Amino Acid Metabolism Lecture PDF

Dr. Ardina Grüber Nanyang Technological University School of Biological Sciences Division of Structural Biology and Biochemistry Singapore 637551 email: [email protected]  Amino acid metabolism: an overview The primary function of amino acids is:...

Dr. Ardina Grüber Nanyang Technological University School of Biological Sciences Division of Structural Biology and Biochemistry Singapore 637551 email: [email protected]  Amino acid metabolism: an overview The primary function of amino acids is: to act as the monomer unit in protein synthesis to be used as substrate for biosynthetic reactions of different compounds like: ~ signaling pathway ❖nitric oxide (NO, a chemical messenger), & made from 2-arganize - ❖hormones, nicotinamide (in coenzymes), epinephrine -synthesized from tyr top (synthesized from toptophan ❖heme (as part of hemoglobin in red blood cells), & a lysine from succingl cost ❖purine and pyrimidine bases (for nucleic acids). & from aspartate glutamate , , lysine Mathews, van Holde, Ahern: Biochemistry 3rd edition  Amino acid source 20 a. n.- 20 biosynthetic pathways => ro degradative pathways a. a. is not stored I in body needs to be supplied by ① &Q & ① ② dietary intake synthesis of amino acid ⑤ degradate of prexist P  Digestion of proteins acidic gastric juice acts as denaturing agent unfold - globular P , & denature them reveals peptide bonds for enzymatic hydrolysis bonds & release peptide an they cleare Lippincott’s Illustrated Reviews: Biochemistry 5th edition I diff enzymes have specificity  Amino acid metabolism: an overview The entire collection of free amino acids throughout the body-amino acid pool- occupies a central position in protein and amino acid metabolism. P turnover McMurry, Ballantine, Hoeger, Peterson: Fundamentals of General, Organic, and Biological Chemistry, 7 th edition  Amino acid metabolism: an overview Amino acids released by digestion pass from the gut through the hepatic portal vein to the liver. Most of the amino acids are used for the synthesis of proteins in the liver and in other tissues. Excess amino acids may be converted to glucose or triacylglycerol. During fasting, amino acids are released from muscle protein. Glutamine is oxidized by various tissues, including the lymphocytes, gut, and kidney. Alanine and other amino acids travel to the liver, where the carbons are converted to glucose and ketone bodies, and the nitrogen is converted to urea, which is excreted by the kidneys. - glutamate Marks Basic Medical Biochemistry: A clinical Approach, 4th edition  Amino acid metabolism: an overview The amino group is removed and incorporated into urea for disposal. The remaining carbon skeleton (α-keto acid) can be broken down to CO2 and H2O or converted to glucose, acetyl-CoA, or ketone bodies.  Amino acid catabolism: fate of nitrogen atom Transamination reaction - removal of the amino group Most aminotransferases use glutamate/α-ketoglutarate as one of the two α-amino/α- keto acid pairs involved; Transamination reaction provides a route for redistribution of amino acid nitrogen Serum glutamate-oxaloacetate transaminase (SGOT) catalyzes the interconversion of oxaloacetate and aspartate, and serum glutamate-pyruvate transaminase (SGPT) catalyzes the interconversion of pyruvate and alanine.  Amino acid catabolism: fate of nitrogen atom ~ in most case scenario All amino acids except lysine, proline and threonine can undergo transamination reaction. Aminotransferases utilize a coenzyme - pyridoxal phosphate (PLP) - derived from vitamin B6.. No free ammonium is released at any stage of the reaction followed oxidative deaminate transaminate by & cytoplasm Mitochondria  Vitamin B6 (pyridoxine,pyridoxal,pyridoxamine) PLP is the prosthetic group for many enzymes that catalyze a variety of reactions involving amino acid such as transamination, racemization, decarboxylation, and side- chain elimination or replacement. Marks Basic Medical Biochemistry: A clinical Approach, 4th edition  Mechanism of transaminases all components are interacta activated w 20 enzyme form substrate Nu attack - Schiff base (covalent interact) Schiff base cleared op pulled - Nr amino grp & trsf it to X-keto acid  Mechanism of transaminases In the first step of virtually all PLP-dependent reactions, the aldehyde group of the coenzyme forms an imine (Schiff base) linkage with a lysine side chain on the enzyme. Generally, the next step is an imine exchange, as the amine nitrogen on the amino acid substrate replaces the enzyme lysine nitrogen in the imine linkage. This substrate-coenzyme adduct is stabilized by a favorable hydrogen bond between PLP and the imine nitrogen.  