Biochemistry - 41 - The Urea Cycle 2023.pdf

The Urea Cycle Lecture 41 Reference: Lieberman and Peet, Chapter 36 Bluefield University – VCOM Campus MABS Program Biochemistry Jim Mahaney, PhD Learning Objectives a. Recall the relationship between ammonia and ammonium ion. Interpret how ammonia toxicity can arise in patients despite the very h...

The Urea Cycle Lecture 41 Reference: Lieberman and Peet, Chapter 36 Bluefield University – VCOM Campus MABS Program Biochemistry Jim Mahaney, PhD Learning Objectives a. Recall the relationship between ammonia and ammonium ion. Interpret how ammonia toxicity can arise in patients despite the very high pKa of ammonia. b. Recall the three sources of free amino acids for the body and the major uses for amino acids. c. Relate nitrogen balance for the body and relate negative versus positive nitrogen balances. d. Recall the two phases of amino acid catabolism and relate how free ammonia is minimized by the transamination reaction or the oxidative deamination reaction. Use the reactions of ALT, AST and glutamate dehydrogenase as examples. e. Relate the glucose-alanine cycle and the gluatamine cycle as major pathways for moving ammonia through the body and for delivery to the liver for excess nitrogen elimination. f. Relate the goal of the urea cycle and recall the source of amino groups for urea synthesis. Identify which amino acids “collect” nitrogen atoms and deliver them to the liver for the urea cycle. g. Recall the steps of the urea cycle pathway. Identify the organ that carries out the urea cycle and identify the cycle steps that take place in mitochondria and in the cell cytoplasm. h. Interpret the critical role of carbamoylphosphate synthetase I (CPS I) in decreasing blood ammonia levels and relate how defects in this enzyme would affect free ammonia / ammonium and blood urea nitrogen (BUN) levels. i. Identify the factors that regulate the urea cycle and interpret of how blood urea nitrogen (BUN) production changes depending on carbohydrate (glucose) consumption versus during fasting (short and long fasts). j. Compare and contrast acquired versus congenital urea cycle deficiency. 2 Nitrogen Metabolism in the Liver • Body protein is in a constant state of turnover through the activity of protein biosynthetic and degradative pathways. • Waste nitrogen is ammonia: an important metabolic intermediate, but toxic when levels get too high! • Ammonia plays a pivotal role in nitrogen metabolism. • Needed for biosynthesis of (but also derived from): • Nonessential amino acids • Nucleic acids • Major function of liver is nitrogen metabolism and ammonia management. Ammonia concentration in liver is 700 mmol/L; >10X higher than plasma 3 Ammonia (NH3/NH4+) Objective A • Waste nitrogen is ammonia: an important metabolic intermediate, but toxic when levels get too high! • The ammonium ion releases a proton to form ammonia by a reaction with a pKa of 9.3. • Therefore, at physiological pH (7.4), the equilibrium favors NH4+ by a factor of 100/1 • Ammonia plays a pivotal role in nitrogen metabolism. • Needed for biosynthesis of (also derived from) nonessential amino acids and other nitrogen-containing compounds • Ammonia (NH3) is produced by all tissues during the metabolism of a variety of compounds and is disposed of primary by the formation of urea in the liver • Cells in the body and bacteria in the gut release the nitrogen of amino acids as ammonia (NH3) because this is the form that can cross cell membranes • Major function of liver is ammonia metabolism (concentration in liver is 700 mmol/L (10X higher than plasma) • In the kidneys most of this ammonia is excreted into the urine as NH4+, which provides an important mechanism for maintaining the body's acidbase balance through excretion of protons 4 Nitrogen Intake: Amino Acids Objective B • Unlike fats and carbohydrates, amino acids are not stored in the body – rather there are free “pools” of amino acids in cells and in the blood. • Amino acids are obtained from: • Diet – dietary protein • Produced from normal protein degradation / turnover • Synthesized de novo • Amino acids are used for: • Protein synthesis • Nitrogen containing compounds (nitrogenous