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Transcript
Amino acid Catabolism Amino Acids Metabolism Introduction • amino acids are not stored by the body, that is, no protein exists whose sole function is to maintain a supply of amino acids for future use. Therefore, amino acids must be obtained from the diet, synthesized de novo, or produced from normal protein degradation. Any amino acids in excess of the biosynthetic needs of the cell are rapidly degraded. • The first phase of catabolism involves the removal of the α-amino groups (usually by transamination and subsequent oxidative deamination), forming ammonia and the corresponding α-keto acid—the “carbon skeletons” of amino acids. • A portion of the free ammonia is excreted in the urine, but most is used in the synthesis of urea, which is quantitatively the most important route for disposing of nitrogen from the body. • In the second phase of amino acid catabolism, the carbon skeletons of the α-ketoacids 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 Key concepts of amino acid metabolism Amino acid Catabolism The amino acids recovered from protein turnover, or obtained from the diet or de novo synthesis, are used to : • Support ongoing protein synthesis in cells. • they are also used as metabolic precursors for numerous biomolecules, including heme groups (hemoglobin and cytochromes), nucleotide bases (purines and pyrimidines) and a variety of signaling molecules (neurotransmitters, hormones, nitric oxide). • Can be used as energy source. • Glucose, fatty acids and ketone bodies can be formed from amino acids. • The liver is the major site of protein degradation in mammals. Amino acid Catabolism • There are three major steps in catabolism of AAs. 1. Removal of amino group; deamination by: I. Transamination : Transfer of amino gp to a-ketoglutarate yielding glutamate II. Oxidative deamination: removal of amino gp from glutamate to release ammonia III. Other deamination processes. 2. Urea Cycle: Conversion of NH3 to urea for excretion 3. Metabolic break down of carbon skeleton to generate common intermediates that can be catabolized to CO2 or used in anabolic pathways to be stored as glucose or fat. Transamination Introduction • The presence of the α-amino group keeps amino acids safely locked away from oxidative breakdown. • Removing the α-amino group is essential step in the catabolism of all amino acids. • Once removed, this nitrogen can be incorporated into other compounds or excreted, with the carbon skeletons being metabolized. Transamination • The first step in the catabolism of most L-amino acids, once they have reached the liver, is removal of the -amino groups, promoted by enzymes called aminotransferases or transaminases. • In these transamination reactions, the α -amino group is transferred to the -carbon atom of α-ketoglutarate, leaving behind the corresponding α-keto acid analog of the amino acid • The effect of transamination reactions is to collect the amino groups from many different amino acids in the form of L-glutamate. • The glutamate then functions as an amino group donor for biosynthetic pathways of nonessential amino acids. or for excretion pathways that lead to the elimination of nitrogenous waste products. • All amino acids, with the exception of lysine and threonine, participate in transamination [These two amino acids lose their αamino groups by deamination]. Serine and Threonine The β–hydroxy amino acids, serine and threonine, can be directly deaminated (a) Overview of catabolism of amino groups (shaded) in vertebrate liver. (b) Excretory forms of nitrogen. Substrate specificity of aminotransferases • Cells contain different types of aminotransferases. • These enzymes are found in the cytosol and mitochondria of cells throughout the body—especially those of the liver, kidney, intestine, and muscle. • Each aminotransferase is specific for one or, at most, a few amino group donors. • Aminotransferases are named after the specific amino group donor, because the acceptor of the amino group is almost always αketoglutarate. (e.g alanine aminotransferase, aspartate aminotransferase). • The reactions catalyzed by aminotransferases are freely reversible . • The two most important aminotransferase reactions are catalyzed by alanine aminotransferase (ALT) and aspartate aminotransferase AST Transamination • Typically, α-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. Aminotransferases – Alanine aminotransferase (ALT): Formerly called glutamate-pyruvate transaminase, ALT is present in many tissues. The enzyme catalyzes the transfer of the amino group of alanine to α-ketoglutarate, resulting in the formation of pyruvate and glutamate. The reaction is readily reversible. However, during amino acid catabolism, this enzyme (like most aminotransferases) functions in the direction of glutamate synthesis. Thus, glutamate, in effect, acts as a “collector” of nitrogen from alanine. – Aspartate aminotransferase (AST): AST formerly called glutamateoxaloacetate transaminase, AST is an exception to the rule that aminotransferases funnel amino groups to form glutamate. During amino acid catabolism, AST transfers amino