Amino Acids - Lindo - Lecture Notes PDF

Living cells produce macromolecules e.g. proteins and nucleic acids- structural components as biocatalysts- as hormones and for the genetic information of a species. These macromolecules are Biopolymers constructed of distinct monomer units/building blocks. For nucleic acids: the monomer units are n...

Living cells produce macromolecules e.g. proteins and nucleic acids- structural components as biocatalysts- as hormones and for the genetic information of a species. These macromolecules are Biopolymers constructed of distinct monomer units/building blocks. For nucleic acids: the monomer units are nucleotides. For Proteins-: L-α-amino acids determined by: 1.The kinds of a.a. present 2.The order in which they are linked together in a polypeptide chain and 3.The spatial relationship of one a.a. to another. α-a.a.- have both an amino and a carboxylic acid function attached to the same –CARBON ATOM CLASSES OF AMINO ACIDS  Diaminomonocarboxylic acid- positively charged e.g. lysine  Monoaminodicarboxylic acid- negatively charged e.g. aspartate  POLAR or NON-POLAR ( hydrophilic/ hydrophobic)  Glycine- R=H has no optical activity  Although D-a.a. occur in cells and in polypeptides (eg. In polypeptide antibiotics elaborated by certain microorganisms). They are not present in proteins.  Many a.a.’s occur in plants or in antibiotics. Over 20 a.a’s occur NATURALLY e.g. D-alanine and D-glutamic acid of certain bacterial cell walls and a variety of D-a.a in antibiotics.  A.A. -trivial names (e.g. glycine, tryptophan)- - systematic chemical names  The Carbon bearing the C=O and NH2 groups is the α– CARBON and the next is β-CARBON. from ammonia ― NH2 from CO2 C=O from H2O ― NH2 from aspartate Amino Acid Structure  Amino Acids- structure of 20 amino acids found in proteins  Building blocks of proteins (basic units)  Soluble in polar solvents eg. H2O;  Soluble in non-polar organic solvents eg. benzene, toluene ― General Structural Formula of Amino Acids H ― NH2 — C ― COOH R Undissociated form COOH- carboxylic group NH2- amino group R- side chain H ― + C COOH ― H3N ― ― At pH 1.5 charge is (+1) OH R → → OH- COOH ⇌ COO + H - + loses a proton +H+ H ― C COO- charge is zero at pH 7.2 ― NH3 ― ― + R ← → OH- +H+ H ― C ― ― NH2 R ― At further pH of 11.0 COO- The overall charge is now -1 At pH 7.2 - 7.3 , amino acid exists in a polar form (“Zwitterion” ion) ⇒ dipolar At pH 7.2 - 7.3 : ⊕ ― H — NH3 ― C R ―COO ⊝ where: COOH ⇌ COO- + H+ NH2 + H+ ⇌ NH3+ The pH of the solution affects the charge on the amino acid – H ― +NH ― 3 - H+ R C — COO – ― ⇌ H3N — C ― COOH — ― ⊕ H add OH The COOH ⇌ COO– + H+ loses a proton R Charge is zero (0) on the molecule At pH 1.5 - acid the COOH group and NH3 groups – the overall charge is +1 At further pH of 11.0 Add OH – H — C — COO ⇌ H – NH2 — C — COO — — R – – — — +NH 3 OH R The overall charge is now -1 Classification of Amino Acids Based on the R-group 1) Non-polar amino acid-hydrophobic-R-groups 2) Polar amino acids (uncharged)- hydrophilic-R-groups 3) Positively charged R-groups 4) Negatively charged R-groups 1. Non-polar R-groups:– Do not like water (hydrophobic) a) Alanine (Ala/A) - least hydrophobic H — — H3N+ — C — COO – CH3 R ≡ CH3 b) Valine (Val/V) H — ― H3N+ — C — COO – R ≡ CH-(CH3)2 CH CH3 c) CH3 Leucine (Leu/L) H ― ― H3N+ — C — COO – ― CH2 CH CH3 CH3 R ≡ CH2-CH-(CH3)2 d) Isoleucine (Ile/I) an isomer of Leucine R ≡ CH2-CH-(CH3)2 H ― ― NH3 e) ⊕ R ≡ CH-CH2-CH3 ― ― CH3 CH3 ― CH2 ― CH — C — COO – CH3 Proline (Pro/P) – a substituted α- amino group – α imono acid found in proteins where the amino group is substituted H2C ― CH2 ― ― H2C C NH2 COO – H ⊕ one of H from CH3 The R group is a substituent in the amino group f) Phenylalanine (Phe/F)- an aromatic amino acid Alanine + have a benzene ring added H ― ― ― CH2 — C — COO – +NH3 R g) Tryptophan (Trp/W) – an aromatic amino acid Indole Acetic Acid (IAA) - a plant hormone H ― ― ― C ― C ― H2C — C — COO– ‖ CH ⊕NH3 N ― H R ≡ indole group h) Methionine (Met/M)- R has a sulfur atom present H ― ― ⋆ CH3 — S ― H2C ― H2C — C — COO ⊕NH3 R Provides methyl groups for metabolism- the initiating amino acid during protein biosynthesis occurs in the interior of proteins i) Glycine (Gly,G) -simplest amino acid – non-polar but can be considered as polar H — ― H3N ⊕ — C — COO H R≡H In an amino acid sequence: in a polypeptide chain the R ≡ H is polar ⊕NH3 COO - eg. Ala ― Gly — Phe In a polypeptide (several amino acids are linked by the peptide bond) the NH3+ group and COO– group are tied up so H+ in Glycine is polar. 2. Polar R-Groups:- more soluble. Their R groups contain neutral uncharged polar functional groups which can be H-bonded with H2O. (uncharged + hydrophilic -loves water) a) Serine (Ser/S) polarity due to their OH– group H ― — HO― CH2 — C — COO R ⊕NH3 OH group is attached to CH3 group of Ala ⇒ allows for H-bonding How amino acid → Proteins by Peptide Bond formation where: H H2N — C — COOH + H2N — C — COOH ― ↓ ― ― ― H [ -H2O] R1 R2 O H ‖ H2N — C — C ― N ― C ― COOH ― ― H H ― ― ― R1 O ‖ C−O−H H N H R2 peptide bond COOH NH2 A simple dipeptide : H2N − Ala − Gly − COOH A Tripeptide eg. H2N −Ala − Gly − Phe − COOH 5-10 amino acid ⇒ oligopeptide >10 amino acid ⇒ polypeptide – common in the body,enzymes, haemoglobin, trypsin etc. Each amino acid is referred to as Residues eg. H2N − Ala − Gly − Phe − Arg….. ↑ Val − COOH N terminus ↓ C terminus b) Threonine (Thr/T) H H ― ― ― ― CH2 ⊕NH3 H3C ― C — C — COO – ― OH R c) - OH group present Tyrosine (Tyr/Y)- similar to phenylalanine – add a OHgroup onto the para-position of benzene ring H — HO― – — ― CH2 — C — COO ⊕NH3 This is an aromatic amino acid d) Cysteine (Cys/C) – occurs in its oxidised form as cystine in proteins. - Important in the formation of Disulfide bonds S-S - In some proteins, eg. insulin disulfide bonds are present with A-chain and B-chain. - Cysteine residues are present in both chains with the two chains linked by the disulfide bridge and if this bridge is broken then it becomes IN ACTIVE. H ― – ― HS ― H2C — C — COO R NH3 ⊕ cysteine – incorporated into proteins during synthesis H — – OOC ― C ― CH ― SH 2 — — H HS ― CH2 ― C ― COO – + — ↓ [ O ] / –H2 +NH3 ⊕NH3 — ― — — ― C ― CH2 ― S = S ― CH2 ― C ― cystine found in blood. S–S e) Asparagine (Asn/N) – polarity due to amide group. First isolated from asparagus H — R C= O an amide group present ― — H2N ― C ― H2C — C — COO ‖ O ⊕NH3 _ NH2 Glutamine (Glu)- related to asparagine (polar amide group) ↓ (an extra CH2 group) f) Function as a H-bond donor H — NH2 — C ― CH2 ― CH2 — C — COO – Four hydrophilic residues occur on the exterior surface of proteins ⊕NH3 CO group functions as a H-bond acceptor Negatively charged R-groups:a) Aspartic Acid (Asp/D) referred to as aspartate when charged 3. monoamino dicarboxylic amino acid — act as proton acceptors H negative charge at physiological COO–― CH — C — COO– 2 If it is not charged pH that is COOH — R ⊕NH3 NH2 COOH aspartic acid b) A cousin is Glutamic Acid (Glu/ E) extra CH2 group H ― — – – COO ― CH2 ― CH2 — C — COO referred to as Glutamate ⊕NH3 These are referred to as Acid Amino Acid – since the R- group is a carboxylic acid group (COO-) 4. Positively Charged R- groups:- (Basic- have a net positive charge) a) Lysine (Lys/K) diamino monocarboxylic amino acid H ― ⊕ — – H3N ― CH2 ― CH2 ― CH2 ― CH2 — C — COO ⊕NH3 R b) Arginine (Arg/R) H ― — NH2 ― C ― NH ― CH2 ― CH2 ― CH2 — C — COO ‖ ⊕NH2 ⊕NH3 R ≡ guanidinium group ⊕ c) Histidine (His/H) H ― H⊕N NH ― ― ― – HC = C ― CH2 — C — COO +NH3 CH R ≡ imidazole group –  A Toxic Amino Acid- eg. hypoglycin :- found in the ackee-responsible for the disease known as Jamaican Vomitting Sickness  Raw or Uncooked causes hypoglycaemia- ie. the blood sugar level is decreased to ~½ ⇒coma due to the brain cells lacking glucose ⇒Death (vomitting stops).The malnourished child will die much quicker CH2 — H ² α ¹ – H2C = C ― CH — CH2― C — COO β ⊕NH3 —  Scientific Name: L-α-amino-β-methylene cyclopropyl propionic acid Hypoglycin-Arilus- part to cook in the ackee (boil)-most of the hypoglycin is in the seed; 10 amino acid ⇒ polypeptide – common in the body,enzymes, haemoglobin, trypsin etc. Each amino acid is referred to as Residues eg. H2N − Ala − Gly − Phe − Arg….. ↑ Val − COOH N terminus ↓ C terminus BC 10A AMINO ACID METABOLISM Lecture 2 AMINO ACID METABOLISM  In higher animals, a.a.’s serve as building blocks of proteins and as precursors of many other important biomolecules such as hormones, purines, pyrimidines porphyrins and vitamins. They also serve as a source of energy particularly when they are ingested in excess. When they are used as fuel the A.A.’s undergo loss of their amino groups, their carbon skeletons having two major fates: 1.oxidation to CO2 via TCA 2.conversion into glucose by gluconeogenesis  Vertebrates actively oxidise both exogenous and endogenous a.a.’s. Digestion  Since the proteins cannot enter the epithelial cells, they must be digested into a.a.’s or di-or tripeptides before absorption can occur.  Digestion involves several stages including: 1. the extraction of proteins from food 2.denaturation of proteins 3. hydrolysis of proteins etc..  Protein is extracted from the food by the process of mastication and by the mechanical activity of the stomach. The low pH of the stomach plays the role of denaturation of proteins thus making it more accessible to the proteolytic enzymes. Digestion  For early stages of digestion four types of enzymes are important:  Pepsin- secreted by serous cells in gastric gland  Trypsin, elastase and chymotrypsin- secreted by acinar cells of pancreas.  These enzymes are synthesized as inactive precursors (zymogens) which contain one or more extra peptides.  Pepsinogen HCl → Pepsin + 5 polypeptides  Pepsinogen produced by the chief cells. Pepsin, an endopeptidase catalyses the hydrolysis of proteins → large polypeptides. The active site of pepsin contains Aspartate which reacts with the carbonyl grps. of peptide bonds via its carboxylate oxygen C=O Digestion → Trypsinogen Trypsin + 6 polypeptide enterokinase (EK)/ trypsin Trypsin can autocatalyse itself and further catalyses Chymotrypsinogen → Chymotrypsin + peptides trypsin /chymotrypsin Pro-elastase → trypsin elastase + peptides  Trypsin, chymotrypsin and Elastase are endopeptidases- catalyse hydrolysis of internal peptide bonds of proteins (A) Digestion of Proteins By Gastric Secretion Begins in the stomach which secretes gastric juice- containing HCl and pepsinogogen. HCl- stomach acid is too dilute (pH 2-3) to hydrolyse proteins. - Function is to kill some bacteria and to denature proteins so more susceptible to proteases. 1. 2. Pepsin- the acid stable endopeptidase secreted by serous cells of the stomach as an inactive zymogen-pepsinogen which is activated to pepsin either by HCl /autocatalytically by other pepsin molecules. - Pepsin releases peptides and a few a.a. from dietary proteins.  Zymogens contain extra a.a.’s preventing them from being catalytically active. Removal of these a.a.’s permit the folding required for an active enzyme. Zymogens → trypsin + hexapolypeptide trypsin EK  Trypsinogen  Chymotrypsinogen → chymotrypsin + peptides trypsin chymotrypsin (B) Digestion of Proteins By Pancreatic Enzymes On entering the small intestines.  