AMİNOASİTLERİN OKSİDASYONU AMİNOASİTLERİN METABOLİZMASI AZOT METABOLİZMASI* ÜRE DÖNGÜSÜ* KARBON İSKELETLERİNİN METABOLİZMASI AZOT METABOLİZMASI Aminoasit katabolizması tüm vücutta gerçekleşen geniş bir süreç olan azot metabolizmasının bir bölümüdür. Azot vücuda besinlerde var olan bileşikler şeklinde girer ve vücudu üre,amonyak şeklinde terkeder. Besinsel protein vücut için en önemli olan aaleri içerir. Besinsel protein Proteinlerin sindirimi midede başlar ve ince barsakta sonlanır Serbest aaler barsak epitel hücrelerinden emilir Bir kısmı karaciğere gelerek metabolize edilir Bir kısmı genel dolaşıma verilir Overview of Amino Acid Catabolis m Metabolic Circumstances of Amino Acid Oxidation Amino acids undergo oxidative catabolism under three circumstances: Protein amino-acid residues from normal turnover are recycled to generate energy and molecular components Dietary amino acids that exceed body’s protein synthesis needs are degraded Proteins in the body are broken down to supply amino acids for catabolism when carbohydrates are in short supply (starvation, diabetes mellitus), The Amino Group is Removed From All Amino Acids First Fates of Nitrogen in Organisms Plants conserve almost all the nitrogen Many aquatic vertebrates release ammonia to their environment Many terrestrial vertebrates and sharks excrete nitrogen in the form of urea Urea is far less toxic that ammonia Urea has very high solubility Some animals, such as birds and reptiles excrete nitrogen as uric acid Passive diffusion from epithelial cells Active transport via gills Uric acid is rather insoluble Excretion as paste allows to conserve water Humans and great apes excrete both urea (from amino acids) and uric acid (from purines) Excretory Forms of Nitrogen Enzymatic Transamination • Typically, -ketoglutarate accepts amino groups • L-Glutamine acts as a temporary storage of nitrogen • L-Glutamine can donate the amino group when needed for amino acid biosynthesis • All aminotransferases rely on the pyridoxal phosphate cofactor Structure of Pyridoxal Phosphate and Pyridoxamine Phosphate • Intermediate, enzymebound carrier of amino groups • Aldehyde form can react reversibly with amino groups • Aminated form can react reversibly with carbonyl groups Pyridoxal Phosphate is Covalently Linked to the Enzyme at Rest • The linkage is made via an nucleophilic attack of the amino group an active-site lysine side chain • After dehydration, a Schiff base linkage is formed • The covalent complex is called internal aldimine because the Schiff base connects PLP to the enzyme Internal Aldimine in Aspartate Aminotransferase (Lys258purple) Chemistry of the Amino Group Removal by the Internal Aldimine The external aldimine of PLP is a good electron sink, allowing removal of -hydrogen PLP Also Catalyzes Racemization of Amino Acids The external aldimine of PLP is a good electron sink, allowing removal of -hydrogen PLP Also Catalyzes Decarboxylation of Amino Acids The external aldimine of PLP is a good electron sink, allowing removal of carboxylate Ammonia in Transported in the Bloodstream Safely as Glutamate • Un-needed glutamine is processed in intestines, kidneys and liver Glutamate can Donate Ammonia to Pyruvate to Make Alanine • Vigorously working muscles operate nearly anaerobically and rely on glycolysis for energy • Glycolysis yields pyruvate that muscles cannot metabolize aerobically; if not eliminated lactic acid will build up • This pyruvate can be converted to alanine for transport into liver Excess Glutamate is Metabolized in the Mitochondria of Hepatocytes The Glutamate Dehydrogenase Reaction Two-electron oxidation of glutamate followed by hydrolysis Net process is oxidative deamination of glutamate Occurs in mitochondrial matrix in mammals Can use either NAD+ or NADP+ as electron acceptor Ammonia is Re-captured via Synthesis of Carbamoyl Phosphate This is the first nitrogen-acquiring reaction Fate of Individual Amino Acids Seven to acetyl-CoA Six to pyruvate Ile, Met, Thr, Val Two to fumarate Arg, Glu, Gln, His, Pro Four to succinyl-CoA Ala, Cys, Gly, Ser, Thr, Trp Five to -ketoglutarate Leu, Ile, Thr, Lys, Phe, Tyr, Trp Phe, Tyr Two to oxaloacetate Asp, Asn Summary of Amino Acid Catabolism Aminoasitlerin Yıkımı(1) C,H,O,N atomlarını içeren moleküllerdir İçerdikleri N depolanamaz ve hücrenin ihtiyacından fazla olan aaler hemen yıkılırlar alfa-amino grupları transaminasyon ve oksidatif deaminasyonla uzaklaştırılarak alfa-ketoasit ve NH3 oluşturulur Amino Acid Oxidation Animals are unable to convert fatty acids into carbohydrate. During times of starvation, amino acids are used to replenish TCA cycle