groups from glutamate to oxaloacetate, forming aspartate, which is used as a source of nitrogen in the urea cycle.The AST reaction is also reversible. COO− COO− COO− CH2 COO− CH2 CH2 CH2 CH2 CH2 HC NH3+ COO− + C O COO− C O + COO− HC NH3+ COO− aspartate α-ketoglutarate oxaloacetate glutamate Aminotransferase (Transaminase) Example of a Transaminase reaction: Aspartate donates its amino group, becoming the α-keto acid oxaloacetate. α-Ketoglutarate accepts the amino group, becoming the amino acid glutamate. CH3 HC COO− COO− CH2 CH2 CH2 NH3+ COO− alanine + C CH3 O COO− α-ketoglutarate C CH2 O COO− pyruvate + HC NH3+ COO− glutamate Aminotransferase (Transaminase) In another example, alanine becomes pyruvate as the amino group is transferred to α-ketoglutarate. Mechanism of action of aminotransferases • All aminotransferases have the same prosthetic group and the same reaction mechanism. • The prosthetic group is pyridoxal phosphate (PLP), the coenzyme form of pyridoxine, or vitamin B6 which is covalently linked to the ε-amino group of a specific lysine residue at the active site of the enzyme through an aldimine (Schiff base) • Pyridoxal phosphate functions as an intermediate carrier of amino groups at the active site of aminotransferases. • Aminotransferases act by transferring the amino group of an amino acid to the pyridoxal part of the coenzyme to generate pyridoxamine phosphate. The pyridoxamine form of the coenzyme then reacts with an α-keto acid to form an amino acid, at the same time regenerating the original aldehyde form of the coenzyme. • It undergoes reversible transformations between its aldehyde form, pyridoxal phosphate, which can accept an amino group, and its aminated form, pyridoxamine phosphate, which can donate its amino group to an keto acid. Ping-Pong reaction of aminotransferases • Aminotransferases are classic examples of enzymes catalyzing bimolecular Ping-Pong reactions in which the first substrate reacts and the product must leave the active site before the second substrate can bind. • Thus the incoming amino acid binds to the active site, donates its amino group to pyridoxal phosphate, and departs in the form of an -ketoacid. • The incoming -keto acid then binds, accepts the amino group from pyridoxamine phosphate, and departs in the form of an amino acid. • Measurement of the alanine aminotransferase and aspartate aminotransferase levels in blood serum is important in some medical diagnoses. 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 Mechanism of Transamination Reaction: role of PLP R H C COO Enz − (C H 2 ) 4 NH2 A m in o a c id N+ O− −O P O HC H2 C H O − O + N H CH3 E n z y m e (L y s )-P L P S c h iff b a s e 1- In the resting state, the aldehyde group of pyridoxal phosphate is in a Schiff base linkage to the ε-amino group of an enzyme lysine side-chain. Enz−Lys−NH2 R H C COO− N+ 2- The α-amino group of a substrate amino acid displaces the enzyme lysine, to form a Schiff base linkage to PLP. O− −O P O HC H2 C H O− O + N H CH3 Amino acid-PLP Shiff base (aldimine) 3- The active site lysine extracts H+, promoting tautomerization, followed by reprotonation &hydrolysis. O Enz−Lys−NH2 4- an amino acid leaves as an α-keto acid. O− −O P O NH2 CH2 H2 C R C COO− α-keto acid OH O + N CH3 H Pyridoxamine phosphate (PMP) The amino group remains on what is now pyridoxamine phosphate (PMP). 5- A different α-keto acid reacts with PMP and the process reverses, to complete the reaction. Mechanism of Transamination reaction: role of PLP •Reactions begin with formation of a new Schiffbase (aldimine) between the -amino group of the amino acid and PLP, which substitutes for the enzyme-PLP linkage. • A quinonoid intermediate is involved in the reaction. • a second -keto acid replaces the one that is released, and this is converted to an amino acid in a reversal of the reaction steps (right to left). Biochemistry in Medicine Assays for Tissue Damage • Analyses of certain enzyme activities in blood serum give valuable diagnostic information for a number of disease conditions. • Alanine aminotransferase (ALT; also called glutamate-pyruvate transaminase, GPT) and aspartate aminotransferase (AST; also called glutamateoxaloacetate transaminase, GOT) are important in the diagnosis of heart and liver damage caused by heart attack, drug toxicity, or infection. • After a heart attack,a variety of enzymes, including these aminotransferases,leak from the injured heart cells into the bloodstream. • Measurements of the blood serum concentrations of the two aminotransferases and of another enzyme, creatine kinase, Provide information about the severity of the damage. Biochemistry in Medicine • The SGOT and SGPT tests are also important in occupational medicine, to determine whether people exposed to carbon tetrachloride, chloroform, or other industrial solvents have suffered liver damage. • Liver degeneration caused by these solvents is accompanied by leakage of various enzymes from injured hepatocytes into the blood. • Aminotransferases are most useful in the monitoring of people exposed to these chemicals,because these enzyme activities are high in liver and can be detected in very small amounts.