bases, catecholamines, etc) • Interconverted to other amino acids or catabolized for energy. • Any amino acids in excess of the biosynthetic needs of the body are converted to fat for energy storage or eliminated from the body. 5 Disposal of Nitrogen BIG Picture Summary Ultimate fate of excess nitrogen is urea synthesis The conversion of excess nitrogen to urea occurs mainly in the liver I. Removal of Nitrogen from Amino Acids • Transamination Reactions • Oxidative deamination of amino acids • Transport of ammonia to the liver II. Waste Nitrogen from Other Sources • Purine nucleotide cycle (brain, muscle) • Gut bacteria III. Urea Cycle • Regulation of the Urea Cycle • Urea Cycle Dysfunction 6 Nitrogen Balance Objective C The healthy human adult is in nitrogen balance which means the amount of nitrogen excreted (mainly through urine) equals the amount consumed (dietary protein). • “Negative” nitrogen balance – The amount of nitrogen excreted is greater than the amount consumed. – Associated with burns, tissue injury, wasting diseases, fevers, periods of fasting. Can be used as part of a clinical evaluation of malnutrition. • “Positive” nitrogen balance – The amount of nitrogen excreted is less than the amount consumed. – Associated with periods of growth, hypothyroidism, tissue repair, and pregnancy. • Typical of growing children: lots of amino acid and protein synthesis. 7 Amino Acid Catabolism Objective D 1st phase of amino acid catabolism • Removal of the α-amino group via transamination or oxidative deamination, converting the amino acid to an α-keto acid (carbon skeleton). If not transferred to another molecule, the amino group is released as ammonia. • Most free ammonia produced is used to synthesize urea, which is the most important route for disposing (excreting) nitrogen. 2nd phase of amino acid catabolism • Carbon skeletons of α-keto acids are converted to common intermediates of energy producing metabolic pathways • These compounds can be metabolized to CO2 and water, glucose, fatty acids, or ketone bodies by the central pathways of metabolism 8 Transamination Reactions aminotransferase Objective D • Obligatory 1st step in the catabolism of most amino acids • General Process: • The amino group from the original amino acid is transferred to α-ketoglutarate, forming glutamate, whereas the original amino acid is converted to its corresponding α-keto acid • All amino acids EXCEPT lysine and threonine have the ability to undergo transamination • Catalyzed by transaminases or aminotransferases aminotransferase • Each aminotransferase is specific for 1 or, at most, a few amino group donors • Aminotransferases are named after the specific amino group donor, b/c the acceptor is almost always α-ketoglutarate • Pyridoxal phosphate (PLP) is a required cofactor for all aminotransferases (derived from Vitamin B6) • Reactions are readily reversible (involved in amino acid synthesis and degradation) 9 ALT and AST Objective D • Aminotransferases are normally intracellular enzymes, with low levels found in the plasma • Elevated plasma levels of aminotransferases indicate damage to cells rich in these enzymes (e.g. physical trauma or disease) • 2 aminotransferases are of particular diagnostic value when they are found in plasma • ALT – alanine aminotransferase • AST – aspartate aminotransferase • Liver disease • Plasma AST and ALT are elevated in nearly ALL liver diseases • Particularly high in conditions that cause cell necrosis, such as severe viral hepatitis, chronic alcoholic cirrhosis, and prolonged circulatory collapse • Serial measurements of AST and ALT (so-called “liver function tests”) are useful in determining the course of liver damage 10 Oxidative Deamination of Amino Acids Objective D … Step 1: Transamination: the funneling of amino groups to glutamate • Step 2: Rapid oxidative deamination of glutamate • RESULT: the liberation of the amino group from glutamate as free ammonia • Glutamate is unique in that it is the only amino acid that undergoes rapid oxidative deamination • Reaction is catalyzed by glutamate dehydrogenase • Can use either NAD+ or NADPH as a coenzyme • NAD+ is primarily used in oxidative deamination (loss