Protein Metabolism Lesson 2 PDF

Protein Metabolism. ) Lesson 2 Removal of Nitrogen. Dr. Mohamed Khomsi Fb: Sept / 2024  Removal of Nitrogen Introduction: - Metabolism of Amino Acids includes its...

Protein Metabolism. ) Lesson 2 Removal of Nitrogen. Dr. Mohamed Khomsi Fb: Sept / 2024  Removal of Nitrogen Introduction: - Metabolism of Amino Acids includes its Anabolism & Catabolism. - The First Step in Amino Acid Catabolism is the Removal of Nitrogen ( -Amino group). - Removal of the -Amino group) is an Obligatory Step in Catabolism of A.As. - The Nitrogen ( -Amino group) is Removed as Ammonia. - The Removal of Nitrogen ( -Amino group) Produces an -Keto Acid (Carbon Skeleton). Pathways: - Nitrogen is Removed by one of the Following Reactions: (1) Transamination, (2) Deamination, (3) Trans-Deamination. Transamination Deamination Trans-Deamination It is the Transfer of It is the Removal of Includes -Amino -Amino Both from A.A to -Keto Acid in the Form of Ammonia Transamination & Deamination - Deamination can be Oxidative or Non-oxidative. - Oxidative Deamination can be Type 1, 2, 3. - Non-Oxidative Deamination can be Dehydration or Hydrolysis. - All amino acid Reactions Require PLP Except Oxidative Deamination. - All amino acid Reactions are Irreversible Except Transamination & Type I Oxidative Deamination. Importance: - Nitrogen Removal is Important for the Following Purposes: (1) Synthesis of Non-Essential Amino Acids. (2) Synthesis of Nitrogen Containing Compounds. (3) Synthesis of -Keto Acids. (4) Synthesis of Glucose & Fatty acids. (5) Production of Energy.  Transamination Definition: - It is the Transfer of -Amino group from a.a to -Keto acid to form a New -keto acid & New a.a. - Transamination Reactions Do Not Form Free Ammonia. Reversibility: - It is a Reversible Reaction. Substrate: - All Amino Acids undergo Transamination Except Threonine, Lysine. - There is No Transamination for Thr & Lys. - Thr & Lys Lose their -Amino Groups by Deamination. Enzyme: - It is Catalyzed by Transaminase. - Transaminase is also called Aminotransferase. - A Transaminase can be Specific for a Single Amino acid or Specific for a Group of Amino acids. - Transaminases are Named after the Specific Amino group Donor. Coenzyme: - It Requires PLP. - PLP is Pyridoxal Phosphate. - PLP is the Active Form of Vitamin B6. - PLP Covalently attaches to Lysine - PLP is Found in Active Site Covalently Bound to Amino Group of Lysine of Transaminase. In Transamination Reactions: - Amino Acids are Amino Group Donors & -Keto Acid are Amino Group Acceptors.  Transamination Examples: Alanine Transaminase (ALT) Alanine + -ketoglutarate Pyruvate + Glutamate Aspartate Transaminase (AST) Aspartate + -ketoglutarate Oxaloacetate + Glutamate PLP Glycine + -ketoglutarate Glyoxlate + Glutamate Cysteine + -ketoglutarate Meracaptopyruvate + Glutamate Tyrosine + -ketoglutarate p-Hydroxyphenylpyruvate + Glut Leucine + -ketoglutarate -Ketoisocaproate + Glutamate Biological Importance: - In Transamination Reactions, - -Ketoglutarate is the Most Frequently Used -Ketoacid. - So Glutamate is the Main Acceptor of Amino Group. - Glutamate Produced by Transamination Can be: - Easily Deaminated into Ammonia, - Or Used as an Amino Group Donor in the Synthesis of: (1) Synthesis of Non-Essential Amino Acids. (2) Synthesis of Nitrogen Containing Compounds. - Transamination Reactions Convert Amino acids to their Respective -ketoacids. - These -Keto Acids Like Pyruvate & Oxaloacetate are Used: - To Form Glucose & Fat or Further Metabolized by TCA Cycle. (3) Synthesis of Glucose, Fatty acid. (4) Production of Energy. - Alanine Transaminase (ALT) = Glutamate Pyruvate Transaminase (GPT). - Aspartate Transaminase (AST) = Glutamate Oxaloacetate Transaminase (GOT).  