Amino Acid Nitrogen Disposal

• Amino acid nitrogen is disposed of in the body

Learning Objectives

• Protein turnover and degradation
• Protein digestion
• Ammonia transfer to the liver
• Urea cycle

Overview

• Lipids are stored as triacylglycerols (TAG) in adipose tissue
• Glucose is stored as glycogen in muscle and liver
• Proteins are degraded to amino acids, then to ammonia and alpha-keto acids
• Transamination or oxidative deamination can degrade proteins
• Free ammonia is excreted in urine, with the remainder used to synthesize urea

Nitrogen Metabolism

• Nitrogen enters the body through food, predominantly as amino acids
• Amino acids come from dietary sources and the breakdown of body proteins
• Nitrogen balance occurs when input to the amino acid pool equals output in healthy individuals

Protein Turnover

• Protein turnover is the replacement of degraded proteins at a sufficient rate by protein synthesis
• A 70 kg man has an average protein turnover rate of 300-400 g per day
• The body amino acid pool is always in a dynamic steady state
• Short-lived proteins are rapidly degraded, while long-lived proteins may take days to weeks
• Structural proteins have half-lives from months to years
• Insulin, androgen, and estrogen have an anabolic effect on protein turnover
• Glucagon and cortisol have a catabolic effect

Protein Degradation

• ATP-dependent ubiquitin-proteasome and ATP-independent lysosomal systems are the two major enzyme systems for protein degradation
• The proteasome system selectively degrades damaged or short-lived proteins
• Lysosomes use acid hydrolases to non-selectively degrade proteins

Lysosomal Protein Breakdown

• Lysosomal protein breakdown occurs at an acidic pH
• Various hydrolyses, including proteases and nucleases, participate in the process
• Degradation of extra/intracellular fragments and phagocytosed microorganisms occurs
• Protein uptake occurs via endocytosis, autophagy, and phagocytosis

Ubiquitin-Dependent Protein Breakdown

• Ubiquitin-dependent protein breakdown occurs at a neutral pH
• It is an ATP-dependent degradation process
• This process is significant for digesting proteins programmed for quick destruction, misfolded proteins, denatured proteins, and abnormal proteins

Chemical Signs for Protein Degradation

• Proteins are degraded at different times due to their different half-lives
• Proteins chemically altered by oxidation or tagged with ubiquitin are preferentially degraded
• Proteins with serine in the N-terminal have a half-life of 20 hours, while proteins with aspartate have a half-life of 3 minutes
• Proline, glutamate, serine, and threonine (PEST) sequence-rich proteins are rapidly degraded

Digestion of Dietary Proteins

• Proteolytic enzymes are secreted as inactive zymogens
• Zymogens are converted to their active form in the intestinal lumen
• Cooking denatures dietary proteins, making them more easily digested
• Proteolytic enzymes are produced by the stomach, pancreas, and small intestine

Proteolytic Enzymes

• Endopeptidases act on peptide bonds inside the protein molecule (e.g., pepsin, trypsin, chymotrypsin, and elastase)
• Exopeptidases act on the peptide bond only at the end region of the chain (e.g., carboxypeptidase and aminopeptidase)

Digestion by Gastric Secretion

• Digestion begins in the stomach
• Hydrochloric acid (HCl) in the stomach activates pepsin to hydrolyze peptide bonds
• HCl kills bacteria and denatures proteins
• Pepsin (chief cells) is an endopeptidase secreted as zymogen
• Pepsinogen is activated by HCl

Digestion by Pancreatic Enzymes

• The optimum pH for pancreatic enzyme activity (pH 8) is provided by alkaline bile and pancreatic juice
• Pancreatic juice contains the endopeptidases trypsin, chymotrypsin, elastase, and carboxypeptidase
• These enzymes are secreted as zymogens (trypsinogen, chymotrypsinogen, and pro-elastase) to prevent the pancreatic acinar cells from being autolyzed
• Cholecystokinin and secretin mediate the release of zymogens, which are polypeptide hormones

Activation of Zymogens

• Trypsinogen is activated by enterokinase (enteropeptidase) on the luminal surface of intestinal mucosal cells of the brush border membrane
• Trypsin is activated by removing a hexapeptide from the N-terminal end
• Once activated, trypsin activates other enzyme molecules

Digestion of Oligopeptides by Enzymes of the Small Intestine

• Aminopeptidases and an exopeptidase cleave N-terminal residues
• These cause oligopeptides to produce even smaller peptides and free amino acids

Abnormalities in Protein Digestion

• Incomplete digestion and absorption of protein and fat can occur in individuals with a deficiency in pancreatic secretion (chronic pancreatitis, cystic fibrosis, or surgical removal of the pancreas)
• Abnormal lipid appearance (steatorrhea) and proteins in feces can be seen
• Celiac disease is a malabsorption of protein gluten due to immune-mediated damage of the small intestine
• Gluten is found in wheat, barley, and rye

Transport of Amino Acids into Cells

• Amino acid absorption occurs mainly in the small intestine
• The proximal tube of the kidney also has common transport
• It is an energy-requiring process, with transport systems that are carrier-mediated and/or ATP sodium-dependent
• There are 5 different carriers for amino acids: neutral, basic, imino acids and glycine, acidic, and beta amino acids

Cystinuria

• Cystinuria is a common genetic error of amino acid transport
• It is a disorder of the proximal tubule's reabsorption of filtered cystine and dibasic amino acids (ornithine, arginine, and lysine)
• Accumulation of cystine leads to the precipitation of cysteine to form kidney stones

