechanism:

    • One nitrogen atom of urea is transferred from aspartate.

    • Another nitrogen atom is derived from free NH₄⁺ (from Glu).

    • Carbon comes from HCO₃⁻ (from CO₂ hydration).

  • Linkage to the Citric Acid Cycle:

  • Urea cycle is connected to citric acid cycle via oxaloacetate/aspartate and fumarate.

    • The overall reaction: The disposal of nitrogen in the urea cycle links nitrogen metabolism with biochemistry of carbon skeleton.

Part 5: Nucleotide Synthesis

Purine Synthesis

  • Formation of AMP and GMP involves:

    • Aspartate, fumarate, Gln, and Glu.

Deoxyribonucleotide Synthesis

  • Formation of deoxyribonucleotides from ribonucleotides through reduction of their 2'-OH.

    • Exception: dTTP synthesized via dUMP ➔ dTMP route.

Pyrimidine Synthesis

  • Pyrimidine ring is assembled before attachment to ribose-5-phosphate from pentose-phosphate pathway.

  • Assembled from bicarbonate and ammonia to form carbamoyl phosphate, which requires ATP.

CTP Synthesis

  • Glutamine serves as the amino donor in CTP synthesis.

Take-home Messages

  • Amino Acid Metabolism:

    • Nitrogen fixation makes N₂ available in the form of ammonia.

    • Transamination reactions are key in amino acid synthesis; Glu and Gln are prominent donors.

    • Amino acid catabolism results in carbon skeleton transformations.

    • Nitrogen is excreted by ileum as urea, uric acid, or ammonia.

    • The urea cycle converts catabolic nitrogen into urea.

  • Nucleotide Biosynthesis:

    • Purine synthesis involves base attachment to ribose phosphate, leading to IMP, AMP, or GMP.

    • Pyrimidines undergo formation before attachment to ribose phosphate.

    • Deoxyribonucleotide synthesis is a reduction-driven process, particularly important in cell division and growth regulation.