Transamination Medical Importance: - Transaminases like ALT & AST are Used in Diagnosis of Disease. - ALT & AST are Predominantly Intracellular Enzyme. - Increase in ALT or AST in Blood Indicates Disease. Disease Diagnosis by Liver Disease (e.g. Hepatitis) ALT & AST Heart Muscle Disease AST Skeletal Muscle Disease AST Kidney Disease AST ** Liver Disease is Diagnosed by Increase in both ALT & AST But ALT is More Specific **  Deamination Definition: - It is the Removal of -Amino group in the Form of Ammonia. - Deamination Reactions Forms Free Ammonia. Types: (1) Oxidative Deamination, (2) Non-Oxidative Deamination. Removal of Nitrogen Transamination Deamination Trans-Deamination Oxidative Deamination Non-Oxidative Deamination Type I Type II Type III Dehydration Hydrolysis  Oxidative Deamination Definition: - It is Deamination Reaction with Oxidation by Removal of Hydrogen. Types: (1) Type 1 Oxidative Deamination, (2) Type 2 Oxidative Deamination, (3) Type 3 Oxidative Deamination. Removal of Nitrogen Transamination Deamination Trans-Deamination Oxidative Deamination Non-Oxidative Deamination Type I Type II Type III Dehydration Hydrolysis  Oxidative Deamination Type I Oxidative Deamination Glutamate Dehydrogenase Glutamate -ketoglutarate NAD/NADP H2o NH3 NADH/NADPH Definition: - It is an Oxidative Deamination with & NAD is the Acceptor. Reversibility: - It is a Reversible Reaction. Substrate: - Glutamate. Enzyme: - It is Catalyzed by Glutamate Dehydrogenase. Coenzyme: - It Requires NAD/NADP Regulation: - It is Allosterically Activated by AMP, NAD & Allosterically inhibited by ATP, NADH+H. - After Ingestion of Protein, Reaction Proceeds in Oxidative Deamination Direction. - When Ammonia Level is High, Reaction Proceeds in Reductive Amination Direction. - Trans-Deamination is Combined Action of Aminotransferase & GDH.  Oxidative Deamination Type II Oxidative Deamination L-a.a Oxidase L-amino acid L- -keto acid FMN H2o NH3 FMNH2 Definition: - It is an Oxidative Deamination with & FMN is the Acceptor. Reversibility: - It is an Irreversible Reaction. Substrate: - L-amino acids. Enzyme: - It is Catalyzed by L-a.a Oxidase. Coenzyme: - It Requires FMN Biological Importance: - It has Low Activity in Liver & Kidney. - It is of Little Importance.  Oxidative Deamination Type III Oxidative Deamination D-a.a Oxidase D-amino acid D- -keto acid FAD H2o NH3 FADH2 Definition: - It is an Oxidative Deamination with & FAD is the Acceptor. Reversibility: - It is an Irreversible Reaction. Substrate: - D-amino acids. Enzyme: - It is Catalyzed by D-a.a Oxidase. Coenzyme: - It Requires FAD. Biological Importance: (1) D-A.As are Efficiently Metabolized by the Liver. - D-A.As are found in Plants and in Cell Walls of Microorganisms, - But are Not Used in the Synthesis of Mammalian Proteins.  Non-Oxidative Deamination Definition: - It is Deamination Reaction Without Oxidation of Amino Acids. Types: (1) Dehydration Non-Oxidative Deamination, (2) Hydrolytic Non-Oxidative Deamination. Removal of Nitrogen Transamination Deamination Trans-Deamination Oxidative Deamination Non-Oxidative Deamination Type I Type II Type III Dehydration Hydrolysis  Non-Oxidative Deamination Dehydration Serine Dehydrate Serine D- Pyruvate PLP H2o NH3 H2o Threonine Dehydrate Threonine - -Keto-butarate PLP H2o NH3 H2o Definition: - It is a Non-Oxidative Deamination by Removal of Water. Reversibility: - It is an Irreversible Reaction. Substrate: - OH-Containing Amino Acids (Serine, Threonine). 