Amino Acid Catabolism and Urea Production PDF

Amino acid catabolism and urea production Marc A. Ilies, Ph. D. Lehninger - Chapter 18 [email protected]; lab 517, office 517A (Tu, Fr 3-5) For questions, comments please use the discu...

Amino acid catabolism and urea production Marc A. Ilies, Ph. D. Lehninger - Chapter 18 [email protected]; lab 517, office 517A (Tu, Fr 3-5) For questions, comments please use the discussion tool in Canvas ©MAIlies2024 1  Amino acids catabolism Amino acid catabolism (through oxidative degradation) occurs under the following circumstances: ► During normal protein degradation/synthesis (protein turnover) inside the cells; aminoacids released are recycled and reused into the new proteins synthesized; excess amino acids are catabolized ► When animal relies exclusively on meat as food (carnivore animals) ► When diet brings more amino acids than the normal body needs for making proteins; surplus is catabolized as amino acids cannot be stored ► During starvation and during uncontrolled diabetes mellitus, when carbohydrates are either exhausted or cannot be utilized (in case of diabetes) Digestion of dietary proteins: Enzymatic hydrolysis to amino acids Entry of food stimulates gastric mucosa cells to secrete gastrin Gastrin stimulates secretion of HCl by parietal cells and secretion of pepsinogen (zymogen) by chief cells; pepsinogen self-cleaves at low pH (1.5-2) to generate active pepsin Pepsin cuts protein into peptides in the stomach. Trypsin and chymotrypsin cut proteins and larger peptides into smaller peptides in the small intestine. Aminopeptidase and carboxypeptidases A and B in the small intestine degrade peptides into amino acids, which are absorbed  Amino acid catabolism Amino acids absorbed can be used for: 1. Making new proteins 2. Energy, through amino acid catabolism: – removal of amino group → NH4+ – entry into central metabolism (glycolysis, citric acid cycle) Amino acid are first converted to NH4+ and α-keto acids (C skeletons), which are catabolized further: ► excess amino acids are sent to the liver, where they are catabolized; this is also a form to “ship” ammonia/ NH4+ ► in the liver, ammonia/ NH4+ is reused, excess excreted directly or transformed in urea or uric acid and excreted ► a key role in nitrogen metabolism is played by glutamate, glutamine, alanine and aspartate (the ones easily converted into TCA intermediates: Glu, Gln → α-ketoglutarate, Ala pyruvate, Asp → oxaloacetate Amino acid catabolism Krebs Bicycle  Fates of Nitrogen in Organisms Plants conserve almost all the nitrogen Many aquatic vertebrates release ammonia to their environment. – passive diffusion from epithelial cells – active transport via gills Many terrestrial vertebrates and sharks excrete nitrogen in the form of urea. – Urea is far less toxic that ammonia – Urea has very high solubility in water Some animals such as birds and reptiles excrete nitrogen as uric acid. – Uric acid is rather insoluble. – Excretion as paste allows the animals to conserve water. Humans and great apes excrete both urea (from amino acids) and uric acid (from purines). Step 1: Removal of the Amino Group Free ammonia is toxic, it cannot be released this way from catabolized amino acids Ammonia is captured by a series of transamination reactions Transaminations allow transfer of an amine to a common metabolite (e.g., α-ketoglutarate) and generate glutamate  Enzymatic Transamination Catalyzed by aminotransferases (also named transaminases) Uses the pyridoxal phosphate cofactor (PLP, vitamin B6) Typically, -ketoglutarate accepts amino groups, generating L-glutamate Vit B6/Pyridoxal Phosphate L-Glutamate acts as a temporary storage of nitrogen L-Glutamate can