Lecture 8. Amino Acid Metabolism I PDF

Biochemistry 1 PHBC 521 Hans-Georg Breitinger Mohamed Z. Gad Sahar Mohamed Sally Ibrahim Ingy Hashad Ulrike Breitinger Raghda Elsabbagh Marina Fam Nancy Turky Noura Ayman Ch...

Biochemistry 1 PHBC 521 Hans-Georg Breitinger Mohamed Z. Gad Sahar Mohamed Sally Ibrahim Ingy Hashad Ulrike Breitinger Raghda Elsabbagh Marina Fam Nancy Turky Noura Ayman Christine Adel Heba Nafea Sally Sobhy Faculty for Pharmacy and Biotechnology Lecture 8 – Protein and Amino Acid metabolism I PHBC 521 Biochemistry I 1  Lecture Contents and Learning Outcomes At the end of this lecture, you should be able to describe … - Digestion of proteins - Amino acid transport - Protein intracellular degradation: Lysosomes Ubiquitin system - Fate of nitrogen of amino acid - Glucose - alanine cycle - Urea cycle, regulation, - Genetic Defects of Urea Cycle - Ways to eliminate excess amino acid from the bod PHBC 521 Biochemistry I 2  Lecture References Reference: Stryer/Berg/Tymoczco 5th edition Chapter 23. Protein Turnover and Amino Acid Catabolism 23.1. Proteins Are Degraded to Amino Acids 23.1.1. The Digestion and Absorption of Dietary Proteins 23.1.2. Cellular Proteins Are Degraded at Different Rates 23.2. Protein Turnover Is Tightly Regulated 23.2.1. Ubiquitin Tags Proteins for Destruction 23.2.2. The Proteasome Digests the Ubiquitin-Tagged Proteins 23.3. The First Step in Amino Acid Degradation Is the Removal of Nitrogen 23.3.1. Alpha-Amino Groups Are Converted into Ammonium Ions by the Oxidative Deamination of Glutamate 23.3.2. Pyridoxal Phosphate Forms Schiff-Base Intermediates in Aminotransferases 23.3.3. Aspartate Aminotransferase Is a Member of a Large and Versatile Family of Pyridoxal-Dependent Enzymes 23.3.4. Serine and Threonine Can Be Directly Deaminated 23.3.5. Peripheral Tissues Transport Nitrogen to the Liver 23.4. Ammonium Ion Is Converted Into Urea in Most Terrestrial Vertebrates 23.4.1. The Urea Cycle Begins with the Formation of Carbamoyl Phosphate 23.4.2. The Urea Cycle Is Linked to the Citric Acid Cycle 23.4.5. Urea Is Not the Only Means of Disposing of Excess Nitrogen PHBC 521 Biochemistry I 3  Degradation of proteins and amino acids Protein degradation is a source of amino acids occurs through - food digestion - intracellular degradation (lysosomes, ubiquitin system) Amino acids are building blocks for proteins and other (N-containing) compounds, e.g. nucleobases Amino acids are not used as energy storage – they are directly funneled into energy metabolism. This usually occurs through the urea cycle. Protein turnover for cellular proteins can range from 10 min to >150 hours. PHBC 521 Biochemistry I 4  Protein Digestion PHBC 521 Biochemistry I 5  Extraction of amino acids from food Pancreatic enzymes Chymotrypsin Trypsin specific transporters Carboxypetidase Elastase Pepsin(stomach) optimum pH 2 PHBC 521 Biochemistry I 6 PHBC 521 Biochemistry I 7  Na+-K+-pump (Na+/K+- Co-transport of amino dependent ATPase) removes acid along Na+-gradient. excess Na+ and pumps K+ into No energy required; cell. Na+ accumulates inside. Requires energy (ATP), electrogenic (1 negative intracellular charge per cycle). 8 PHBC 521 Biochemistry I  g-Glutamyl cycle PHBC 521 Biochemistry I 9  g-Glutamyl cycle 1) The cycle functions mainly in the kidney, particularly the renal epithelial cells. 2) The enzyme g-Glutamyl transpeptidase is located in the cell membrane and suttles Gluathione (GSH ) to the cell surface to interact with an amino acid. 3) Reaction with the amino acid liberates cysteinyl-glycine and generates a g-Glutamyl amino acid which is transported into the cell and hydrolyzed to release the amino acid. 4) 3 ATP molecules are utilized in this reaction. PHBC 521 Biochemistry I 10  Protein degradation – proteases and lysosomes Proteins are degraded by proteases. Four major groups of proteases are known: - Serine proteases (trypsin, chymotrypsin, elastase): Ser in catalytic centre O O H2N O C – NH C–O C – OH HO HO - Aspartate proteases (pepsin, lysosomal proteases, HIV protease): Asp in catalytic centre - Zinc proteases (carboxypeptidase, metalloproteases): Zn2+ in active site - Cysteine proteases (papin, cathepsins, calpains, caspases): SH Cys attacks peptide C=O. Proteases are usually synthesised as preproteins; activation occurs by limited proteolysis. In compartments where their activity is unwanted, proteases are inactivated by protease inhibitors. Lysosomes are organelles of protein degradation. They have an acidic medium (ATP-driven proton transport) and contain various proteases. Proteins are marked for lysosomal degradation by a signal sequence (KFERQ), or glyosylation signals. PHBC 521 Biochemistry I 11  Ubiquitin-dependent protein degradation Every protein has a specific half-life, ranging from minutes to years (mostly ~10 – 150 min). Defective and misfolded proteins need to be degraded. Proteins to be degraded are labeled with a specific marker. This is ubiquitin, a small protein (76 residues) that is covalently linked to the target protein. Ubiquitin is highly conserved among eukaryotes. Between yeast and human ubiquitin only 3 in 76 residues are different! Linkage is between the side chain – NH2 of a lysine residue and the C(OO-) terminus of ubiquitin; Lys 48 of ubiquitin allows the binding of a second ubiquitin. Eventually, the target protein is marked by a chain of ubiquitins. If this chain contains 4 or more ubiquitin molecules, the protein is targeted for destruction in the proteasome. Some proteins have a “destruction box”, a sequence that is recognised by the ubiquitin ligase, making ubiquitination even easier. PHBC 521 Biochemistry I 12  Attachment of a ubiquitin chain Ubiquitin Target protein PHBC 521 Biochemistry I 13  When are proteins degraded – N-end rule - Defects (misfolding, oxidation, hydrolysis,...) “proofreading“ - N-terminus determines half-life of a ‚normal‘ (not damaged or misfolded) protein  N-end rule - Specific sequences may destabilise a protein (PEST – proteins rich in Pro , Glu , Ser , Thr are rapidly degraded; destruction boxes facilitate ubiquitination) PHBC 521 Biochemistry I 14  Where are proteins degraded – the proteasome Binds ubiquitin- conjugated substrate Isopeptidase cleaves ubiquitin from protein Regulatory units Substrate is unfolded (ATP-requiring process) Catalytic unit Proteasome contains serine- and threonine- peptidases 15 PHBC 521 Biochemistry I  Impaired protein degradation can cause disease Human Papillomavirus (HPV) encodes a spezific E3-ligase. This ligase destroys p53, a tumor supressor protein (several functions, controls DNA repair). Virus-induced loss of p-53 often leads to cancer due to inefficient DNA repair. Accumulation of misfolded proteins in Alzheimer‘s disease, and spongiforme encephalopathies (BSE – mad cow disease, Creutzfeld-Jacob) lead to formation of insoluble protein aggregates and eventual degradation of neurones. Several Parkinson’s disease-causing mutations have been identified in genes encoding for ubiquitin-mediated degradation pathway proteins. PHBC 521 Biochemistry I 16  Amino acid pool Free amino acids distributed through the body are called amino acid pool. Sources of the amino acid pool: – Dietary proteins (absorbed amino acids) – Hydrolysis of body proteins – Synthesis of non essential aminoacids PHBC 521 Biochemistry I 17  Fate of amino acid pool Catabolic Anabolic pathway pathway Alpha ketoacids are Synthesis Synthesis Removal of further Synthesis of of small NH2 from metabolized to of proteins specialized peptides amino acids be completely by oxidized into  Enzymes, products  CO2, H2O or transaminatio hormones creatine, Glutathion n and converted into heme, etc. e deamination glucose, fatty acids and Ketone bodies PHBC 521 Biochemistry I 18  Dietary Proteins Synthesis of non- Body essentioal amino proteins acids Amino Synthesis of acid Synthesis specialized products of body pool proteins Glucose R-CO- COOH NH3 Urea Ketones , Fatty Acids CO2 H2O PHBC 521 Biochemistry I 19  Degradation of amino acids Amino acid degradation must take care of two molecular entities: nitrogen and the C-skeleton. The amino group of amino acids is transferred onto a-ketoglutarate