2 Enzyme: - It is Catalyzed by Dehydratase. Coenzyme: - It Requires PLP.  Non-Oxidative Deamination Hydrolysis Asparaginase Asparagine - Aspartate PLP H2o NH3 Glutaminase Glutamine - Glutamate PLP H2o NH3 Histidinase Histidine - Urocanate PLP H2o NH3 Definition: - It is a Non-Oxidative Deamination by Hydrolysis of Amino group in Side Chain. Reversibility: - It is an Irreversible reaction. Substrate: - It occurs to Asparagine, Glutamine, Histidine Enzyme: - It is Catalyzed by Hydrolase. Coenzyme: - It Requires PLP.  Ammonia Name: - Ammonia. Formula: - NH3. pH: - Basic. Blood Levels: - Normal Amount of Ammonia in Blood is 10-20 g/dl. Sources: (1) Catabolism of Amino Acids by Deamination & Trans-Deamination **Main Source** (2) Glutaminase Action on Glutamine (Liver). (3) Catabolism of Nitrogen Compound like Purines & Pyrimidine. (4) Absorbed From the Gut (Intestine Bacterial Urease Breaks Urea into Ammonia). Fate: (1) Detoxification in Liver by Urea Cycle **Main Fate** (2) Synthesis of Glutamate & Glutamine. (3) Excreted in Urine. Medical Importance: - Ammonia is Toxic in Excess Especially to the CNS. - It has Neurotoxic Effect.  Ammonia Transportation: - A.A Nitrogen (Ammonia) Flow to Liver as: (1) Alanine & Glutamine, (2) Other Amino Acids, (3) Free Ammonia (from Portal Blood). - Ammonia is Transported from Tissue to Liver in a Non-Toxic Form. - There are Two Transportation Mechanisms: (1) Glutamine Transportation System. (2) Alanine Transportation System. Glutamine Transportation System: - In Most Tissues, Glutamine Synthase Combines Ammonia with Glutamate to Form Glutamine. - Glutamine is a Non-Toxic Transport Form of Ammonia from Tissue (Brain) to Liver. - In Liver, Glutaminase Cleave Glutamine to Glutamate and Free Ammonia. Glutamine Synthetase Glutamate Glutamine NH3 ATP ADP Glutaminase Glutamine Glutamate H2o NH3 Alanine Transportation System: - This System Uses Glucose-Alanine Cycle. - Alanine is a Non-Toxic Transport Form of Ammonia from Muscle to Liver. - In Liver, Transdeamination Releases Free Ammonia. - Alanine & Glutamine are the Most Abundant amino acids in blood. - In Most Terrestrial Animals: - Gln Carries NH3 to Liver & Kidney, Where it is Hydrolyzed for Excretion as Urea. - Under Conditions of Starvation: - The Liver Exports Gln for Use in Other Tissues, - Gln Serves as Amine group Donor for Synthesis of Many Other Molecules like: - Alanine, Glycine, Histidine, Tryptophan, - Carbamoyl-phosphate, Glucoseamine-6-P, AMP, CTP,  Ammonia Muscle Amino acids: - Muscle Proteins Contain Different Amount of Amino acids. - 7-10 % of Amino acids in Muscle Proteins is Ala. - 6 % of Amino acids in Muscle Proteins is Gln. - In the Post-Absorptive State, AA are Released from Muscle. - 30% Of the Total AA Released by Muscle is Ala. - 25% of the Total AA Released by Muscle is Gln. - Both Alanine & Glutamine Represent > 50% Total AA Released. - The ALA + GLN Output (Released) is More Abundant than Muscle Content. Where does this Extra ALA & GLN Come From? Sources of Alanine from Muscle: In Muscle: Ala + AA -Keto acids NH4 + Pyruvate Ala. - Therefore, Total Ala Released > Ala Derived from Proteins. - The Extra Ala Released is Made from Other AA. - Ala Serves as a Vehicle for Transport of NH4+ from Muscle to Liver. Sources of Glutamine from Muscle: In Muscle: Gln + AA -Keto acids Gln. - Therefore, Total Gln Released > Gln Derived from Proteins. - The Extra Gln Released is Made from Other AA. - Gln Serves as a Vehicle