Metabolic Biochemistry: Nitrogen Metabolism and the Urea Cycle

Nitrogen Metabolism

Overview of Nitrogen Metabolism

• Humans depend on other organisms to convert atmospheric nitrogen into usable forms.
• $N_2$ is unusable by most organisms due to the triple bond between nitrogen atoms (bond energy of $930kJ/mol$).
• Nitrogen must be "fixed" into ammonium $(NH<em>4)$ or nitrate $(NO</em>3)$ ions for biological use.
• Nitrogen fixation is performed by bacterial nitrogenases, forming reduced nitrogen $(NH_4^+)$, used by organisms to form amino acids.

Nitrogen Fixation

• Atmospheric nitrogen is reduced by adding hydrogen atoms, breaking down the bonds between nitrogen atoms ($N_2 → N ≡ N$).
• Equation: $N<em>2 + 3H</em>2 → 2NH_3$
• Nitrogen fixation requires:
  • Nitrogenase complex (enzyme system).
  • A strong reducing agent (dinitrogenase reductase).
  • ATP to transfer hydrogen atoms to dinitrogen.
• Dinitrogenase needs eight high-potential electrons from reduced ferredoxin, generated by photosynthesis or oxidative processes.
• Two ATP molecules are hydrolyzed for each electron transferred; at least 16 ATP molecules are hydrolyzed per $N_2$ molecule reduced.
• Overall reaction:
  $N<em>2 + 10H^+ + 8e^- + 16ATP → 2NH</em>4^+ + 16 ADP + 16Pi + H_2$

Nitrogen Cycle

• The nitrogen cycle involves the conversion of nitrogen between various chemical forms. Key components include:
  Nitrogen ($N<em>2$), Ammonia ($NH</em>3$ and $NH<em>4$), Nitrate ($NO</em>3$).

Amino Acids

• Amino acids are the building blocks of proteins.
• They are precursors for biologically active molecules like neurotransmitters, local mediators, energy-related metabolites, heme, and DNA bases (purines).
• Amino acids serve as an energy source during fasting, diabetes, and high-protein diets.
• Some amino acids regulate gene expression and cellular signaling.

From Amino Acids to Proteins

• About 300 types of amino acids exist in nature, but only 20 are protein building blocks.
• Proteins are polypeptides with > 20-30 amino acids joined by peptide bonds.
• The chemical properties of amino acids determine a protein's biological activity.
• Proteins catalyze reactions and control cellular processes.

Amino Acid Classification

• Amino acids are classified into three groups:
  • Essential amino acids.
  • Nonessential amino acids.
  • Conditional amino acids.

Essential vs. Nonessential Amino Acids

• Essential amino acids: Valine, Methionine, Histidine, Leucine, Phenylalanine, Threonine, Isoleucine, Lysine, Tryptophan.
• Nonessential amino acids: Alanine, Asparagine, Aspartate, Glutamate.
• Conditional amino acids: Arginine, Cysteine, Glutamine, Glycine, Proline, Serine, Tyrosine, Ornithine.

Dietary Sources of Amino Acids

• Animal sources: meats, dairy products, fish, and eggs.
• Vegan sources: whole grains, pulses, legumes, soy, and nuts.

Dietary Sources of Essential Amino Acids

• Histidine: soy protein, eggs, parmesan, sesame, peanuts
• Isoleucine: eggs, soy protein & tofu, whitefish, pork, parmesan
• Leucine: eggs, soy protein, whitefish, parmesan, sesame
• Lysine: eggs, soy protein, whitefish, parmesan, smelts
• Methionine: eggs, whitefish, sesame, smelts, soy protein
• Cysteine: eggs, soy protein, sesame, mustard seeds, peanuts
• Phenylalanine: eggs, soy protein, peanuts, sesame, whitefish
• Tyrosine: soy protein, eggs, parmesan, peanuts, sesame
• Threonine: eggs, soy protein, whitefish, smelts, sesame
• Tryptophan: soy protein, sesame, eggs, winged beans, chia seeds
• Valine: eggs, soy protein, parmesan, sesame, beef

Digestion of Dietary Proteins

• In the stomach (pH 1-2), pepsin breaks down dietary proteins into polypeptides and amino acids.
• In the small intestine (pH 7), pancreatic enzymes (elastase, carboxypeptidase, trypsin, chymotrypsin) further digest polypeptides into oligopeptides and amino acids.
• Aminopeptidases continue the digestion into amino acids.