Large polypeptides produced in the stomach by the action of pepsin are further degraded to oligopeptides and a.a. by pancreatic proteases. 1. Specifically e.g. Trypsin cleaves only when the C=O gp of the peptide bond is contributed by Arg/Lys. 2. Release of zymogens-by the secretion of Cholecystokinin and secretin-two polypeptide hormones of the digestive tract. 3. Activation of zymogens- enteropeptidase (enterokinase), enzyme synthesized by and present on the luminal surface of Intestinal mucosal cells of the brush border membrane converts the pancreatic zymogen. Peptidases  The product of thee proteolytic enzymes are peptides of various sizes. These are further degraded by the action of various peptidases that remove terminal a.a.’s.  Carboxypeptidases A and B- hydrolyse a.a.’s sequentially from the carboxyl end of peptides.  Amino peptidases hydrolyse sequentially a.a.’s from the amino end of the peptides.  Dipeptidases → dipeptides → a.a’s  The pro-enzymes contain one or more extra peptide bond which prevent the formation of a three dimensional structure which possesses the catalytic activity. The extra bonds are removed by peptide bond clevage. In the stomach the acidic pH promotes pepsinogen → pepsin + peptide.  The brush border cells of the small intestine secrete small amounts of a proteolytic enzyme called enteropeptidase (enterokinase) which initiates the reaction  These proteolytic enzymes are proteinases i.e. they hydrolyse links in the middle of the peptide bonds. Absorption  This occurs in the jejunum and ileum  The transport of a.a. from the lumen into the cell is an active process like glucose. A specific carrier in the brush border of the absorptive cell combines with the a.a. and the Na+ ion and conveys them to the inner face of the membrane where they are released to the LIVER via superior mesentric and portal veins. Absorption of AA and peptides  Intestinal cells take up free a.a. and some di- and tripeptides by special transport proteins.  All of the peptides are hydrolysed to free a.a.’s in enterocytes before their release into the hepatic portal vein Proteins  Proteins are never stored in the system due to the N- hence eliminated or used in the synthesis of purines and pyrimidines.  N- is toxic to the system- urea/uric acid (ureate)  Most of the urea comes from metabolic breakdown of a.a.  Can store the C- skeleton but not the amino grps. So use this to produce nitrogenous products and excess →NH3 and urea Balance between N  N (coming in as proteins) = N ( going out as urea)  If Intake < Excretion -ve N balance seen in starvation and wasting diseases.  If Intake > Excretion + ve N. balance seen in the Normal state in the growth of man Essential and Non-essential a.a.  Essential a.a- Arg, his, ile, leu, lys, met, phe, thr, tryp, val. (PVT TIM HALL)  Non- essential- not needed by the system.  Arg is essential only to children since needed for Growth but not in adults. The system can produce Arg later in life but NOT in children as there is an excess amt. needed.  Is essential for children Types of A.A.’s  Ketogenic – give ketonene bodies C=O  Glucogenic- give CHO  Some a.a’s are both Classes of enzymes 1) Oxidase 2) Transferase 3) Hydrolase 4) Ligase 5) Isomerase 6) Lyase GENERAL REACTIONS INVOLVING AMINO ACIDS: 2 MAJOR REACTIONS: 1. Amino Acids Oxidase Reactions a.a. oxidase α keto acid + NH3 (essential) A.A. ammonia O2 H2O2 catalase } – not very essential H2O Transamination  During the catabolism of at least 20 a.a., the α-amino group is enzymatically removed by transamination. During this process the α-amino group is transferred to α-carbon atom of an α-keto acid. Leaving an α-keto analogue of the incoming A. A. and causing the amination of the α-keto acid to form an a.a. ― NH3+ H — C — COO − + O ‖ C — COO − ― R2 α-amino acid α-keto acid ― ― R1 ⇌ O ‖ C — COO− R1 + new α-keto ― NH3 H — C — COO− ― R2 new α-amino Transamination  The α-amino group of 20 a.a.’s found in proteins are removed at some stage in their oxidative degradation if not re-used these amino groups are collected and converted into a single excretory group-UREA. This removal of the amino group is promoted by transaminases/amino transferase.  During transamination, the α-amino group of an amino acid is enzymatically removed from α-a.a. and transferred to αCarbon atom of α-ketoglutarate (KG) leaving a corresponding α-keto acid and causing the amination of α-KG. 2.  Transaminase Reactions -Transamination The transfer of an α-NH2 group from an α-amino acid to an α-keto acid. The enzymes are known as Transaminases/amino Transferases. It is involved in: a) Synthesis of non-essential a.a. b) Degradation of most a.a.’s c) Exchange of a.a. group  There are TWO a.a.’s - SERINE and THREONINE which do NOT go through this reaction because they have hydroxyl (OH-) groups present hence use DEHYDRATASE enzyme.  