yielding glutamate. Glutamate can then be deaminated and the resulting ammonia converted to urea and excreted. PHBC 521 Biochemistry I 20 Amino acid degradation – nitrogen removal Nitrogen of amino acids is removed by: – Transamination – Deamination - Oxidative - Non oxidative - Hydrolytic – Transdeamination PHBC 521 Biochemistry I 21  Transaminase reaction Transfer of an a-Aminogruppe onto an a-keto acid is catalysed by aminotransferases, also known as transaminases. eg: Aspartate-aminotransferase (OAA) Alanine-aminotransferase (Pyruvat) Note: transaminations are reversible and can also be used to synthesise amino acids from a-keto acids. PHBC 521 Biochemistry I 22 Alanine Transaminase (ALT) PHBC 521 Biochemistry I 23 Aspartate Transaminase (AST) PHBC 521 Biochemistry I 24  Diagnostic importance of liver transaminases Transaminases are intracellular enzymes Damage to cells producing these enzymes will lead to elevated levels of transaminases in the blood Elevated ALT and AST suggest damage to the liver tissues PHBC 521 Biochemistry I 25 Pyridoxal phosphate (PLP) – the cofactor of all transaminases Phenolic hydroxyl group, slightly acidic, pKA = 9.9 Pyridine ring, slightly basic pKB = 9.8 PHBC 521 Biochemistry I 26  Pyridoxal phosphate (PLP) – the cofactor of all transaminases Aldehyde group is vital for transamination since it can form Schiff bases with primary amines. R–CH=O + R‘–NH2  R–CH=N–R‘ + H2O Schiff Base Easy proton transfer between pyridine ring and phenolic OH. PHBC 521 Biochemistry I 27 Pyridoxal phosphate (PLP) – the cofactor of all transaminases PLP is the cofactor of transaminases. The aldehyde group is available for the binding of external amino acids. PHBC 521 Biochemistry I 28  PLP forms Schiff base with transferred amino groups Lys from active site of Amino acid to be deaminated transaminase enzyme. Note: Schiff base from aromatic aldehyde is stabilised by resonance. 29 PHBC 521 Biochemistry I Conjugated Conjugation Conjugation shifted broken 30 PHBC 521 Biochemistry I  The second reaction of transamination A second reaction sequence can now take place proceeding in the reverse direction. The amino group of pyridoxamine is now transferred onto an a-keto acid. PLP is regenerated and a new amino acid formed. PHBC 521 Biochemistry I 31  Transamination After transamination, we have two products: - the carbon skeleton of the amino acid, an a-keto acid - glutamate, which contains the amino group PHBC 521 Biochemistry I 32  Oxidative deamination Glutamate dehydrogenase oxidises the C – NH2 bond to form a Schiff base. This Schiff base is not stable and is hydrolyzed by water to give a-ketoglutarate and ammonia, NH4+. Glutamate dehydrogenase PHBC 521 Biochemistry I 33  34 PHBC 521 Biochemistry I  The net result of transamination and oxidative deamination Excreted a-amino acid + a-KG + NAD+ + H2O  a-keto acid + glutamate + NH4+ + NADH Ammonia can also be bound by glutamate, a reaction catalysed by glutamine synthetase: glutamate + NH4+ + ATP -------> glutamine + ADP + Pi + H+ This reaction is used to transport ammonia amongst peripheral organs (mostly from organs to the kidneys). Ammonia is transported in a non-ionic, non-toxic form. In the kidney, glutaminase cleaves glutamine to regenerate glutamate and ammonia (excreted). glutamine + H2O -------> glutamate + NH3 35 PHBC 521 Biochemistry I  Non-Oxidative deamination This occurs for hydroxylated aliphatic amino acids serine, threonine without removal of hydrogen (non-oxidative). Coenzyme is PLP Also: serine- threonine dehydratase PHBC 521 Biochemistry I 36  Hydrolytic deamination 1. Side chain deamination of Glutamine and Aspargine. 2. Both are hydrolytically deaminated by glutaminase & asparaginase, respectively. 3. Glutaminase enzyme is present in the kidney. It produces NH 3 which is used for regulation of acid base balance in the kidney. H H2O NH3 H O O - - OOC C C C OOC C C C H2 H2 Glutaminase H2 H2 NH3+ NH2 NH3+ OH Glutamine Glutamate PHBC 521 Biochemistry I 37  Glucose – Alanine Cycle 1) The glucose-alanine cycle allows skeletal muscle to eliminate nitrogen while replenishing its energy supply. 