Mechanism of transaminases L conjugated double bords restored The first step is abstraction of the alpha-proton from the PLP- amino acid adduct. In the transaminase reactions this initial deprotonation step is immediately followed by a reprotonation hydrat at what was originally the aldehyde carbon of PLP (step 2 above), which results in a new carbon-nitrogen double bond between the α-carbon and the nitrogen of the original amino acid. This imine is then hydrolyzed (step 3) - this is the step where the nitrogen is removed from the amino acid to form an α-keto acid, which can be degraded further. The coenzyme, which now carries an amine group is called pyridoxamine phosphate (PMP).  Mechanism of transaminases Schiff transfer of amino grp betw base OLP & E to X-ketoglutarate restored -no amino gop released http://www.wiley.com/college/fob/quiz/quiz20/20-7.html Amino acid catabolism: fate of nitrogen atom DEAMINATION following transaminat ❖ the removal of amino group from the amino acids as NH3 ❖ results in the liberation of ammonia for urea ❖ may be either oxidative or non-oxidative NON-OXIDATIVE deamination (minor part) Serine, threonine, cysteine/cysteine and histidine undergo this type of deamination to form corresponding α-keto acid Dehydratase catalyze the deamination of serine and threonine (PLP dependent) Desulfurases are responsible for deamination of cysteine/ cysteine (PLP dependent) Histidase catalyze deamination of histidine  Amino acid catabolism: fate of nitrogen atom OXIDATIVE deamination oxidatively removes the glutamate amino group as ammonium ion to giveback a-ketoglutarate. Reaction is catalyzed by enzyme glutamate dehydrogenase Enzyme use NAD+ and NADP+ as coenzyme This takes place mostly in liver and kidney reversible rxth in hepatocytes , glutamate transported from cytosol to mito where it undergoes Tot deaminate  Amino acid catabolism: fate of nitrogen atom In degradation, the aminotransferase works in concert with glutamate dehydrogenase, as exemplified by the degradation of alanine: Combined action of an transaminase and glutamate dehydrogenase referred to as transdeaminase. © 2016 Pearson Education, Ltd.  Alternative pathway of deamination ❖ L-amino acid oxidase (FMN) has higher activity ❖ D-amino oxidase (FAD) ~ than L form ❖ Both enzyme present in liver and kidney localised in peroxisomes where they generate H202 ❖ Not acting on hydroxyl and dicarboxylic amino acid Summary of the sources of NH4 + for the urea cycle can fuse through cell memb , e.. g face aross pass into X e memb NH3 can urine from kidney tubules & ↓ acidity ~ ~ Ourire by binding Ht Inf + NH 4 e physiological pl , eqm favours + NH4 -Toxic Marks Basic Medical Biochemistry: A clinical Approach, 4th edition  Excess ammonia is transported to the liver for excretion as urea (G(u) (f(n) transaminate © 2016 Pearson Education, Ltd.  The Krebs–Henseleit urea cycle & removal of NHp" in liver citrulline transporter 1. Ornithine transcarbamoylase ① 2. Arginosuccinate synthetase 3. Arginosuccinate lyase ② 4. Arginase transporter - ④ ③ transported to kidney  The Krebs–Henseleit urea cycle - Step 1 Carbamoyl phosphate synthetase (CPS-I), catalyzes the reaction that Carbamoyl phosphate synthetase I converts ammonia from glutamate into carbamoyl phosphate, which then Cactivated form of NH ++) combines with ornithine to form citruline. Ornithine transcarbamoylase This reaction is catalyzed by ornithine transcarbamoylase.  The Krebs–Henseleit urea cycle - Step 2 This reaction is driven by conversion of ATP to AMP and pyrophosphate followed by the additional exergonic Argininosuccinate hydrolysis of pyrophosphate. givee synthetase to phosphate Cenergy released Enzyme which catalyze this reaction is uxt* condensate called argininosuccinate synthetase. (pyrophosphate  The Krebs–Henseleit both are N now atom urea cycle - Step 3 carried arginive by Argininosuccinate is cleaved nonhydrolytically to form arginine and fumarate in an elimination reaction catalyzed by argininosuccinate lyase. S Arginine is immediate precursor of urea. Argininosuccinase  The Krebs–Henseleit urea cycle - Step 4 Finally, the guanidinium group is hydrolytically cleaved to form ornithine and urea in a reaction catalyzed by arginase. Arginase S  of Mrs/M + ↑ rate pat , ~ ↑rate urea of format The urea cycle is regulated by substrate availability (fed Regulation of the urea cycle is via the enzyme carbamoyl phosphate synthetase (CPS-I). Mammals have two versions of in mito this enzyme: CPS-I mitochondrial version & NHat carrier of atom and ast on