There are TWO amino acids e.g. Serine and Threonine which do     NOT go through this TRANSAMINASE reaction because of the hydroxyl (OH-) group present hence they use DEHYDRATASE enzyme. They do not have to go through the UREA CYCLE but all others have to. SERINE → PYRUVATE + NH4+ THREONINE → KETO BUTYRATE + NH4+ Pyruvate + NH4+ α - Ketobutyrate + NH4+  Serine  Threonine H2O — Serine dehydration Serine dehydratase C=O + NH4+ = — CH2OH NH3 ― C ― H H2O COO- — — NH3 ― C ― H COO- — COO- CH2 CH3 (deamination) pyruvate This involves pyridoxal phosphate and schiff base – more of a transaminase reaction but not readily reversible. The enzyme is called a dehydratase because dehydration precedes deamination a) Most transaminase use Glutamic Acid or Keto Glutarate as one of the reactant. Glutamic Acid + keto acid → α-KG + a.a. This is called the Glutamate Transaminase. Another transaminase - Alanine transaminase uses keto acid- pyruvate. For some reactions which are reversible the transaminase reactions are REVERSIBLE. b) a.a. + pyruvate → k.a. + Alanine a) Glutamate Transaminase CH2 R1 COOH + NH3 ― C ― H + CH2 ― ― CH2 ― ― CH2 a.a + ― ― ― H H3N ― C COO ― ― C=O ― C=O ― COO ― COOH ― R1 COOH Glutamate COOH α KG b) Alanine Transaminase ― CH3 Glutamate ― COO H ― C ― NH3 Alanine - Pyruvate ― C=O CH3 ― α KG COO (transaminase) aminotransferase - Alanine + SUCH REACTIONS: e.g. A) Glutamate transaminase a.a. + KG → K.A. + Glutamate B) Alanine transaminase a.a. + pyruvate → K.A. + Alanine  For some reactions the transaminase reactions are REVERSIBLE.  These two transaminases funnel –amino groups from a variety of compounds to GLUTAMATE for conversion to AMMONIA.  The amino group of most a.a.’s are funneled into the formation of GLUTAMATE  NH3 and aspartate are the two amino group donors to the urea cycle.  Alanine transaminase and glutamate dehydrogenase-must give glutamine which leads to the urea cycle. A combination of transamination and deamination to give – keto acid, NH4+ + free a.a. COO- COO― ― aminotransferase COO- Aspartate α KG glutamate C=O ― ― α KG + H ―C ― NH3+ CH2 ― ― CH2 COO- oxaloacetate + Glutamate  In a.a. metabolism all these reactions if they are to be degraded must go through GLUTAMATE DEHYDROGENASE → UREA Synthesis.  The amino group of most a.a. are funneled into the formation of Glutamate or Aspartate which are interconverted by GlutamateAspartate amino transferase.  Glutamate + Oxaloacetate → α -KG + Aspartate  Oxidative Deamination of Glutamate → NH3 + α KG  There is no NET loss of NET AMINO GROUP in these reactions. The whole purpose of transamination is to collect the amino group from ALL OVER to form GLUTAMATE. The products must end in GLUTAMATE for transamination → NH3 + UREA Ala + KG → pyruvate + Glutamate 2. Asp + KG→ OAA + Glutamate 3. Leu + KG → Ketoisocaproate + Glutamate 4. Tyrosine + KG → p-phenylpyruvate + Glutamate 1.  Ammonia is formed from Glutamate by OXIDATIVE DEAMINATION. The enzyme responsible is GLUTAMATE DEHYDROGENASE , a mitochondrial enzyme-occurs in the mitochondria.  GLUTAMATE + NAD+/NADP+ + H2O → NH4+ + α-KG + NADH/NADPH + H+  NAD+/NADP+ in this reaction is serving as a co-factor. The oxidation is transfer of a hydride ion.  READ MECHANISM- The prosthetic group of all transaminases is PYRIDOXAL PHOSPHATE – a derivative of pyridoxine (Vitamin B6).  OXIDATIVE DEAMINATION  TRANSAMINATION is followed by oxidative deamination catalyzed by pyridine linked glutamate dehydrogenase. Ammonia is discharged after being collected by glutamate. Cannot let NH4+ build up in tissue: 1) Affects the pH 2) Affects important reaction that is effect of hyperammonaemia Liver is the only place can turn NH4+ → UREA, it is necessary to carry NH4+ from tissue→LIVER. It is not good to transport a lot of NH4+ in the blood as it interferes with the body system so it is carried as GLUTAMATE to form GLUTAMINE. In the Liver: reconvert it: GLUTAMINE → GLUTAMATE + NH4+ H - C - COO- H - C - COOGlutaminase (liver) CH2 ― CH2 C COO- NH3 + Glutamine + ― This reaction is in the TISSUE ― Glutamate CH2 ― ― COO- CH2 ― ― CH2 Glutamine Synthetase (tissue) H - C - COO- ― ― NH4+ / ATP CH2 H2O ― ― NH3 + NH3 ― + NH3 Glutamate IN THE LIVER NH4+ IN UREOTELIC ORGANISMS- urea is synthesized by the UREA CYCLE. (from ammonia) NH2 (from CO2 ) C=O (from H2O) NH2 (from Aspartate)  One of the N- atoms of UREA comes from NH4+ and the other from ASPARTIC ACID (ASPARTATE). The C atom is derived from CO2. “Ornithine” is the CARRIER of these N and C atoms of the UREA CYCLE. UREA CYCLE  Since it takes place in the liver some of the NH4+ formed in the breakdown of a.a.’s is consumed in the biosynthesis of nitrogen compounds. The excess NH4+ is converted into UREA and EXCRETED.  Urea Cycle was the first metabolic pathway to be discovered. One of the nitrogen atoms of urea in this pathway comes from NH3 (ammonia). Whereas the other one comes from aspartate. The Carbon –atom is derived from CO2 and oxygen is from H2O.  The first free amino group entering the UREA CYCLE arises from the GLUTAMATE which is oxidatively deaminated to an – keto glutarate in the LIVER. GLUTAMATE + NAD+/NADP+ + H2O → NH4+ + α-KG + NADH/NADPH + H+  The free ammonia so formed is now utilized to form CARBAMOYL PHOSPHATE after combining with CO2 in the MITOCHONDRIAL MATRIX. This reaction is catalysed by CARBAMOYL PHOSPHATE SYNTHETASE I and utilizes 2ATP’s. WHAT HAPPENS TO NH4+ in the Liver? UREA CYCLE Fumarate arginino succinase arginase H2O O= Arginine Urea H2N ― C ― NH2 Ornithine ornithine transcarbamoylase Arginino succinate O= arginino succinate synthetase Aspartate cytosol NH2 ― C ― P Citrulline carbamoyl phosphate CO2 + NH4+ In mitochondria  Urea –a neutral electrolyte compound. We generate UREA by a cycle of reactions. The UREA cycle was worked out before the TCA Cycle. THE REACTIONS:  The formation of carbamoyl phosphte (in the mitochondria) P  2ATP + CO2 + NH4+ + H2O→ NH2-C=O + 2ADP + Pi + H+  The consumption of 2 molecules of ATP makes this synthesis IRREVERSIBLE. The enzyme requires N-ACETYL GLUTAMATE for activity. Carbamoyl Phosphate is a high energy compound =O 1. THE FORMATION OF CARBAMOYL PHOSPHATE - in mitochondria OH ― carbamoyl O= C―O ― P ―O ―n z + ADP ― CO2 + ATP + enzyme phosphate synthetase I O- enzyme complex one phosphate used to form the complex NH+4 (NH3) O= O= ― NH2 ― C ― O ― P ― O ― O- ATP ADP + Pi + enzyme nʓ =O =O carbamoyl phosphate carbon dioxide OH ammonia ӏ regulatory enzyme 2 ATP + CO2 + NH+3 + H2O NH2―C―O―P―OH + 2ADP + Pi + H+ carbamoyl phosphate synthetase I Carbamoyl phosphate high energy comp’d The rxn is Irreversible  The carbamoyl phosphate generated in the mitochondria now donates its carbamoyl group to ORNITHINE. Ornithine is usually formed in the CYTOSOL but enters the mitochondria via its specific transport system. This reaction is catalysed by ORNITHINE CARBAMOYL TRANSFERASE/ORNITHINE TRANSCARBAMOYLASE/ CITRULLINE SYNTHETASE of the mitachondrial matrix. The citrulline now formed leaves the mitochondria and passes into the cytosol where the remaining reactions of the UREA CYCLE take place. Citrulline is formed therefore by the transfer of the carbamoyl group from its phosphoric anhydride (CO) to the δ–amino group of Ornithine. 2. THE FORMATION OF CITRULLINE – (in the mitochondria) ammonia ― ― ornithine carbamoyl carbon dioxide transferase ― H ― C ― NH2 ― L-ornithine CH2 ― COOH CH2 ― ― α H ― C ― NH2 Mg ++ δ CH2 ― β CH2 NH ― ― carbamoyl phosphate + ɤ CH2 C=O ― δ CH 2 C=O ammonia NH2 ― ― ― NH2 O ― PO3H2 [ or citrulline synthetase] NH2 COOH L-citrulline + Pi THE FORMATION OF ARGININO SUCCINATE  The second amino group required for urea synthesis arrives from     aspartate which is formed from GLUTAMATE by ASPARTATE aminotransferase in the cytosol. The amino group of aspartate condenses with carbamoyl carbon atom of CITRULLINE in the presence of ATP to form ARGININO SUCCINATE. The reaction is synthesized by arginino succinate synthetase. This is a condensation reaction of aspartate and citrulline and is driven by the cleavage of: ATP → AMP and PPi. The third and final molecule of ATP is consumed. AMP and PPi are also formed. PPi + H20 → 2 Pi PPi is hydrolysed by pyrophosphatase to inorganic phosphate thus pushing the reaction to the right. 3. THE FORMATION OF ARGININO SUCCINATE ammonia = AMP + PPi ― H ― C ― NH2 ― COOH citrulline CH2 CO2 CH2 H — C — NH2 — ― CH2 CH2 — aspartate H — CH2 CH2 — ― COOH ― ― CH2 arginino succinate synthetase — H ―C ― NH2 — + HN ― C ― N ― C ― H — NH COOH ― CH2 ATP ― ― C=O ― ― COOH — NH2 + NH2 COOH arginino succinate COOH aspartate 4. THE FORMATION OF ARGININE In the next step, argininosuccinate undergoes β-elimination by the action of Arginino succinate lyase to give free arginine and FUMARATE which returns to the citric acid pool. COO- CH2 ― CH2 ― ― CH2 CH2 H − C − NH3 ― ― H − C − NH3 Arginino succinate COO- asp ― Fumarate ― NH ― CH2 ― ― aspartate C ― NH2 CH ― ― COO- CH2 elim’n + = ― ― ― NH CH = lyase C ― N―C―H CH2 + NH2 COO- ― COO- ― = carbamoyl H ― + NH2 COO- Arginine 5. THE FORMATION OF UREA  Arginine formed becomes the intermediate precursor of UREA whereas FUMARATE returns to tricarboxylic acid (TCA) cycle. The UREA CYCLE is completed by the hydrolysis of Arginine → Ornithine and Urea. The enzyme used is ARGINASE ammonia aspartate C=O ― ― Arginase NH H2O 2 β Urea CH2 + α H ― C ― NH3 ― ― CH2 ɤ CH ― ― CH2 2 ― ― NH2 + δ CH ― C ― NH2 from H2O NH2 ― = NH2 ― + NH2 COO- + H ― C ― NH3 Ornithine ― CH2 ― COO- Arginine CHEMICAL BALANCE OF UREA SYNTHESIS  NH4+ + CO2 + 2ATP + H2O→ CarbamoylPO4 + 2ADP + Pi  CarbamoylPO4 + Ornithine → Citrulline + Pi  Citrulline + ATP + Aspartate → ArgininoSuccinate + AMP + PPi  ArgininoSuccinate → Arginine + Fumarate  Arginine + H2O → Urea + Ornithine OVERALL BALANCE AFTER CANCELLING BOTH SIDES: NH4+ + CO2 + 3ATP + 2H2O → UREA + 2ADP +AMP + PPi + 2Pi + Fumarate AA. glutamate via GDh OVERALL REACTION: Urea cytosol ornithine H2O NH3 2ATP + CO2 + H2O arginine fumarate ornithine 2 ADP + Pi Carbamoyl phosphate arginino succinate AMP + PPi Pi citrulline citrulline ATP Aspartate NH3 Transamination glutamate cytosol inner membrane mitochondria UREA CYCLE AND TCA CYCLE  The stoichiometry of Urea synthesis is:  NH4+ + CO2 + 3ATP + 2H2O → UREA + 2ADP +AMP + PPi + 2Pi + Fumarate  The synthesis of fumarate is important as this links the UREA CYCLE to the TCA Cycle. Fumarate is hydrated to malate which is in turn oxidized to oxaloacetate Relationship between Urea Cycle and TCA Cycle α k acid Aspartate Citrulline α a.a. CO2 + NH4+ OAA Arginino succinate Ornithine carbamoyl PO4 (lyase) Arginine Malate Fumarate Urea UREA CYCLE TCA CYCLE RELATIONSHIP BETWEEN UREA CYCLE & TCA CYCLE DEFECTS OF UREA CYCLE ENZYMES:  Any enzymatic defects in the urea cycle causes HYPERAMMONAEMIA that is high levels of NH4+ ions are TOXIC to human resulting in COMA and DEATH shortly after birth. Partial deficiency of these enzymes causes MENTAL RETARDATION. DEFECTS OF UREA CYCLE ENZYMES: It is thought that the toxicity caused by high NH4+ occurs as follows: α-KG NH4+ GDH * GLUTAMATE NH4+ Glutamine synthetase * GLUTAMINE  Excess NH4+ leads to the depletion of α–KG which is used in Glutamate production. The reaction goes further in the presence of excess NH4+ ions to give Glutamine.  The depletion of α–KG, a TCA cycle intermediate leads to a decrease in the rate of formation of ATP. The toxicity results since the brain is highly vulnerable to decreases in ATP level.  Since the TCA cycle is affected by α–KG , ONLY ONE NAD is produced, no FAD or the other two molecules of NAD thus leading to less ATP production. HYPERAMMONAEMIA: Inherited hyperammonemia:  Genetic defects of various enzymes of urea cycle have been observed in many humans. High levels of NH3 are toxic to humans.  A complete block of any of the steps is INCOMPATABLE with life because there is no other way by which urea can be formed.  