intermediates and as precursors for gluconeogenesis. In addition, organisms with a diet high in proteins can catabolize excess amino acids as fuel. Unlike carbohydrates or lipids, amino acids are not stored. They are either used or burned. For animals, amino acids (in the form of polypeptides) represent their major source of nitrogen, and amino acids are used to make use nitrogenous compounds. Amino Acid Oxidation There is a key difference between amino acids and the other 2 types of oxidizable molecules: the nitrogen. The first step in their degradation is usually the removal of the amino group. This is carried out by aminotransferases (a.k.a. transaminases), which catalyze the transfer from an amino acid to an keto acid (usually -ketoglutarate): amino acid + -Kg –> -keto acid + Glu The coenzyme pyridoxal phosphate is Pyridoxal phosphate Catalyzes many reactions with amino acids functional end = aldehyde attached to hydroxypyridine derivative Mechanism: 1. 2. 3. 4. 5. formation of a Schiff's base with substrate amino group (stabilized by internal H-bond) abstraction of H+ from -C (stabilized by resonance; N of pyridoxine acts as e- sink) donation of H+ to C-4' shifts Schiff's base (C=N) to -C resolution depends upon enzyme eventually the Schiff's base will be hydrolyzed to regenerate an amine and a carbonyl Can also catalyze decarboxylation of amino acids (lose CO2 instead of H+ in step 2; otherwise similar). Amino Acid Oxidation The amino groups are thus collected in the form of Glu. They can be removed by glutamate dehydrogenase (in mito): Glu + NAD(P)+ -iminoglutarate + NAD(P)H The imine is then spontaneously hydrolyzed: -iminoglutarate + H2O –> -Kg + NH4+ The combined action of transaminases and Glu DH to remove amino groups as ammonia is sometimes called transdeamination. The logic The amino groups are harvested from the various amino acids that are in excess and collected as Glu. Thus, glutamate serves as a universal Ncarrier. For example, Glu can serve as an indicator of intracellular N supply, as well as a donor of amino groups. If there is an excess of amino groups in the system, then Glu DH removes them as ammonia. (You do not need a separate enzyme for each a.a.) Then the carbon skeletons can be attacked. But what does the cell do with the excess ammonia? If it is not a liver cell, it must excrete it, but not as NH3. It uses glutamine synthetase to incorporate it as Gln: Glu + NH3 + ATP –> Gln + ADP + Pi The carboxylate of Glu is first activated by forming an acid anhydride with phosphate (You will see this mechanism again!) But what does the cell do with the excess ammonia? The Gln can then be excreted to the circulatory system and reach the liver, where it is hydrolyzed by glutaminase: Gln + H2O –> Glu + NH4+ What to do with the carbon skeletons All 20 of the amino acids can be catabolized to either acetyl-CoA or TCA cycle intermediates. They can be classified into 5 categories based upon the end product: 1. 2. 3. 4. 5. -ketoglutarate: Glu, Gln, Pro, Arg, (His) oxaloacetate: Asp, Asn Succinyl-CoA: Val, Ile, Met Pyruvate: Ala, Ser, Gly, Thr, (Cys) Acetoacetyl-CoA: (Lys, Trp, Phe, Tyr), Leu Note that this is an oversimplification, since several amino acids have more than one end product. (We will discuss in detail all but the ones in parentheses.) Aminoasitlerin Yıkımı(1) NH3 İDRARLA ATILIR ÜRE DÖNGÜSÜNE* GİRER *Azotun vücuttan uzaklaştırılması için kullanılan en önemli mekanizma üre döngüsüdür. Transaminasyon Bir çok aminoasitin yıkımındaki ilk aşama α-amino gruplarının α-ketoglutarata aktarılmasıdır Ürünler:α-keto asit ve glutamat α-ketoglutaratın aa metabolizmasındaki tek rolü glutamat şekline geçerek diğer aalerden amino gruplarını toplamaktır Glutamat:oksidatif deaminasyon veya esansiyel olmayan aalerin sentezinde –NH3 vericisi olarak Transaminasyon Her aminotransferaz bir ya da birkaç amino grubu vericisine özgündür aminotransferazlar özgün amino grup vericisine göre adlandırılırlar(örn/alanin) amino grubunun alıcısı her zaman α-ketoglutarattır Transaminasyon En önemli iki aminotransferaz reaksiyonu ALT ve AST tarafından katalizlenir ALT veya GPT alaninin -NH3 grubunu α-ketoglutarata aktarır piruvat ve glutamat oluşur Transaminasyon AST veya GOT glutamatın amino grubunu okzaloasetata transfer eder aspartat oluşur aspartat bir azot kaynağı olarak üre döngüsüne girer Transaminasyon Tüm aminotransferazların koenzimi piridoksal fosfattır aminotransferazlar,bir aminoasitin -NH3 grubunu piridoksale transfer ederek piridoksamin fosfat oluştururlar piridoksamin fosfat bir α-ketoasit ile reaksiyona girerek bir aa oluştururken kendisi de orijinal aldehit formuna yeniden dönüşür Oksidatif Deaminasyon Oksidatif deaminasyon amino grubunu serbest NH3 şeklinde açığa çıkarır Glutamat,hızlı bir oksidatif deaminasyona giren tek aa dir Enzim: glutamat dehidrogenaz Koenzim: NAD NADP Oksidatif Deaminasyon glutamat dehidrogenaz Degradation of Amino Acids 1. 