as N CPS-II cytosolic version. N N-acetyl-glutamate art on glutamate as carrier of atom synthetase CPS is subject to allosteric activation by N-acetylglutamate, produced from the combination high [arg) in cytosol contribute high [ornithire] which is of acetyl-CoA and glutamate, as to & used in mito well as increased concentrations transported to step , of area incle of arginine. Marks Basic Medical Biochemistry: A clinical Approach, 4th edition  Summary of the Krebs–Henseleit urea cycle Part of the cycle takes place in the mitochondrion and part in the cytosol. Fumarate and aspartate are the direct links to the citric acid cycle. The synthesis of fumarate is a link between the urea cycle and the citric acid cycle. Fumarate is an intermediate transaminati of the citric acid cycle, and it oxaloacetate & f can be converted to om catabolism oxaloacetate. A transamination reaction can convert oxaloacetate to aspartate, providing another link between the two cycles. Both pathways were discovered by the same person, Hans Krebs. Lehninger Principels of Biochemistry, 5th edition The Urea Cycle-Disorders toxic to nervous system levels glutamine in circulate , & is a ketoglutarate not regenerated removal of -wHa by from glutamine  7 main intermediates Amino Acid Catabolism: Fate of Carbon Atom Amino acids that form acetyl CoA or acetoacetyl CoA can contribute to the formation of fatty acid and ketone bodies are called ketogenic. Amino acids that are degraded to pyruvate or citric acid cycle intermediates are called glucogenic. Some of amino acids are both glucogenic and ketogenic because different parts of their carbon chains form different products.  Biosynthesis of Amino Acid can be synthesized in cannot be synthesized ~ human body ~ in body Lehninger Principels of Biochemistry, 5th edition  Amino acids can be synthesized from intermediates in glycolysis, the > pentose phosphate pathway, or the citric acid cycle © 2016 Pearson Education, Ltd.  Biosynthesis of amino acids Four metabolic intermediates like pyruvate, oxaloacetate, glutamate and α-ketoglutarate are the precursors for synthesis of the nonessential amino acids. We can organize amino acids into families based on common precursors. - precursor Mathews, van Holde, Ahern: Biochemistry 4rd edition  Biosynthesis of amino acids Glutamate can be made from NH4+ and a-ketoglutarate by reductive amination Reaction requires NADH/NADPH Glutamate dehydrogenase enzyme carries out the reaction Glutamate is a major donor of amino groups in reactions, and α-ketoglutarate is a major acceptor of amino groups. Biosynthesis of nonessential amino acids PLP dependent transamination glutamine ~ Asparagin Glutamin synthetase synthetase glutamate Mathews, van Holde, Ahern: Biochemistry 4rd edition  Synthesis of nonessential amino acids 20) feedback inhibit 1. OH is oxidized to a keto group forming 3- phosphohydroxypyruvate 2. 3-phosphohydroxypyruvate undergoes transamination with glutamate to form 3-phosposerine and α-ketoglutarate 3. 3-phosposerine is hydrolyzed to give serine and Pi  Synthesis of nonessential amino acids Serine is the major source of glycine via a reversible reaction catalyzed by serine hydroxymethyltransferase. -create another ring 7 transfer - CHO4 into tetrahydrofolate convert it to The serine hydroxymethyltransferase reactions requires two cofactors: the prostetic group PLP and the cosubstrate tetrahydrofolate. Vitamin B9 (folic acid, folate) carrier - one carbon group 2012 Pearson Education, Inc. Folate has three main components: 1. pterin (2-amino-4-oxopteridine) 2. p-aminobenzoic acid moiety (PABA) 3. glutamate residue  Synthesis of nonessential amino acids Bacteria and plants can synthesize cysteine by incorporation of H2S, with serine providing the carbon skeleton. Some bacteria can condense H2S with serine directly, via a pyridoxal phosphate-dependent enzyme. Plants and most microorganisms use O-acetylserine as the substrate reacting with H2S.  Synthesis of nonessential amino acids The amino acid tyrosine is classified as nonessential because we can synthesize it from phenylalanine, an essential amino acid: biosynthesis of a a. non-reversibile exte in only. ~ Phenylalanine hydroxylase > - X accumulate of these related to 2 pots is brain damage leads to mental retardate Phenylketonuria (PKU) Synthesis of nonessential amino acids transaminate uvea cycle Mathews, van Holde, Ahern: Biochemistry 4rd edition  Genetic disorders of amino acid metabolism Marks Basic Medical Biochemistry: A clinical Approach, 4th edition  One week Two days before exams before exams Right after exams Examination results