Inherited disorders caused by partial block cycle enzymes leading to high levels of NH3 in the blood. Such high levels could lead to COMA or DEATH. Other symptoms are mental retardation, lethargy and vomiting. Treatment:  Low protein diet and α-Keto analogues. α-keto analogues take up NH3 and convert it to GLUTAMATE thereby lowering the NH3 content. Reasons:  The brain is highly vulnerable. A high concentration of NH4+ shifts the equilibrium of GDh reaction to the formation of Glutamate. Glutamate results in the depletion of α-keto glutarate.  This leads to the decrease in the formation of ATP via TCA cycle. Glutamate can further form GLUTAMINE by an addition of NH4+. Pyrimidine derivatives are increased and appear in the urine. Portal System Encephalopathy (PSE):  This type of hyperammonaemia is an acquired type. It is as a result of development of collateral circulation around the liver due to cirrhosis or hepatitis.  As a result of this shunting the circulatory access to the liver is severely impaired and detoxification of NH3 is impaired. Blood ammonia levels increase drastically (500 µg/mL). Normal: 4-5 µg/mL CATABOLISM OF AA’s  The CARBON atoms of degraded a.a. form major metabolic intermediate that can be converted into Glucose or be oxidized by the TCA.  The C-atoms of the 20 a.a.’s are converted into SEVEN MOLECULES. They are: *Pyruvate *Acetyl CoA *Acetoacetyl CoA *α-KG *Succinyl CoA *Fumarate *Oxaloacetoacetate (OAA)  AA degraded to Acetyl CoA or Acetoacetyl CoA are termed KETOGENIC to give Ketone bodies  Those degraded to the others are GLUCOGENIC to give Glucose and all others. Some a.a.’s are PURELY KETOGENIC, PURELY GLUCOGENIC or can be either one. One is truly ketogenic- LEUCINE.  The acetyl CoA and Acetoacetyl CoA cannot be converted to Glucose as there is NO ENZYME present for this but this enzyme is present in plants. Glucose Alanine Glycine Cysteine Serine Threonine Both ! Ketogenic + Glucogenic Isoleucine Leucine Tryptophan Purely ketogenic Leucine Lysine Phenyl Ala Tyro. Tryp. PEP Pyruvate Asparagine + Aspartate Acetyl CoA ⇌ Aceto acetyl CoA oxaloacetate citrate α -KG fumarate α- KG Tyrosine Phenyl Alanine Aspartate Both glucogenic and ketogenic Succinyl CoA Isoleucine Methionine Threonine Valine aceto acetyl CoA acetyl CoA glutamate glutamine Hist Proline Arginine Isoleucine Lysine Phenylalanine Tyrosine Tryptophan DEGRADATION OF PHENYLALANINE  To Show Inborn Errors Of Phenylalanine (involves Tyrosine Metabolism) To Show Inborn Errors of Phenylalanine (Catabolism)- Degradation of Phenylalanine COO- − − − + H3N − C − H quinoide dihydrobiopterin (oxidised) (reduced) Tetrabiopterin + H3N − C − H CH2 phenyl alanine H2O *Lack of of hydroxylase leads to =O + PKU phenylketonuria) - C=O C=C - Aceto acetate H2O ~ H − fumaryl acetoacetase CH2 H (trans) Fumarate COO H+ − -OOC − C = C − C − CH − C − CH COO2 2 H O O 4 fumaryl acetoacetate (trans) mAA isomerase O 4 maleyl acetoacetate (4 mAA) dihydro ascorbate (cis) Homogentisate 1,2 dioxgenase or homo oxidase (2 oxygens) defect of this enzyme → alcaptonuria − = H O ascorbate − = − H O2 hydroxylase HCO-3 OH − C = C − C − CH2 − C − CH2COO- − -OOC p-OH phenyl pyruvate OH = = − trans Tyr transaminase defect of this causes Tyrosinemia COO- − − − - H3C − C − CH2 − COO COO Tyrosine (one oxygen added) OH Phe hydroxylase or Phe-4-mono oxygenase H CH2 − O2 − − − COO- OH CH2COO- To Show Inborn Errors Of Phenylalanine (involves Tyrosine Metabolism) (due to melanin) Phenylalanine Lack of hydroxylase leads to PKU (phenylketonuria) transaminase * phe hydroxylase Melanine tyrosine hydrolase Tyrosinase Dopadecarboxylase 3,4 dihydroxyphe (DOPA) defect leads to tyrosine accumulation ⇒ albinism p-OH phenyl pyruvate ↷ defect of this Tyrosine causes Tyrosenemia Tyrosine Albinism CO2 - OH Homogentisate HO- -CH2-CH2 HO homogentisate oxidase 1,2 dioxygenase 4 Maleylacetoacetate (MAA) (cis) 4 Fumaryl Acetoacetate (trans) Fumarate Acetoacetate (Defect of this enzyme ⇒ Alcaptonuria) dopamine-βhydroxylase [O] Norepinephrine - hydroxylase Dopamine NH2 Epinepherine Melanin Synthesis  Melanin is a dark pigment found in the skin, hair and the choroid coat of the eye.  It is formed from phenylalanine and tyrosine and is present in the skin (melanoblasts)  The Synthesis: phenylalanine to tyrosine using tyrosine hydroxylase then to DOPA (dihydroxyphenylalanine using tyrosinase and oxygen) to dopaquinone which is a fast reaction then to the formation of leuco compound and then hallochrome –red and then polymerises) to melanin PHENYLKETONURIA (PKU)  This is an inborn error of phenylalanine metabolism.  Untreated patients with phenylketonuria are severely retarded.  1% of patients in mental hospitals are phenylketonurics.  The weight of the brain is below normal, myelination of the nerves is defective and their reflexes are hyperactive.  The life expectancy is shortened. Half are dead by age 20 and ¾ by age 30. PHENYLKETONURIA (PKU) cont’d  This is the most prominent disease caused by the deficiency of an enzyme of a.a. metabolism phenylhydroxylase/phenylalanine 4 monooxygenase which converts Phe to Tyr or more rarely by the absence of the tetrahydrobiopterin cofactor.  As a result phenylalanine cannot be converted to tyrosine and it accumulates in all body fluids. Hence the phenylalanine is transaminated to phenyl ketone. Thus there is excretion of phenylpyruvate (a phenyl ketone) in the urine.  Phenyl pyruvate is detected as a green colour with ferric chloride (FeCl3). PHENYLKETONURIA (PKU) cont’d  The deficiency is caused by an autosomal recessive gene.  