for Transport of NH4+ from Muscle to Gut & Kidneys. - In Gut: Liver) - In Kidneys:  Ammonia Transportation of Ammonia:  Urea Cycle Name: - Urea Cycle. Pathway: - Major Pathway. - 80-90% of Ammonia is Detoxified by Urea Cycle. Definition: - It is a Detoxification Cycle that Converts Toxic Ammonia to Non-Toxic Urea. Site (Organ): - Urea Cycle Occurs in the Liver. - Urea Cycle occurs in Periportal Hepatocytes. Site (Cell): - Urea Cycle Occurs in Both Mitochondria & Cytosol. - Urea Cycle Begins in Mitochondria & Ends in the Cytosol. - Urea Cycle Begins Mitochondria Because it Needs Co 2 & ATP from Krebs Cycle. Substrate: - NH3 + Co2 + Aspartate. End Product: - The End Product is Urea. - The Chemical Formula for Urea is: (1) One Nitrogen is Supplied by Free Ammonia. (2) Other Nitrogen is Supplied by Aspartate. (3) Carbon & Oxygen are Supplied by Carbon Dioxide.  Urea Cycle Steps: - 5 Steps. - First Two Reactions (Steps) of Urea Cycle Take Place in Mitochondria. - The Remaining Reactions in Cytoplasm of the Hepatocyte. Energy: - It Requires 3 ATP (4 High Energy Phosphates). - Carbomyl-Phosphate Synthetase I Requires 2 ATP (2 Pi). - Argininosuccinate Synthetase Requires 1 ATP (1 PPi). Equation: - The Net Reaction of the Urea Cycle, NH3 + Co2 + Aspartate + 3 ATP Urea + Fumarate + 2 ADP + 1 AMP + 2 Pi + 1 PPi NH4 + + HCO 3- + Aspartate + 3 ATP Urea + Fumarate + 2 ADP + AMP + 4 Pi  Urea Cycle Key Enzyme: - Carbomyl-Phosphate Synthase I (CPS-I). - Is the Commitment Step of Urea Cycle. Regulation: - Urea Cycle is Activated by: (1) High Protein Diet, (2) Starvation, (3) A.As, (4) Arginine, (5) Glutamate. - Urea Cycle is Regulated in Two Ways: (1) High Protein Diet & Starvation Leads to Increased Synthesis of, All Five Enzymes of Urea Cycle & N-Acetylglutamate Synthase. (2) Arginine Activates of N-Acetylglutamate Synthase, - N-Acetylglutamate Synthase Converts Glutamate to N-Acetylglutamate, - N-Acetylglutamate Allosterically Activates CPS-I. - N-Acetylglutamate is the Most Important Activator. N-Acetyl glutamate Synthase Glutamate + Acetyl CoA N-Acetyl glutamate CoA - A Specific Hydrolase Removes N-AcetylGlu. - CPS-I is Completely Inactive in the Absence of N-AcetylGlu. - A Genetic Deficiency in NAcetylGlu Synthase Can Cause Lethal Defect in Urea Cycle.  Urea Cycle Interaction: - Urea cycle & Kreb's cycle are Synergetic & Connected by Many Intermediates. Urea Cycle Provides krebs Cycle Provides Fumerate ATP, Co2, Aspartate - The Aspartate Consumed in the Urea Cycle, Can be Regenerated from the Fumerate, - Fumarate Enters TCA Cycle & Converted to Malate & Later Oxaloacetate, - Oxaloacetate is Transaminated to Form Aspartate, - Aspartate Enters Urea Cycle. - This Process Uses Both Cytosolic & Mitochondrial Enzymes. Fate of Urea: - Urea Travels in Blood from Liver to Kidney & Intestine. - In the Kidney, it is Excreted in Glomerular Filtrate/Bladder (Urine). - In the Intestine, it is Excreted in Feces. - In the Intestine, Intestinal Bacteria Containing Urease Enzyme Break Urea into Ammonia & Co 2. Compound Excreted % of Nitrogen Excreted Urea 80-90% Creatinine 3-4% NH4+ 2.5-4.5% Uric Acid 1-2% Amino Acid 1-2% Blood Levels: - Normal Blood Urea Nitrogen is 7-18 mg/dl. Urea Decreases with: (1) Hepatic Failure. Urea Increases with: (1) Increase A.A Catabolism, (2) Increase Glutamate & N-Acetyl Glutamate, (3) Renal Insufficiency  Urea Cycle Biological Importance: - Urea Cycle is the Main Route of Detoxification & Disposal of Ammonia. - Urea Cycle is Quantitatively the Most Important Disposal Route for Ammonia. - Urea Synthesis Provides an Efficient Mechanism for Land Animals to Remove Excess Nitrogen from Body. Medical Importance: - Problems with Urea Cycle or the Liver Will Cause Hyperammonemia.  