of ammonia coupled with the oxidation of the carbon skeleton) • NADPH is used in reductive amination (gain of ammonia coupled with the reduction of the carbon skeleton) • Direction of reaction depends on the relative concentrations of glutamate, αketoglutarate, ammonia, and the ratio of oxidized to reduced coenzymes • Ingestion of high protein meal à ↑glutamate levels.. Reaction proceeds in direction of amino acid degradation • ↑ammonia levels…. Reaction proceeds in direction to glutamate synthesis Objective d 11 Transport of Ammonia to the Liver Objective E Glucose-alanine cycle Glutamine cycle • Deamination of amino acids produces free NH4+ that must be reincorporated or eliminated. • Free NH4+ will lead to free NH3 Also from Kidney and Brain 12 The Urea Cycle Objective F • Urea is the major disposal form of amino groups derived from amino acids and accounts for about 90% of the nitrogen-containing components of urine. • Two amine groups are incorporated into one urea molecule: • 1 amino group is supplied by free ammonia • 1 amino group is supplied by aspartate • Glutamate is the immediate precursor of the free ammonia (through oxidative deamination) and aspartate carries an amino group from the transamination of oxaloacetate. • The carbon and oxygen of urea are derived from CO2 (as HCO3-) • Urea is produced in the liver and is then transported in the blood to the kidneys for excretion in the urine. 13 Reactions of the Urea Cycle Objective G • Step 1: Formation of carbamoyl phosphate • NH3, bicarbonate, and 2 ATP react to form carbamoyl phosphate (carbamoyl phosphate synthetase I – CPS I) • Step 2: Formation of citrulline • The carbamoyl portion of carbamoyl phosphate is transferred to ornithine by ornithine transcarbamoylase (OTC) as the high-energy phosphate is released (Pi) [Citrulline is transported across the mitochondrial membrane in exchange for cytoplasmic ornithine and enters cytosol] • Step 3: Synthesis of argininosuccinate • Argininosuccinate synthetase combines citrulline with aspartate to form argininosuccinate [driven by cleavage of ATP] • Step 4: Cleavage of argininosuccinate • Five steps [2 in mitochondrial matrix/ 3 in cytosol] • Urea is eliminated from the cell and eliminated from the body via urine • Goal: convert free NH3 to urea Location: Liver • Argininosuccinate is cleaved by argininosuccinate lyase to yield arginine and fumarate* • Step 5: Cleavage of arginine to ornithine and urea • Arginase hydrolyzes arginine to ornithine and urea 14 Carbamoylphosphate Synthetase I Objective H • Carbamoyl phosphate synthetase I (CPS-I) catalyzes the first step of the urea cycle. • “inducible” enzyme that can respond to the needs of the system. • Consumes a free ammonia molecule, thereby decreasing the free ammonia level in the liver. • High blood ammonia in newborns is often a sign of CPS-1 deficiency. • Notice the use of ATP: one to provide a phosphate group and the second to provide energy to drive the reaction. For later: CPS-II is an enzyme in pyrmidine synthesis that does the same reaction using glutamate to provide the amino group. 15 Overall Stoichiometry of the Urea Cycle Objective G Aspartate + NH3 + HCO3- + 3 ATP + H2O à urea + fumarate + 2 ADP + AMP + 2 Pi + PPi • Because 4 high energy phosphate bonds are consumed in the synthesis of each molecule of urea, the synthesis of urea is irreversible (large –ΔG) • 1 nitrogen of the urea molecule is suppled by free ammonia (NH3) while the other is supplied by aspartate • Glutamate is the intermediate precursor of both ammonia and aspartate nitrogen • In effect, both nitrogen atoms of urea arise from glutamate, which in turn gathers nitrogen from other amino acids 16 Regulation of the Urea Cycle Objective I • In general, the urea cycle is regulated by substrate availability • The ↑ rate of NH3 production, the ↑ rate of urea formation • “Feed-forward” regulation • 2 other types of regulation control • Allosteric activation of CPSI by N-acetylglutamate (NAG) • Induction/repression of urea-cycle enzyme synthesis 17 Regulation of the Urea Cycle Objective I • N-acetyl glutamate (NAG) is formed specifically to activate carbamoyl phosphate synthetase (CPSI) (no other known