In these patients phenylketonurics-severe physiological symptoms, low Intelligent Quotient since the enzyme is impaired, tyrosine metabolism is affected too leading to MENTAL RETARDATION, light colour of skin, eyes (albinism).  Phenylacetate, phenyl lactate,o-phenyl lactate are derived from phenyl pyruvate SKIN (HYPOPIGMENTATION):  Phenylketonurics have lighter skin and hair colour than their siblings. The hydroxylation of tyrosine by tyrosinase, which is the FIRST step in the formation of MELANIN is COMPETITIVELY BLOCKED by high levels of phenylalanine in the phenyl ketonurics, and less melanin is formed.  Mental retardation is an enigma.  Phe a competitive inhibitor of tyrosine hydroxylase → MELANIN SYNTHESIS. PHENYLKETONURIA (PKU) cont’d  CNS SYMPTOMS: Mental retardation, failure to walk or talk, seizures, hyperactivity, tremors and failure to grow.  SYMPTOMS: Vomiting, retarded growth  DIAGNOSIS AND TREATMENT: Phenylketonurics appear normal at birth but are severely defective AT THE AGE OF ONE if untreated. The therapy of such patients is a LOW PHENYLALANINE DIET. The aim being to provide just enough Phe to meet the growth needs. This diet must be started after BIRTH to avoid IRREVERSIBLE brain damage.  EARLY DIAGNOSIS: is essential to test the urine of new borns with FeCl3 which would give a green olive colour in the presence of phenylpyruvate.  Phenylalanine levels in the blood is a more appropriate test. The measurement of intravenous phenylalanine from blood is a more definite test.  High level of phenylalanine in pregnant women results in the abnormal development of the foetus. This is a striking example of MATERNAL FOETAL RELATIONSHIP at the MOLECULAR LEVEL.  CLINICAL MANIFESTATIONS: Severely mentally retarded. This is not a very well understood neurological disorder.  It is clearly related to the accumulation of phenylalanine rather than tyrosine deficiency since dietary control of phenylalanine levels prevents MENTAL RETARDATION.  Phe may compete and inhibit the transport of other A.A.’s to the brain during the development of growth and maturation.  CLINICAL MANIFESTATIONS: Severely mentally retarded. This is not a very well understood neurological disorder.  It is clearly related to the accumulation of phenylalanine rather than tyrosine deficiency since dietary control of phenylalanine levels prevents MENTAL RETARDATION.  Phe may compete and inhibit the transport of other A.A.’s to the brain during the development of growth and maturation. CLINICAL MANIFESTATIONS:  Mental retardation  Poor psychomotor maturation  Tremors, vomiting  Seizures  Hypopigmentation eczema  I.Q. score less than 50  Urine odour ( phenylacetate) TYROSINAEMIA:  The deficiency of Tyrosine transaminase is responsible for the accumulation of Tyrosine and other metabolites including nacetyl tyrosine, p-OH phenyl pyruvate, tyramine.  The disease is characterized by eye and skin lesions and sometimes MENTAL RETARDATION.  The deficiency of p-OH phenyl pyruvate oxygenase/ hydroxylase is believed to be responsible for neuro tyrosinaemia which is usually a temporary condition which in some cases respond to ASCORBIC ACID. ALCAPTONURIA:  The deficiency of Homogentsatic oxygenase.  Most of the ingested tyrosine is excreted as homogentisatic acid (hydroquinone) in the urine. This is colourless but on standing oxidizes itself (autooxidises) to the corresponding quinone which polymerises to form an intensely dark colour.  Homogentisate is slowly oxidized to pigments that are deposited in the bones, connective tissues and various organs. This is responsible for arthritis. ALBINISM:  In this condition, tyrosinase is deficient and MELANIN is not formed.  Lack of pigment in the skin makes albinos sensitive to sunlight which may cause CARCINOMA (cancer) of the skin in addition to burns.  A lack of pigmentation in the eye (skin) causes photophobia. The lack of pigment does not mean there is a problem with SIGHT but does not have the ability to look directly at sunlight (uv). The pupils are not dark but PINK. PARKINSON’S DISEASE (related to Phenylalanine)  This usually affects people over 60 years of age but occasionally in much younger people.  Tremors may develop that gradually interfere with motor function of various muscle groups.  The condition is named Parkinson’s Disease-the deficiency is established as degeneration of cells in certain small areas of the brain-sustantia nigra and locus coelureus.  The brain cells are NEVER REPLACED. Cells in these domains produce DOPAMINE ( a neurotransmitter) and the amount released decreases as a function of the # of surviving cells hence TREMORS. PARKINSON’S DISEASE  The administration of DOPA- can be used to relieve the symptoms (NOT PERMANENT) of tremors.  The elucidation of the major biochemical abnormality has not led to complete control of Parkinson’s Disease (that is dopamine becomes toxic after a certain concentration) FATES OF CARBON ATOMS OF DEGRADED AMINO ACIDS The strategy of Amino Acid degradation is to form major metabolic intermediates that can be converted to glucose or be oxidised by TCA cycle. Carbon skeletons of 20 amino acids are funneled into seven molecules i.e.Pyruvate, acetylCoA, acetoacetyl CoA, α-KG, succinyl CoA, fumarate and OAA. Ketogenic Amino Acids  These are those amino acids which are degraded into acetyl CoA or acetoacetyl CoA and they give rise to ketone bodies.  Glucogenic Amino Acids:  These are those AA which are degraded to:  Pyruvate, α-KG, succinyl CoA, fumarate and oxAA.  