Removal of Nitrogen from Amino Acids

• The alpha-amino group keeps amino acids locked away from oxidation
• Removing the amino group is essential for producing energy from amino acids
• Once removed, nitrogen can be incorporated into other compounds or excreted as urea
• Transamination and oxidative deamination are two ways to remove nitrogen

Transamination: Funneling of Amino Groups to Glutamate

• Amino acids are degraded in the liver
• An amino group is transferred from an amino acid to an alpha-keto acid, usually alpha-ketoglutarate
• The reaction is catalyzed by a transaminase or aminotransferase
• A new amino acid, usually glutamate, and a new alpha-keto acid are formed
• Glutamate produced by transamination can be oxidatively deaminated or used as an amino donor for the synthesis of non-essential amino acids
• Almost all amino acids participate in transamination

Oxidative Deamination

• Liver mitochondria contain glutamate dehydrogenase (GDH)
• GDH deaminates glutamate to alpha-keto-glutarate plus ammonia
• Amino acids are first transaminated to glutamate, which is then deaminated (transdeamination)
• These reactions primarily occur in the liver and kidney, and provide alpha-keto-glutarate to be used in different pathways

Transfer of Ammonia to the Liver

• Ammonia is highly toxic and should be eliminated or detoxified when formed
• Ammonia is produced by almost all cells, including neurons
• Intracellular ammonia is trapped by the combination of NH3 with Glutamate to form Glutamine
• Glutamine is transported to the liver while the reaction is reversed by the glutaminase enzyme
• Glutamate dehydrogenase is available primarily in the liver
• Alanine is formed by the transamination of pyruvate
• The Glucose-Alanine cycle involves transporting Alanine to the liver, where is converted to pyruvate then later to glucose. The resulting glucose is then sent to muscle tissue

Urea Cycle

• Urea is the major disposal form of amino groups

• It accounts for 90% of the nitrogen-containing components of urine

• The urea cycle is the sole source of endogenous production of arginine

• Urea formation takes place in the liver, while excretion occurs through the kidney

• Provides 25-30 g of urea daily for urine formation in the kidneys

• 6 amino acids participate in urea formation: Ornithine, Citrulline, Aspartic acid, Arginosuccinic acid, Arginine, and N-Acetyl-Glutamate

• Urea synthesis is a cyclic process

• The first two reactions occur in the matrix of the mitochondrion

• The remaining reactions occur in the cytosol

• Five enzymes catalyze the reactions of the urea cycle

• There is no net loss or gain of Ornithine, Citrulline, argininosuccinate, or arginine because Ornithine consumed in the 2nd reaction is regenerated in the last reaction

• Ammonium ion, CO2, ATP, and aspartate are, however, consumed

Step 1: Formation of Carbamoyl Phosphate

• This step occurs in the mitochondria when ammonium ion reacts with CO2 from the citric acid cycle
• Ammonia is primarily provided by the oxidative of glutamate by glutamate dehydrogenase
• Carbamoyl phosphate synthetase 1 (CPS1) is strongly activated by N-acetyl glutamate, which controls the rate of urea production

Step 2: Formation of Citrulline

• The carbamoyl group of carbamoyl phosphate is transferred to ornithine
• The transport of ornithine into the mitochondria and citrulline out of the mitochondria involves mitochondrial inner membrane transport systems
• Citrulline is continuously exported, and ornithine is taken up across the inner mitochondrial membrane

Step 3: Formation of Arginosuccinate

• Argininosuccinate synthase (ASS) links L-Aspartate and citrulline via the amino group of aspartate-providing the second nitrogen of urea
• The reaction requires ATP, and the third and last ATP in the cycle is used in this step

Step 4: Cleavage of Arginosuccinate

• Argininosuccinate lyase (ASL) catalyzes the cleavage of argininosuccinate
• The cleavage proceeds with the retention of nitrogen in arginine and the release of the aspartate skeleton as fumarate

Step 5: Cleavage of Arginine

• Liver arginase (ARG1) catalyzes the hydrolytic cleavage of the guanidino group of arginine, releases urea,

• The other product, ornithine, reenters liver mitochondria for additional rounds of urea synthesis

• Both Ornithine and lysine competitively inhibit arginase with arginine

• Water addition to fumarate forms L-malate, which can enter the TCA cycle

• L-malate can be oxidized to oxaloacetate for gluconeogenesis

• Oxaloacetate can be converted to aspartate (transamination)

Fate of Urea

• Urea diffuses from the liver and is translocated in the blood
• The blood is headed to the kidneys, where it filters, while a portion diffuses from blood to intestines where it is cleaved to CO2 and NH3
• In patients who experience kidney failure, they will likely have increased levels of plasma urea

Regulation of Urea Cycle

• N-acetylglutamate (NAG) is an essential activator for CPS I, a rate-limiting step
• The cycle is also regulated by substrate availability

Metabolism of Ammonia

• Sources of ammonia include glutamine, bacterial degradation of urea in the intestine, amines obtained from diet, and the degradation of purines and pyrimidines

• The transported forms of ammonia are urea and glutamine

• Hyperammonemia, a medical emergency, results from liver disease and genetic defects of the urea cycle

• It could be acquired or congenital

• Ammonia intoxication causes slurring of speech, cerebral edema, blurring of vision, coma, and death

• Arginase catalyzes the formation of free urea during the urea cycle

• N-acetyl glutamate is required to initiate the urea cycle

• Histidine is not involved in the urea cycle

• Carbamoyl phosphate and aspartate are the direct source of ammonia in the urea cycle