2) Glycolysis produces pyruvate which can undergo transamination to Alanine. 3) Additionally, during periods of fasting, skeletal muscle protein is degraded for the energy value of the amino acid carbons; alanine is a major amino acid in protein. 4) Alanine enters the blood stream and is transported to the liver where it is converted back to pyruvate, a substrate for gluconeogenesis. 5) The newly formed glucose can then enter the blood for delivery back to the muscle. 6) The amino group transported from the muscle to the liver in the form of alanine is converted to urea in the urea cycle and excreted. PHBC 521 Biochemistry I 38 PHBC 521 Biochemistry I 39 Transport of nitrogen from peripheral tissues to the liver Transaminase PHBC 521 Biochemistry I 40  Ammonia Ammonia is a toxic substance especially to the CNS The main pathway by which the body can get rid of ammonia is formation of Urea Urea cycle takes place exclusively in liver Elevated glutamine and glutamate would affect osmosis in the brain In hyperammonemia ammonia will react also with alpha ketoglutarate to form glutamate which might lead to depletion of alpha ketoglutarate (Krebs cycle) PHBC 521 Biochemistry I 41  42 PHBC 521 Biochemistry I  In the liver, ammonia is removed via the urea cycle ornithine transcarbamoylase Carbamoyl phosphate synthetase-I Mitochondrion Cytosol PHBC 521 Biochemistry I 43 PHBC 521 Biochemistry I 44  Metabolic integration of the urea cycle The urea cycle, the citric acid cycle, and the transamination of oxaloacetate are linked by fumarate and aspartate. PHBC 521 Biochemistry I 45  Genetic defects of the urea cycle (urea cycle defects, UCDs) Defects of the urea cycle usually have devastating consequences, as there are no alternatives for the excretion of ammonia.  Defects of the urea cycle result in elevated levels of NH4+ in blood (hyperammonemia). Frequent symptoms: irreversible brain damage, coma. The actual reason for the toxicity of ammonia is still unclear. Possibly the accumulation of glutamine causes the toxic effects. There are therapeutic strategies to compensate for some defects of the urea cycle: - Antibiotics can be administered to kill intestinal ammonia producing bacteria. - Dietary supplementation with arginine or citrulline can increase the rate of urea production in certain UCDs. - Sodium benzoate and sodium phenylacetate can be administered to covalently bind glycine (forming hippurate) and glutamine (forming phenylacetylglutamine), respectively. These latter compounds, which contain the ammonia nitrogen, are excreted in the feces. PHBC 521 Biochemistry I 46  Defects of the urea cycle Argininosuccinase deficieny Ornithine transcarb- amoylase deficiency Carbamoylphosphate Synthetase deficiency PHBC 521 Biochemistry I 47 Two ways to eliminate excess amino acids from the body PHBC 521 Biochemistry I 48  Summary Dietary protein is degraded in stomach and intestine and taken up in form of amino acids. Proteins inside the body are degraded (i) to remove unnecessary enzymatic activity, (ii) to remove misfolded and damaged proteins (proofreading). The half-life of proteins ranges between ~ 10 minutes and 2-3 hours; some proteins can exist much longer. There are two major patways of protein degradation: in lysosomes and via the proteasome. Proteins may contain certain sequences that marks them for rapid degradation (death sequences, also sequences rich in proline, glutamate, serine, threonine – PEST proteins). Actual degradation happens by proteases (four families, Ser/Thr proteases, Zinc proteases, Asp proteases, Cys proteases). Ubiquitin is a highly conserved marker for protein degradation in eukaryotes. It is attached to the w-C of lysine. A chain of 4 ubiquitins is the signal for degradation of the protein in the proteasome. Amino acids are deaminated by transaminases and oxidative deamination. Ammonia is converted to urea in the urea cycle and excreted. Defects in the urea cycle lead to serious illness since no other way to remove ammonia from the body exists. PHBC 521 Biochemistry I 49