These can be then converted to GLUCOSE via phosphoenolpyruvate (PEP) THE C 3 FAMILY: Ala, serine and cysteine  These AA are converted to pyruvate  Eg. Alanine to pyruvate  Glycine  serine pyruvate deamination The C4 Family: Asp, asparagine  These are converted to oxAA  Aspartate + α-KG  Ox AA + Glutamate trans  Asparagine   aspartate NH3 OAA transamination α -KG The C5 Family: Glutamine, Proline, Arg, His  Eg  Glutamine Glutamate α-KG BRANCHED CHAIN A.ALeucine, valine , Isoleucine  This leads to maple syrup disease –is caused by the blocking of oxidative decarboxylation of these AAwhich are essential AA  These AA share certain structural analogies and have common degradative pathways.  In afflicted individuals the levels of these Aas and their corresponding α-keto acids are elevated.  The biochemical defect is the absence or greatly reduced activity of α- keto acid decarboxylase enzyme. This enzyme catalyses the conversion of all these 3 AAs to acyl CoA esters and carbon dioxide.  This enzyme catalyses the conversion of all these three a.a.’s to acyl CoA esters and carbon dioxide Maple Syrup Disease The most striking feature of the hereditary disease is the odour of the urine-maple syrup or burnt sugar. These patients suffer from branched chain ketonuria. ( branched chain amino acids accumulate in and spillover in the urine Although the new born appears to be normal, characteristic signs of the disease appear within a week after birth. The infant is difficult to feed and vomits- mental retardation if not corrected Maple Syrup Urine Disease (MSUD) or Branched Chain ketoaciduria  This disease is caused by the blocking of oxidative decarboxylation of these a.a –Leu,Ile, Val which are essential a.a’s.  These a.a’s share certain structural analogies and have common degradative pathways.  In afflicted individuals the levels of these a.a’s and their corresponding α–keto acids are elevated.  The biochemical defect is the absence or greatly reduced activity of α–keto acid decarboxylase enzyme. Maple Syrup Disease  becomes lethargic.  Hypoglycaemic  Convulsions and rigidity  Hypotonia- that is diminished tone of skeletal muscle Treatment and Diagnosis :  This can be done by the enzymatic analysis  Extensive brain damage results if the treatment is not given at the right time within a year. MAPLE SYRUP DISEASE Further to mental retardation there is a:  deficiency of myelination and  Reduction in brain lipid content The toxic agent which interferes with brain function is high leucine levels. The metabolites responsible for urine odour are α-ketoisocaproic acid which interferes with myelination Treatment: Strict dietary control of these amino acids ISOVALERIC ACIDEMIA  This is a rare genetic disorder in the degradative pathway of Leucine. It is presumed to be due to a block in the conversion of Isovaleric acid to β –methyl crotonic acid catalysed by isovaleryl CoA dehydrogenase ( Step 3 absent)  As a result isovaleric acid accumulates in the blood and urine and this gives rise to an odour “sweaty feet” –(rotten cheese) Symptoms: ( within a week) Vomitting, acidosis,tremors, lethargy, coma Surviving patients develop mental retardation Treatment: strict dietary control of leucine. Methyl Malonyl Aciduria  This is caused by the high levels of methyl malonic acid in the blood. In healthy persons this is not detectable. This disease is caused by the failure to convert methyl malonyl CoA to succinyl CoA and the defective enzyme is methyl malonyl CoA mutase  From valine: to methyl malonyl CoA to succinyl CoA using the enzyme mutase B12  As a result of this defect large amounts of methyl malonic acid are excreted in the urine (1 g/day). Normal healthy people excrete less than 5 mg/day Goitre and Tyrosine  The thyroid hormones thyroxine (T4) and 3,5,3’ tri-      iodotyrosine (T3) are synthesized in the thyroid gland from tyrosine residues in thyroglobulin, a glycoprotein. The tyrosine residues of thyroglobulin are first iodinated 3mono iodo tyrosine (MIT) Further iodination of MIT forming 3,5 di-iodotyrosine (DIT) and Coupling of two di-iodotyrosine to form thyroxine. This involves the reaction: 2I +2H++H2O2 I2 +H2O ( peroxidase) monoidotyrosine (MIT) Goitre and Tyrosine Goitre  The thyroid hormones thyroxine (T4) and 3,5,3’ triidotyrosine (T3) are synthesized in the thyroid gland from tyrosine residues in thyroglobulin, a glycoprotein. The tyrosine residues of thyroglobulin are first iodinated forming 3,5 diidotyrosine (DIT) and 3 monoidotyrosine (MIT) Caused by: long acting thyroid stimulation which is a gammaglobulin. Level of Thyroid Stimulating Hormone (TSH) in blood is normal indicating TSH is not responsible for the formation of excess thyroid hormones. The iodine content is lower than normal. Proportion of tri iodotyrosine to thyroxine (T4 ) is greater  The TSH, or thyroid-stimulating hormone, comes from the pituitary gland and stimulates your thyroid to release both T3 and T4.  1.  2.  3.  4. Iodination of tyrosyl residues in proteinmonoidotyrosine Further iodination of monoiodoform to form di iodootyrosine Coupling of two di-iodotyrosine to form thyroxine Coupling of one mono to di-iodotyrosine to form tri-iodotyrosine Symptoms: hyperthyroidism, thyrotoxicosis, goitre, more common in women Nervousness, restlessness, tremor, tiredness ,undue sweating breathlessness.. Symptoms The thyroid gland elaborates two hormones T3 and T4 which only differ in their iodine content The requirement of iodine is met by a highly efficient mechanism for concentrating the halogen – 40 times in the gland The iodine ingested in the diet is rapidly taken up by the tyrosyl residues and are iodinated These processes are regulated by TSH secreted by the Anterior Pituitary Prevention  Iodised salts  0.5 g KI to 1 kg of NaCl GOOD LUCK FOR YOUR EXAMS!!