Protein Degradation

• Protein digestion occurs via enzymes secreted by the pancreas.
• Free amino acids and dipeptides are absorbed by epithelial cells; dipeptides are hydrolyzed in the cytosol before entering portal circulation.
• Amino acids transport into cells via active transporters.

Amino Acid Composition of Proteins

• The amino acid composition affects protein properties:
  • Size (molecular weight) depends on the number and size of amino acids.
  • Charge (isoelectric point) depends on the proportion of basic and acidic amino acids.
  • Hydrophobicity depends on the levels of hydrophobic and hydrophilic amino acids.

Basic Structure of an Amino Acid

• All amino acids have a basic structure, differing only in the R-group or side chain.
• Glycine is the simplest amino acid with a hydrogen (H) as its R-group.

Biosynthesis of Amino Acids

• Amino acid biosynthesis pathways are diverse.
• Carbon skeletons come from intermediates of glycolysis, pentose phosphate pathway, or citric acid cycle.
• Amino acids are grouped into six biosynthetic families based on these starting materials.

Biosynthesis of Amino Acids - Starting Materials

• Oxaloacetate: Aspartate, Asparagine, Methionine, Threonine, Lysine, Isoleucine.
• α-Ketoglutarate: Glutamate, Glutamine, Proline, Arginine.
• 3-Phosphoglycerate: Serine, Cysteine, Glycine.
• Pyruvate: Alanine, Valine, Leucine.
• Phosphoenolpyruvate + Erythrose 4-phosphate: Phenylalanine, Tyrosine, Tryptophan.
• Ribose 5-phosphate: Histidine.

Synthesis of Amino Acids

• Amino acids are synthesized primarily by amination and transamination.

Amination

• Ammonia reacts with α-ketoglutaric acid to form glutamic acid.
• $NH<em>4^+ + α-ketoglutarate + NADPH + H^+ → L-glutamate + NADP^+ + H</em>2O$

Transamination

• The α-amino group of most amino acids comes from glutamate.
• Glutamic acid forms other 17 amino acids through transamination.
• Amino acids consist of a carboxyl group (-COOH) and one or more amino groups (-NH2).
• Transamination transfers the amino group from one amino acid to the keto group of a keto acid. Transaminase is the enzyme responsible.

Example of Transamination

• α-Ketoglutarate + L-Amino acid
• Pyridoxal phosphate (PLP) is an intermediate carrier of amino groups.

Nitrogen Storage

• Humans cannot store nitrogen like fats and carbohydrates.
• The recommended protein intake to maintain nitrogen balance is about 50g per day.

Protein Turnover

• Protein turnover involves the constant degradation and re-synthesis of proteins in cells.
• Muscle proteins last about three weeks, while liver enzymes turn over in a couple of days.
• Some regulatory enzymes have half-lives measured in hours or minutes.

Protein Turnover

• Most amino acids released during protein degradation are re-incorporated into new proteins.
• Net protein synthesis uses less than one-third of dietary amino acid intake.
• Most ingested protein is oxidized for energy.
• Surplus nitrogen is excreted, mainly as urea.

Nitrogen Balance

• Nitrogen Balance = Nitrogen intake - Nitrogen loss.
• Nitrogen intake sources: meat, dairy, eggs, nuts, legumes, grains, and cereals.
• Nitrogen loss examples: urine, feces, sweat, hair, and skin.

Nitrogen Balance

• Dietary protein intake is balanced against nitrogen losses from the body.
• Nitrogen equilibrium: intake = output.
• Positive nitrogen balance: intake > output.
• Negative nitrogen balance: intake < output.

Amino Acid Deficiency

• Amino acid deficiency can lead to malnutrition and health problems.

Kwashiorkor

• A severe form of protein-energy malnutrition with sufficient calorie intake but insufficient protein consumption.

Marasmus (PEM) vs. Kwashiorkor

• Marasmus: Severe deficiency of all nutrients and inadequate caloric intake.
  • Absent peripheral edema and hair changes.
  • Dry and wrinkled skin without dermatosis.
  • Voracious appetite.
  • Absent subcutaneous fat.
  • Uncommon fatty liver.
  • Better prognosis.
• Kwashiorkor: Severe protein deficiency but normal caloric intake.
  • Present peripheral edema and hair changes.
  • Dermatosis with flaky paint appearance of skin.
  • Poor appetite.
  • Reduced subcutaneous fat.
  • Common fatty liver.
  • Worse prognosis.