2. 3. 4. Discuss in general terms the use of amino acids for the synthesis of nitrogen-containing compounds Discuss the various functions of glutamine Discuss the roles of transamination Discuss the glutamate dehydrogenase (GDH) & regulation Amino Acid Degradation Degradation : → removal & disposal of amino group → utilization of the carbon skeleton for energy and gluconeogenesis ALA & GLN → non-toxic vehicles for transport of NH4+ from the periphery to the liver for AA catabolism Most nitrogenous waste is disposed of as → urea. N is also disposed of as NH4+, uric acid & creatinine . Transamination Transamination 15 N AA free exchange among AA (except THR & LYS) (not true of the carbon portion) Enzyme = transaminase or aminotransferase Quantitatively most important reaction of AA metabolism Involved in: Synthesis NEAA Degradation most AA Redistribution Transamination Reaction 1. 2. 3. 4. 5. 6. There are many transaminases Coenzyme is pyridoxal PO4 (PPal) formed from vitamin B6 All AA except THR &v LYS can undergo transamination with α ketoglutarate Equilibrium of reaction is close to 1 therefore reaction direction depends on the [reactants] which are directed by other cellular processes Directionality removal/addition of products of AA pool Urea Synthesis provides direction by withdrawing amino groups from the AA pool increase deamination and AA catabolism Transaminases – Clinical Use Transaminases – Clinical Use 1. 2. 3. 4. ASP & ALA transaminases are the most abundant Several are present in both cytosol and mitochondria (isoenzymes) ASP aminotransferase is one of the most frequently assayed enzymes in the clinical laboratory. Its determination in serum diagnostic acid especially for assessing liver disorders Nomenclature of transaminases is confusing: same enzyme = aspartate – glutamate transaminase aspartate transaminase glutamate – oxaloacetate transaminase SGOT (clinical literature) Role of Transamination (i) Redistridution of amino groups to balance AA pool -- dietary proteins provide a mixture of AA whose proportions differ from AA pool required by body correct imbalance (ii) AA synthesis / degradation performed in conjunction with glutamate dehydrogenase (GDH) GDH can remove or add amino groups to the AA pool Most amino groups glutamate due to the action of transaminases. When there is a surplus of AAs in the pool, the amino groups can be funneled through glutamate and released as NH4+ Coupling The release of amino groups as NH4+ is catalysed by glutamate dehydrogenase through oxidative deamination. Since the reaction is reversible it can also synthesize amino groups. GDH Requirements 1. The enzyme is the principal source of NH4+ in the body. GDH is a mitochondrial enzyme located in matrix, present in liver cells and most tissues. 2. Important for three reasons: (i) Link between TCA cycle & metabolism of AA ( keto acids are TCA cycle intermediates). (ii) In mammals, only reaction in which an inorganic molecule (NH4+) can be fixed to a C skeleton. Therefore essential AA could be provided in the diet as keto acids and the amino groups as NH4+ because NH4+ glutamate other AA by transamination. (iii) GDH is the major AA oxidative pathway and the major source of NH4+ Also provides directionality to transamination/GDH. In vivo, [GLU] , NAD+ & removal of NH4+ drive deamination of glutamate. With excess NH4+ (bacterial metabolism in intestine), glutamate can be formed. Glutamate Dehydrogenase 1. Driving Force: necessity to maintain low levels of ammonia which is toxic. Therefore Transaminase + GDH mediates α amine NH3 urea 2. Glutamate: link between transamination and Urea synthesis Transamination funnels amino groups through glutamate & a single dehydogenase suffices therefore activity of GDH is key Regulation of GDH Regulation of GDH: allosteric control through diverse substances. Major: (i) energy is there enough? If not oxidize AA (ii) AA load surplus? Therefore degrade (even when energy is high) Energy: a) GTP (& ATP) inhibit GDH. When GTP (TCA cycle) & ATP (glycolysis / oxid. phosphorylation) are , energy index cell therefore GDH b) Conversely ADP and GDP , energy therefore GDH active in order to produce Keto acids TCA cycle to produce ATP/GTP c) NAD H inhibits GDH AA LOAD: Excess AA: override inhibition caused with energy therefore AA themselves can GDH activity.