function) • NAG increases the affinity of CPSI for ATP • NAG is synthesized from glutamate and acetyl CoA by N-acetylglutamate synthase in a reaction that is activated by arginine • Thus, as arginine levels increase… *CPSI is the rate-limiting step of the urea cycle • ↑ NAG à ↑ rate of carbamoyl phosphate production • ↑ ornithine (via arginase) 18 Regulation of the Urea Cycle Objective I The induction of urea –cycle enzymes occurs in response to conditions that require increased protein metabolism (i.e. Highprotein diet or prolonged fasting) • In fasted state, the liver maintains blood glucose levels by using amino acids from protein degradation in gluconeogenesis • In summary: • Amino acid carbon is converted to glucose • Amino acid nitrogen is converted to urea • Urinary excretion of urea is high during fasting • Brain eventually begins using ketone bodies, sparing blood glucose (↓ muscle cleaved, ↓ urea) 19 Urea Cycle Dysfunction Objective J • In a normal system, the capacity of the hepatic urea cycle exceed the normal rates of ammonia generation (low levels of serum ammonia) • However, when liver function is compromised, due to genetic defects in urea cycle OR liver disease, blood ammonia levels can rise > 1,000µmol/l = hyperammonemia • Hyperammonemia is a medical emergency because ammonia has a direct neurotoxic effect on the CNS • ↑ plasma NH3 à ammonia intoxication à tremors, slurring of speech, somnolence (drowsiness), vomiting, cerebral edema, and blurring of vision • ↑↑ plasma NH3 à coma and death • 2 major types of hyperammonemia: acquired and congenital 20 Acquired Hyperammonemia Objective J • Liver disease is a common cause of hyperammonemia in adults and may be due to many conditions: • Hepatitis (inflammation of the liver) due to parasites, bacteria or viruses • Damage to liver by chronic alcohol consumption-metabolism • Damage to liver by toxic substances, e.g., acetaminophen overdose • As a result of liver disease and damage, portal blood is shunted directly into the systemic circulation and does not have access to the liver. • Therefore, the conversion of ammonia to urea is severely impaired, leading to elevated levels of ammonia 21 Congenital Hyperammonemia Objective J Defects in ANY urea-cycle enzyme lead to elevated ammonia (hyperammonemia) with the inability to synthesize urea (low BUN) • (1) CPS I: High blood NH3, Low blood orotate, citrulline, and arginine. • (2) Ornithine transcarbamoylase (OTC): High blood and urine orotic acid (orotate), high blood NH3, low citrulline and arginine. (most common defect) • (3) Argininosuccinate synthetase: Highly elevated blood citrulline, high blood NH3, low blood arginine • (4) Argininosuccinate lyase: Elevated blood citrulline, high NH3, high argininosuccinate, low blood arginine • (5) Arginase: high blood arginine, moderately high NH3 22 Urea Cycle Thought Problem Five newborn infants who appeared normal at birth developed hyperammonemia after 24 hrs. Given the following information, determine which urea cycle enzyme might be defective in each case. All infants had low levels of blood urea nitrogen (BUN). Normal citrulline levels are 10-20 µM. Some of these are easy to see… others are not obvious and need additional clinical data. This is just FYI. 23 Answers: What’s High, What’s Low? Infant I II III IV V Enzyme deficiency CPS I Argininosuccinate synthetase Arginase OTC Argininosuccinate lyase 24 Question Growing children often retain most of the nitrogen they consume in their diet to be used for growth and as a result, will eliminate very little nitrogen. Which of the following terms describes the nitrogen balance for this situation? a. b. c. d. e. Balanced nitrogen Negative balance Positive balance Congenital nitrogen balance deficiency Acquired nitrogen balance deficiency 25 Answer Growing children often retain most of the nitrogen they consume in their diet to be used for growth and as a result, will eliminate very little nitrogen. Which of the following terms describes the nitrogen balance for this situation? a. b. c. d. e. Balanced nitrogen Negative balance Positive balance Congenital nitrogen balance deficiency Acquired nitrogen balance deficiency 26 Thank You! 27