Kwashiorkor

• Literal translation: "the sickness the baby gets when the new baby comes."
• Develops after weaning from breast milk onto a high-carbohydrate, protein-deficient diet.
• Symptoms: edema of hands and feet, irritability, anorexia, rash, hair discoloration, and fatty liver.
• Treatment: gradual reintroduction of protein into the diet.

Fate of Amino Acids

• Excess amino acids cannot be stored.
• The α-amino group is removed, and the carbon skeleton is converted into a major metabolic intermediate.
• Most amino groups of surplus amino acids are converted into urea through the urea cycle.

Transport of Nitrogen

• Nitrogen is transported from muscle to the liver, either as glutamine or alanine.
• Nitrogen in the liver is converted to urea, which is then transported to the kidneys before excretion.

Glucose-Alanine Cycle

• Alanine carries amino groups from muscle to the liver.
• In muscle amino group of glutamate is transferred to pyruvate to form alanine by the action of alanine aminotransferase, which is released into the blood.
• The liver takes up the alanine and converts it back into pyruvate by transamination. The pyruvate can be used for gluconeogenesis and the amino group eventually appears as urea. This transport is referred to as the alanine cycle.
• Requires 6 ATP in the liver and generates 2 ATP in the muscle, for a net consumption of 4 ATP.

Amino Acid Supplements

• Protein supplements are often consumed by bodybuilders to:

  • Improve performance and training
  • Promote muscle growth
  • Reduce muscle breakdown
  • Increase resistance to fatigue during exercise
  • Support fat loss.

• Little scientific support for these claims.

• Liver damage from supplements has tripled, often involving bodybuilding supplements with unlisted steroids.

Summary of Part I

• Animals need nitrogen as a vital component of amino acids.
• Atmospheric nitrogen is inert and needs to be ‘fixed’ as ammonia ($NH<em>3$) or ammonium ion ($NH</em>4^+$).
• Nitrogen balance is the balance between nitrogen intake and output.
• Amino acids are synthesized by transamination.
• Amino acids are transported via glutamine and alanine.

Part 2: The Urea Cycle

Objectives for Part 2

• Explain the fate of amino acids.
• Understand the urea cycle.
• Explain the catabolism of carbon skeletons.
• Explain key diseases related to the urea cycle.

Amino Acids Metabolism: Disposal of Nitrogen

• Catabolism of amino acids has two phases:
  • Phase I: Removal of α-amino group, forming $NH_4^+$ and α-keto acid via transamination and oxidative deamination.
    • $NH_4^+$ is recycled in biosynthesis or excreted as urea.
  • Phase II: Carbon skeleton of the α-keto acids are converted into common intermediates of energy producing metabolic pathway.
    • Carbon skeletons are transformed into acetyl CoA, acetoacetyl CoA, pyruvate, or citric acid cycle intermediates.
    • Fatty acids, ketone bodies, and glucose can be formed from amino acids.

Amino Acids Metabolism: Oxidative Deamination

• α-amino groups of amino acids are transferred to α-ketoacids, catalyzed by transaminases.
• The reaction is catalyzed by glutamate dehydrogenase, with $NAD^+$ and $NADP^+$ as cofactors.

Amino Acid Catabolism

• Most amino acid degradation occurs in tissues other than the liver (e.g., muscle during exercise and fasting).

Amino Acid Catabolism - Removal of Nitrogen

• The first step is the removal of nitrogen from the amino acid.
• Nitrogen is released and converted into urea by the liver.
• Nitrogen is transported from muscle to the liver as glutamine and alanine.
• Excess nitrogen is transported to the kidney and excreted as urea.

The fate of $N_2$

• Urea: 86%
• Creatinine: 5%
• $NH_4^+$: 3%
• Other: 6%

Urea Cycle

• In most terrestrial vertebrates, excess $NH_4^+$ is converted into urea and excreted.
• The urea cycle is the central pathway in nitrogen metabolism.
• Proposed by Hans Krebs and Kurt Henseleit in 1932.
• Purpose: remove ammonia by converting it to a less toxic substance (urea).

The Urea Cycle

• A cyclic pathway in the liver that converts ammonia into urea.
• Occurs in both the mitochondrial matrix and the cytosol.

Reactions of the Urea Cycle

• Extra-hepatic tissues export their surplus nitrogen to the liver principally as the amino acids alanine and glutamine.