Hyperammonemia Name: - Hyperammonemia. Other Name: - Ammonia Intoxication. Definition: - It is the Elevated Level Blood of Ammonia. Types Hyperammonemia: (1) Congenital Hyperammonemia & (2) Acquired Hyperammonemia Congenital Hyperammonemia Acquired Hyperammonemia Deficiency of Enzyme Liver Disease (Commonest Cause) Since Birth Later in Life Hyperactivity, Tremor, Slurred speech, Mental Retardation Blurred vision, Vomiting - Congenital Hyperammonemia = Hereditary Hyperammonemia = Genetic Hyperammonemia. - Acquired Hyperammonemia is Caused by Liver Diseases like: - Cirrhosis Caused by Alcoholism, Hepatitis, Biliary Obstruction, Results in - Flow of Portal Blood Directly into Systemic Circulation. - Detoxification of Ammonia is Severely Impaired.  Hyperammonemia Congenital Hyperammonemia: - Depending on the Deficient Enzyme there are Five Types: (1) Hyperammonemia Type I, (2) Hyperammonemia Type II, (3) Citrullinemia, (4) Argininosuccinurea, (5) Argininemia. Disease Name Enzyme deficiency Hyperammonemia Type I CPS-I Hyperammonemia Type II OTC Citrullinemia Argininosuccinate synthetase Argininosuccinurea Argininosuccinase Argininemia Arginase - In Citrullinemia, Plasma & Cerebrospinal Fluid Citrulline Levels are Elevated. - In Citrullinemia, 1-2 g of Citrulline are Excreted Daily. - In Argininemia = Hyperargininemia, Plasma & CFS Arginine Levels are Elevated. Effect Hyperammonemia: - Ammonia in Excess is Toxic to the CNS so it Causes, - Brain Damage & Neurological Problems. - It Causes Brain Damage by One of the Following Mechanisms: Decrease or Depletion in Energy Increase Intracranial Pressure - Increase in Ammonia Depletes of Glutamate. - Increase in Ammonia Depletes All -KG - Increase in Ammonia Increases Glutamine. - This impairs TCA & decreases energy - Glutamine Causes Direct Damage to Neurons. - Decrease in Energy Stop Function of Neurons - Glutamine is Osmotically Active. - Causes Brain Edema, Increase ICP, Coma & Death - Cerebral Edema = is an Increase in the B Water Content.  Hyperammonemia Treatment of Hyperammonemia: (1) Stop Protein Intake (2) Increase carbohydrate intake (3) Drugs (4) Dialysis Drugs Include: Antibiotics, L-Arginine, L-Citrulline, Na-Benzoate, Na-Phenylbutarete. - Antibiotics: Kill Intestinal Bacteria that Use Urease to Form Ammonia. - Na-Benzoate, Na-Phenylbutarete: Increase Ammonia Excretion by Excretion of A.A in Urine. - Na-Benzoate Excretes Glycine in Urine as Benzoyl-Glycine (Hippuric Acid). - Na-Phenylacetate Excretes Glutamine in Urine as Phenyl-Acetyl Glutamine. - When Glycine & Glutamine are Excreted the Body Uses Blood Ammonia to Synthesize Them. - This Decreases Blood Ammonia. - Na-Benzoate, Na-Phenylbutarete Drug is Commercially Called Ammonul.  Hyperammonemia Clinical Case History: - Male, Infant, Born Healthy Weighing 2.9 Kg at Birth. - On Day 4 he Starts to Show Seizure. - Mother has a History of Aversion to Meat, Vomiting & Lethargy Investigation: Investigation Results Result Normal Rage Plasma NH4+ 340 µM High 25-40 µM pH 7.5 Mild Alkalosis 7.35 7.45 Plasma amino acids Gln 2400 µM High 350-650 µM Ala 750 µM High 8-25 µM Arg 5 µM Low 30-125 µM Cit Undetectable Low ------- Urinary Orotic acid 285 µM/mg High ----- Creatinine Normal Normal 0.3-10 Diagnosis: - Hyperammonemia. Treatment: - Oral Therapy Initiated: L-arginine & Sodium benzoate Result: - Patient improved after 7 days & plasma NH4+ normal again.