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BMB 3110 Lecture 30: Amino Acid Degradation and the Urea Cycle

Outline of Lecture

  • Removing nitrogen from amino acids
  • Processing of ammonium
  • Using carbons from amino acids
  • Essential Problems: See problems 4, 6-13, and 15 at the end of Chapter 30.

Learning Goals

At the end of this lecture, you should:

  • Know how proteins are earmarked for degradation
  • Be familiar with the enzymes that are used in the degradation of amino acids
  • Be able to outline the steps in the urea cycle
  • Know key enzymes that participate in the urea cycle
  • Understand mechanisms by which nitrogen is transported in the blood
  • Know metabolic products of amino acid degradation and the pathways they enter

Protein Turnover

  • Regulation of Protein Turnover

    • Dietary proteins are broken down by specific proteases (as discussed in Chapter 14).
    • Cellular protein degradation is tightly controlled, whereby proteins targeted for destruction are ubiquitylated.
    • Ubiquitin is a small protein consisting of approximately 80 amino acids.
    • Ubiquitin is attached to proteins through an isopeptide bond.
    • A three-enzyme system is responsible for attaching ubiquitin to lysine residues in proteins.

  • Ubiquitin-Dependent Proteolysis

    • Proteins tagged with ubiquitin are cleaved by the proteasome.
    • Cleavage results in the generation of fragments generally ranging from 7 to 9 residues, which are further cleaved by other proteases.

Amino Acid Degradation

Removal of Nitrogen
  • Overview of Nitrogen Removal
    • Excess amino acids are primarily degraded in the liver, with branched-chain amino acids being exceptions utilized as an energy source in muscle.
    • Nitrogen removal serves as the initial step in amino acid degradation.
    • Key enzymes involved in nitrogen removal include:
    • Aminotransferases (or transaminases) (most amino acids)
      • Function: Transfer NH3 to α-ketoglutarate to produce glutamate.
      • Reaction:
        ext{Amino Acid} + ext{α-ketoglutarate}
        ightarrow ext{Glutamate} + ext{α-keto acid}
    • Dehydrogenase (specifically for glutamate)
    • Dehydratase (for serine and threonine).
Mechanism of Action of Aminotransferases
  • General Mechanism
    • Aminotransferases usually transfer NH3 to α-ketoglutarate leading to the formation of glutamate.
    • Similar enzymes also play roles in synthesizing amino acids and require a prosthetic group known as pyridoxal phosphate for the transfer of nitrogen.
Example Reactions of Aminotransferases
  • Alanine Aminotransferase (ALT)
    • Converts alanine to pyruvate through the reaction:
      ext{Alanine} + ext{α-ketoglutarate}
      ightarrow ext{Pyruvate} + ext{Glutamate}
  • Aspartate Aminotransferase (AST)
    • Converts aspartate to oxaloacetate:
      ext{Aspartate} + ext{α-ketoglutarate}
      ightarrow ext{Oxaloacetate} + ext{Glutamate}
    • The products of these reactions can enter various pathways of carbon metabolism and are essential for energy production.
Production of Ammonium

  • Excess nitrogen results in production of ammonium (NH4+) in the mitochondria.

  • Clinical Insight: Elevated blood concentrations of transaminases indicate liver damage, as these enzymes leak into the bloodstream from damaged tissue.

  • Overall Reaction for Ammonium Production

    • Involves both transaminase and dehydrogenase activities:
    1. NH3 is removed from glutamate, releasing ammonium (NH4+).
    2. The process requires electron acceptors (NAD+ or NADP+) and occurs predominantly in liver mitochondria.

  • The net reaction includes additional notable details:

    • Glutamate dehydrogenase is allosterically regulated by energy charge in mammals, where ATP and GTP serve as inhibitors, and ADP and GDP function as activators.

Transport of Nitrogen to the Liver

  • Branch-chain amino acids including leucine, valine, and isoleucine cannot be directly metabolized in the liver.
  • These amino acids are utilized as fuel in muscle tissue, where nitrogen is transported back to the liver using the glucose-alanine cycle.
Glucose-Alanine Cycle
  • Process In the Muscle and Liver:
    • In muscle, amino acids transfer nitrogen to α-ketoglutarate, forming glutamate.
    • Glutamate further transfers the nitrogen to pyruvate, forming alanine, which is transported via the bloodstream to the liver.
    • In the liver, nitrogen from alanine is transferred to regenerate glutamate.
Alternative Pathway for Nitrogen Transport
  • Nitrogen can also be transported as glutamine:
    • Glutamine Synthetase converts NH4+ and glutamate to glutamine, which is subsequently processed in the liver to generate urea.