donate back the amino group when needed for amino acid biosynthesis (reaction can occur in reverse when needed) 8  Enzymatic transamination: ping-pong mechanism COO COO COO COO H 3N C H H C NH 3 + C O O C R1 CH2 CH2 R1 + CH2 L--amino acid CH2  - keto acid COO COO L-glutamate - ketoglutarate  - amino acid  - keto acid Exemplified for alanine catabolism: Glutamate-Pyruvate Transaminase (GPT) COO COO or Step 1 H 3N CH3 C H Alanine Transaminase (ALT) O C CH3 Pyruvate Alanine  - amino acid  - keto acid N H3 H Enz-Lysine C O CH2 HO OPO3 O PO3 H3C N H 3C N H Pyridoxal Phosphate H Pyridoxamine Phosphate (PLP) Vit B6 COO COO H C NH3 C O Glutamate-Pyruvate Transaminase (GPT) CH2 or CH2 CH2 Alanine Transaminase (ALT) CH2 COO COO Step 2 L-glutamate  - amino acid - ketoglutarate  - keto acid 9  Transaminases and liver function tests - Alanine transaminase ALT(also known as glutamate-pyruvate transaminase GPT) and aspartate transaminase AST (also known as glutamic-oxaloacetic transaminase GOT) are often measured in the serum in order to assess liver function. These liver enzymes are one component of a group of lab tests known as LFTs (liver function tests). - Sometimes lab liver enzyme results are presented as SGOT or SGPT (S = serum) - Elevated levels of ALT, AST indicate liver dysfunctions (cirrhosis, hepatitis) COO COO COO Aspartate COO Transaminase C O H C NH3 H3N C H C O (AST or GOT) CH2 CH2 CH2 CH2 PLP C O CH2 C O CH2 COO OH COO OH Aspartate a-ketoglutarate Oxaloacetate L-glutamate -amino acid -keto acid -keto acid -amino acid 10  Ammonia is safely transported in the bloodstream as glutamine - the ammonia is normally recycled (reused) inside the cell; excess ammonia is transported to the liver (and to a lower extent to specialized cells in small intestine, kidneys) via glutamine, to be transformed into urea: from tissues transport to liver via blood  Ammonia collected in glutamate is further removed by glutamate dehydrogenase in mitochondria matrix Oxidative deamination occurs within mitochondrial matrix. Can use either NAD+ or NADP+ as electron acceptor Ammonia is processed into urea for excretion. Pathway for ammonia excretion; transdeamination = transamination + oxidative deamination Transaminases and liver function tests 13  Ammonia toxicity BUN: Blood Urea Nitrogen – measures total nitrogen (under various forms e.g. urea, NH4+, uric acid, etc) Azotemia (excessive levels of nitrogenous materials in the blood (high BUN), Azoturia (high levels of nitrogenous materials in urine), usually due to kidney failure NH4+ is not permeable through membranes but is in equilibrium with NH 3 (neutral, can pass through the membranes) Brain is very sensitive to NH3 because excess NH3 is converted into glutamate, an excitatory neurotransmitter, via glutamate dehydrogenase; excess glutamate can be normally converted into glutamine by astrocytes, but in case of liver failure (due to serious dysfunctions, cirrhosis, hepatitis) excess glutamate builds up in neurons; part of it is converted into GABA (inhibitory neurotransmitter, modulates muscle tone) excess glutamate cause Asterixis (flapping tremor, liver flap) and coma - Treatment of asterixis: administration of lactulose (galactose+fructose disaccharide) which forms organic acids (RCOO-) in the colon that bind excess NH4+ and eliminate it in the feces) 14  Ammonia toxicity: Asterixis https://www.youtube.com/watch?v=sEnp2ss8VoA https://www.youtube.com/watch?v=9Oog_w2xNq0 15  Ammonia toxicity - ammonia is particularly toxic for the brain: excess ammonia due to liver failure can cause hyperammonemia, excess glutamate accumulation in the brain, hepatic encephalopathy, asterixis (liver flap) and coma: GABA Oxidative Deamination increases