The Urea Cycle Begins

• Ammonia generated in the liver mitochondria reacts with $CO_2$ produced by mitochondrial respiration to form carbamoyl phosphate in the matrix which is catalyzed by carbamoyl phosphate synthetase 1.

Carbamoyl Phosphate and Ornithine React to form Citrulline

• Carbamoyl phosphate and ornithine react to form citrulline, catalyzed by ornithine transcarbamylase.

Citrulline and Aspartate Combine to form Arginosuccinate

• Citrulline and aspartate combine to form arginosuccinate, catalyzed by argininosuccinate synthetase which requires ATP.

Cleavage of Arginosuccinate to form Arginine & Fumarate

• Arginosuccinate is cleaved to form arginine and fumarate, catalyzed by argininosuccinate lyase.

Cleavage of Arginine Releases Urea and re-forms Ornithine

• Arginine is cleaved to release urea and reform ornithine, catalyzed by arginase.

The Urea Cycle Is Linked to the Citric Acid Cycle

• The urea cycle is linked to the citric acid cycle through fumarate, which is an intermediate in both cycles.

The Urea Cycle

• A cyclic pathway in the liver that converts ammonia into urea.
• Occurs in both the mitochondrial matrix and the cytosol.

Fate of Amino Acids - Carbon Skeletons

• Glucogenic: Intermediates for glycolysis or used to make glucose.
• Ketogenic: Ketone bodies or intermediates in citric acid cycle.
• Uric Acid
• A couple of Amino Acids can do both.

Fate of Amino Acids

• Diagram showing the catabolic fates of the carbon skeletons of amino acids, converging on various metabolic intermediates.

Fate of Amino Acids

• Glucogenic: Alanine, Asparagine, Aspartate, Cysteine, Glutamate, Glutamine, Glycine, Proline, Serine, Arginine, Histidine, Methionine, Valine.
• Glucogenic and Ketogenic: Isoleucine, Threonine, Tyrosine, Tryptophan, Phenylalanine.
• Ketogenic: Leucine, Lysine.

Genetic errors of amino acid catabolism

• Phenylketonuria
  • Caused by a block in the conversion of phenylalanine into tyrosine.
  • Readily diagnosed.
  • Treated by removing phenylalanine from the diet.

Defects in the Urea Cycle

• All defects lead to an elevated level of $NH_4^+$ in the blood (hyperammonemia).
• Blockage of carbamoyl phosphate synthesis or of any of the four steps of the urea cycle has devastating consequences because there is no alternative pathway for the synthesis of urea.

Urea cycle disorders (UCDs)

• A urea cycle disorder is a genetic disorder caused by a mutation that results in a deficiency of one of the enzymes in the urea cycle.
• In milder/partial urea cycle enzyme deficiencies, ammonia accumulation may be triggered by illness or stress at almost any time of life, resulting in multiple mild elevations of plasma ammonia concentration; the hyperammonemia is less severe and the symptoms may be subtle.
• In individuals with partial enzyme deficiencies, the first recognized clinical episode may be delayed for months or years.
• The severity of the disorder depends which part of the last reaction of the urea cycle.

Urea cycle disorders (UCDs)

• Based on clinical, biochemical, and genetic data
• A plasma ammonia concentration of 150 mmol/L or higher is a strong indication for the presence of a UCD.
• Plasma quantitative amino acid analysis can be used to diagnose a specific urea cycle disorder
• Liver biopsy

Urea cycle disorders - Treatment

• Balancing dietary protein intake so body receives the essential amino acids responsible for cell growth and development, but not so much protein that excessive ammonia is formed.
• Medications (ie sodium phenylbutyrate) to provide alternative pathways for the removal of ammonia from the blood.
• Amino acid formulas developed specifically for urea cycle disorders.
• At the most extreme end of the spectrum, liver transplants.

Urea cycle disorders - Acquired

• Liver disease such as cirrhosis or hepatitis
• High Protein Diet

Uric Acid

• Uric acid is a product of the metabolic breakdown of purine nucleotides (Adenine and Guanine).
• Consequence of making too much Uric Acid (hyperuricemia)….
• Uric acid is not water soluble
• Uric Acid crystals form
• These travel to the lowest part of the body (toes and feet)
• Results in Gout

Summary of Part II

• Catabolism of amino acids
• Urea cycle
• Catabolism of carbon skeletons
• Diseases related to urea cycle