The Urea Cycle: Overview

  • Ureotelic organisms, including most terrestrial vertebrates, primarily dispose of excess NH4+ in the form of urea.
  • Urea synthesis incorporates:
    • Free ammonium (NH4+)
    • Bicarbonate (HCO3-)
    • Aspartate (source of NH3).

Urea Cycle: Mitochondrial Reactions

  • Carbamoyl Phosphate Synthetase (CPS I) combines NH4+ and bicarbonate in the mitochondria:
    • Involves the hydrolysis of two ATP molecules, making it irreversible.
    • Activated by N-acetylglutamate (indicating the presence of free amino acids).
  • Formation of Carbamoyl Phosphate:
    • This product then combines with ornithine to produce citrulline, notable as non-canonical amino acids (not part of genetic code).

Urea Cycle: Cytoplasmic Reactions

  • Citrulline Transport: Citrulline is transported to the cytoplasm in exchange for ornithine.

  • Argininosuccinate Synthetase Activity: Combines citrulline and aspartate, using ATP hydrolysis for the reaction.

  • Argininosuccinate then undergoes conversion to produce arginine and fumarate, resulting in regeneration of ornithine.

Energetics of the Urea Cycle

  • The incorporation of two NH4+ (including aspartate) into urea requires the investment of four phosphate bonds (converting three ATP to two ADP and one AMP).
  • Fumarate produced in the cycle can enter the citric acid cycle where it can lead to:
    • Synthesis of glucose (gluconeogenesis), or
    • Conversion to aspartate via transamination.

Clinical Insights: Defects in the Urea Cycle

  • Defects across any urea cycle steps are typically lethal due to the absence of alternative pathways for urea formation.
  • Such defects result in hyperammonemia (elevated NH4+ in the bloodstream), impacting approximately one in every 15,000 births.
    • Alcohol Consumption Impact: Excessive consumption can lead to hyperammonemia due to increased NADH production, which damages liver tissue and can cause cirrhosis.

Alternative Strategies for Nitrogen Disposal

  • Most terrestrial vertebrates excrete nitrogen as urea (ureotelic).
  • Aquatic animals tend to release nitrogen in the form of ammonium (ammoniotelic) due to rapid dilution capabilities.
  • Birds eliminate nitrogen as uric acid (uricotelic), enabling them to excrete it in a more energy-demanding manner (as pellets or paste).
  • Notable Behavior in Hibernators: Bears produce urea even while hibernating, which is released into their intestines for bacterial utilization.

Processing of Amino Acid Carbon Skeletons

  • General Conversion Process: Carbon skeletons are transformed into various key metabolic intermediates, including:
    • Pyruvate
    • Acetyl CoA
    • α-ketoglutarate
    • Succinyl CoA
    • Fumarate
    • Oxaloacetate
  • Some carbon skeletons can function in gluconeogenesis (precursors for glucose) or are classified as ketogenic (convert to ketones).

Example Cases in Amino Acid Conversion

  1. Example of Direct Conversion:
    • Alanine and aspartate transaminases directly convert to pyruvate and oxaloacetate, respectively.
  2. Glutamate Conversion:
    • Several five-carbon amino acids are turned into glutamate for further entry into the citric acid cycle.
    • Histidine conversion relies on tetrahydrofolate.
  3. Branched-Chain Amino Acid Degradation:
    • Degradation leads to acetyl CoA, acetoacetate, and succinyl CoA through several enzymatic reactions.
  4. Aromatic Amino Acids:
    • Require molecular oxygen for degradation; for example, phenylalanine hydroxylase is involved in converting phenylalanine to tyrosine.

Defects in Amino Acid Degradation

  • Phenylketonuria (PKU):
    • Resulting from defects in phenylalanine hydroxylase, leading to cognitive disabilities and severe health outcomes if untreated.
    • Occurrence is approximately 1 in 10,000 births.
    • Main treatment involves dietary restrictions of phenylalanine.

Summary of Key Concepts

  • Cells lack mechanisms to store excess amino acids.
  • Key enzymes in amino acid degradation include:
    • Aminotransferases
    • Dehydrogenases
    • Dehydratases
  • Essential for understanding the metabolic role and transformation of amino acids into relevant substrates post-nitrogen removal.
  • Understanding the urea cycle along with its energetics and links to gluconeogenesis is critical for comprehending nitrogen metabolism.
  • Recognize gluco- and ketogenic amino acids and respective pathways, along with genetic defects impacting amino acid metabolism.