GAD Catabolism - degradation B6 NADH + H+ NAD+ Urea Cycle CO2 COO COO (in liver failure) H C NH3 C O CH2 Glutamate Dehydrogenase CH2 + NH4+ NH3 CH2 Mitochondria CH2 ammonium ammonia COO COO + L-glutamate NADP + NADP H + H - ketoglutarate increases Anabolism-synthesis ATP +NH4+ glutamine VERY ACTIVE IN BRAIN synthetase TO DETOXIFY AMMONIA Glutaminase ADP liver COO H C N H3 (in liver failure) CH2 CH2 NH4+ C Urine O NH2 Urea cycle Glutamine 16 The Glucose-Alanine Cycle Vigorously working muscles operate nearly anaerobically and rely on glycolysis for energy. Glycolysis yields pyruvate; part of it converted to lactate (can build up) Another part of pyruvate is converted to alanine for transport into the liver. Alanine can transport amino groups and pyruvate to the liver in a non-toxic form. Liver will reconvert alanine to pyruvate and use it into gluconeogenesis; ammonia is excreted as urea Note that glutamate can also be converted to glutamine in muscle for transport of NH4+ to liver as presented before  Ammonia is first converted into carbamoyl phosphate NH4+ generated in mitochondria enters the urea cycle The first nitrogen-acquiring reaction of the urea cycle is conversion of ammonia into carbamoyl phosphate via carbamoyl phosphate synthase 1 (CPS1)  The Urea Cycle Enzymes of the urea cycle: 1. CPS1: Ammonium to Carbamoyl Phospate. 2. Ornithine Transcarbamoylase (OTC): Carbamoyl PO4 + Ornithine to Citrulline. 3. Synthetase: Aspartate + Citrulline to Argininosuccinate. 4. Lyase: Argininosuccinate to Arginine + Fumarate 5. Arginase: Arginine to Urea + Ornithine  Use the equivalent of 4 ATP/Urea Arginase Lyase OTC Synthetase NH4+ from transdeamination equivalent to 2ATP→ADP +2P in mitochondria CPS1 NAG 20  Essential vs. Nonessential and Conditionally Essential Amino Acids Essential amino acids TABLE 18-1 Nonessential and Essential Amino Acids for Humans and must be obtained as the Albino Rat dietary protein. Nonessential Conditionally essentiala Essential Alanine Arginine Histidine Nonessential amino acids Asparagine Cysteine Isoleucine Aspartate Glutamine Leucine are easily made from Glutamate Glycine Lysine central metabolites. Serine Proline Methionine Tyrosine Phenylalanine Threonine Consumption of a variety Tryptophan of foods supplies all the Valine Required to some degree in young, growing animals and/or sometimes during essential amino acids. a illness. PVT TIM HALL  Pathways of amino acid degradation - the catabolism of amino acid carbon skeletons account for 10% of the body’s energy production. It is a convergent process with 5 products which enter the TCA: 2 1 3 Glucogenic and Ketogenic 5 4 Isoleucine Phenylalanine Tryptophan Threonine Tyrosine Ketogenic Only Leucine Lysine Minor route in humans 22  Cofactors of enzymes involved in amino acid catabolism - carriers of “one carbon units”: Aka SAM also referred to as THF or Folic Acid A sulfonamide This ring is fully reduced (tetrahydro) - Sulfonamide antibiotics are PABA analogs and act as competitive inhibitors of dihydropteroate synthase and bacterial folic acid synthesis 23  Various one-carbon units are carried by THF: - folate is a vitamin (B9) 24  Fighting bacterial infections - an antibiotic combo of a sulfonamide drug (sulfamethoxazole) plus trimethoprim (TMP) (Bactrim®) is commonly used: it affects the folate pathway of bacteria in 2 places: - biosynthesis of tetrahydrofolate: Sulfonamides such as sulfamethoxazole inhibit folate synthesis O O N O S N H H2N Folate Reductase Dihydrofolate Reductase NH2 OCH3 N H2N N OCH3 OCH3 Trimethoprim (TMP) inhibits bacterial Dihydrofolate Reductase 50,000 times more than human enzyme (much lower Km) 25  Catabolism of phenylalanine and phenylketonuria - normal metabolism of phenylalanine: Alternate route for phenylalanine metabolism BUT a major route in patients with PKU BH4 PAH decrease in activity due to mutations results in PKU -Phenylketonuria - symptoms: heart problems, small head (microcephaly), low birth weight, intellectual disabilities (“phenylpyruvic oligophrenia”), mental disorders, Phenyl ketones seizures; newborns genotyped at birth for PAH mutations - treatment: diet with reduced Phe; synthetic BH4 (co- factor in amino acid metabolism similar to pterin of THF) available as Sapropterin (Kuvan®) for treatment of PKU.  Methylation and S-adenosylmethionine SAM also called SAM or SAMe Hyperhomocystinemia Associated with coronary artery disease COO- Suggestion : increase folate and B12 to enhance Low SAM levels have been associated with B +S H 3N C H the conversion of homocysteine to methionine 6erine depression. The suggestion here is to increase intake of exogenous SAM while and also increase B6 intake to enhance conversion to CH2 simultaneously increasing Folate and B12 in cysteine. SH an effort to increase SAM. Cysteine 27  Catabolism of Branched Chain Amino Acids (BCA) - occurs mainly in extra-hepatic tissues: An oxidative decarboxylation Similar to PDH and -KGDH in Krebs 28 Catabolism of methionine, isoleucine, threonine and valine Also product of Defective in ODD CHAIN FATTY ACIDS Methylmalonyl acidemia catabolism (MMA) 29  * * * PAH Ornithine * Transcarbamoylase Deficiency 0.8 Urea Synthesis OTC hyperammonia 30  Goals and Objectives Upon completion of this lecture at minimum you should be able to answer the following: ►When does amino acid catabolism occur? ►Which are the main steps of digestion of proteins, which enzymes are involved, how it occurs and where? ►How are amino acids catabolized, where it occurs, what enzymes are involved, what is the fate of nitrogen and of ketoacids? ►What is the role of PLP in different reactions, which enzymes are catalyzing these reactions? Explain the ping-pong mechanism of transaminases and their implication in the diagnostic of liver disease/failure. ►What is transdeamination, where it occurs, what enzymes are involved, what is the outcome of this process? Explain ammonia toxicity at the brain level upon liver failure, disease generated, remedies available ►Explain the glucose-alanine cycle, what implications does it have? ►Which are the main steps, processes, intermediates and enzymes involved in the urea cycle? What are the sources of urea’s C atoms and two N atoms and what is the energetics of it? ►How is urea cycle regulated, where it occurs, and how it is connected with TCA cycle ? ►Which are the essential and the non-essential amino acids, which ones are ketogenic, glucogenic or mixed ketogenic/glucogenic? ► What co-enzymes are involved in amino acid catabolism, which are the particularities of each of them, enzymes associated with, diseases/disfunctions, and drugs associated ? ► Which are the human genetic disorders associated with amino acid catabolism, what 31 are their main characteristics and how can we treat/alleviate them?  Drugs and Diseases ►Diseases: hepatitis, cirrhosis (assessed by ALT(aka GPT) and AST (aka GOT), kidney failure, azoturia, azotemia (high BUN), asterixis (flapping tremor, liver flap), phenylketonuria (phenylpyruvic oligophrenia), hyperhomocystinemia, depression, maple syrup urine disease, methylmalonic acidemia, ornithine transcarbamoylase deficiency ►Drugs and vitamins: pyridoxal phosphate (PLP, vitamin B6), lactulose, essential amino acids (PVT TIM HALL), folate (vitamin B9), sulfonamide antibiotics (e.g. sulfamethoxazole), trimethoprim, combo of the last two: Bactrim®, Sapropterin (Kuvan®), cobalamin (vitamin B12)