BIOCHEMISTRY Protein Metabolism BIOB111 CHEMISTRY & BIOCHEMISTRY Session 25
Session Plan Digestion & Absorption of Proteins Amino Acid Utilization Amino Acid Degradation Transamination Oxidative Deamination The Urea Cycle Amino Acid Carbon Skeletons Amino Acid Biosynthesis B Vitamins & Protein Metabolism
Protein Digestion & Absorption Protein digestion starts in the stomach involves denaturation & hydrolysis of peptide bonds. Dietary protein entering the stomach promotes release of hormone Gastrin stimulates secretion of Pepsinogen & HCl. HCl has 3 functions in the stomach: Denatures proteins, exposing peptide bonds Kills most bacteria (ph = 1.5-2.0) Activates Pepsinogen (inactive) to Pepsin (active) Pepsin (enzyme) hydrolyzes about 10% peptide bonds
Protein Digestion & Absorption Small batches of acidic chyme containing large polypeptides enter the small intestine (SI) & stimulate secretion of hormone Secretin Secretin promotes pancreatic production of bicarbonate ions (HCO 3- ) help neutralize acidic chyme SI ph = 7-8 allows activation of pancreatic enzymes Trypsin, Chymotrypsin & Carboxypeptidase. Proteolytic enzymes in the SI: Break peptide bonds in proteins, liberating amino acids Trypsin, Chymotrypsin, Carboxypeptidase & Aminopeptidase are produced in inactive forms as zymogens & are activated at their site of action. Trypsin, Chymotrypsin & Carboxypeptidase produced by the pancreas Aminopeptidase secreted by intestinal mucosal cells The free amino acids are absorbed via intestinal wall into bloodstream.
Summary of protein digestion in the human body Stoker 2014, Figure 26-1 p9541
Amino Acid Utilization AAs produced from protein digestion enter the amino acid pool in the body the total supply of free AAs available for use in the human body. The amino acid pool is derived from 3 sources: Dietary protein Protein turnover = a repetitive process in which proteins are degraded & re-synthesized within the human body Biosynthesis of non-essential AAs in the liver
Nitrogen Balance The state that results when the amount of nitrogen taken into the human body as protein equals the amount of nitrogen excreted from the body in waste materials. In a healthy adult the nitrogen intake equals the nitrogen excretion. 2 types of nitrogen imbalance can occur in human body: Negative nitrogen balance protein degradation exceeds protein synthesis the amount of nitrogen in urine exceeds the amount of nitrogen ingested (dietary protein), leading to tissue wasting (starvation, protein-poor diet, wasting illness). Positive nitrogen imbalance protein synthesis (anabolism) exceeds protein degradation (catabolism) results in large amounts of tissue synthesis (during growth & pregnancy).
Amino Acids There is no specialized storage form of AAs in the body, hence a constant source of AAs is needed to maintain normal metabolism. The AAs from the AA pool are used for: Protein synthesis about 75% of AAs are used to continuously replace old tissues (protein turnover) & to build new tissues (growth). Synthesis of non-protein N-containing compounds (purine & pyrimidine bases, haeme, neurotransmitters & hormones). Synthesis of non-essential AAs Energy production as AAs are not stored in the body, any excess is degraded each AA has a different degradation pathway. All degradation pathways involve the removal of N atom & its excretion as urea. The remaining carbon skeleton is broken down into CMP intermediates & used for energy production or storage.
Possible fates for amino acid degradation products Stoker 2014, Figure 26-3 p955
Amino Acid Degradation AA degradation takes place in the liver in 2 stages: Removal of the NH 2 group Degradation of the remaining carbon skeleton Removal of the NH 2 group is a 3 step process: 1. Transamination 2. Oxidative Deamination 3. The Urea Cycle
Transamination Transfer of the NH 2 group of an α-aa to an α-keto acid. Involves 2 AA (1 as a reactant & 1 as a product) & 2 keto acids (1 as a reactant & 1 as a product) 2 keto/amino acid pairs are involved, each pair has a common C-chain base. 2 most encountered keto/amino acid pairs: α-ketoglutarate / Glutamate Oxaloacetate / Aspartate Stoker 2014, p958
Key keto/amino acid pairs encountered in Transamination reactions Stoker 2014, Figure 26-4 p957
Generalized Transamination Reaction Stoker 2014, p956
Transamination Catalyzed by enzyme Transaminase / Aminotransferase. Transamination involves several steps & requires pyridoxal phosphate (coenzyme derived from Pyridoxine).
Glutamate Production via Transamination The most important transamination reaction involves conversion of α-ketoglutarate to glutamate. There are at least 50 aminotransferases they are highly specific to the keto acid substrates they accept. Most aminotransferases accept α-ketoglutarate, others oxaloacetate, producing glutamate & aspartate, respectively. The effect of transamination = to collect the NH 2 group from a variety of AAs onto just 1 AA = glutamate, which acts a NH 2 donor for further processing of NH 2 group. Glutamate is further processed via 2 nd transamination with oxaloacetate forming aspartate or via oxidative deamination forming ammonium ion (NH 4+ ) both are NH 2 group carriers participating in the Urea cycle.
Glutamate Production via Transamination Stoker 2014, p959
Aspartate Production via Transamination Glutamate (AA) reacts with Oxaloacetate (keto acid) forming Aspartate (AA) & regenerating α-ketoglutarate. Aspartate now carries N atom into the Urea cycle. Stoker 2014, p959
Oxidative Deamination The removal of the NH 2 group from Glutamate in the form of ammonium ion (NH 4+ ) & α-ketoglutarate is regenerated for transamination. Occurs in liver & kidney mitochondria. Catalyzed by Glutamate dehydrogenase. Requires NAD + as coenzyme forming NADH enters ETC & forms ATP.
The Urea Cycle Urea A series of biochemical reactions, in which urea is produced from NH 4 + & Aspartate as nitrogen sources. The NH 4+ produced in oxidative deamination is relatively toxic it enters the Urea cycle (in mammals) & is converted to Urea. Urea cycle occurs in the liver urea is transported in the blood to the kidneys & eliminated from the body via urine. Urea is highly water-soluble but doesn t contribute to the odour or colour of urine). An adult with normal metabolism excretes about 30g of urea daily in urine, although the exact amount varies with dietary protein intake.
3 AA intermediates involved in the Urea cycle: Arginine Ornithine Citruline
Carbamoyl Phosphate The fuel for the Urea cycle. 1 molecule of carbamoyl phosphate is produced from NH 4+, CO 2, H 2 O & 2 ATP. Carbamoyl phosphate contains a high-energy phosphate bond. This reaction occurs in the mitochondrial matrix.
Steps of the Urea Cycle Part of the UC occurs in the mitochondrion & part in the cytosol. Ornithine & Citruline must be transported across the IMM. The Urea cycle is a series of 4 steps: 1) Transfer of carbamoyl group 2) Citrulline Aspartate condensation 3) Cleavage of arginosuccinate 4) Hydrolysis of arginine The 1 st step occurs in the mitochondrial matrix. Steps 2,3 & 4 take place in the cytosol.
Step 1: Carbamoyl Group Transfer Carbamoyl phosphate transfers its carbamoyl group to Ornithine to from Citruline, releasing P i. Catalyzed by Ornithine transcarbamoylase.
Step 2: Citrulline Aspartate Condensation Citrulline is transported into cytosol & reacts with Aspartate (from transamination of Glutamate) to produce Argininosuccinate utilizing ATP. Catalyzed by Arginosuccinate synthase.
Step 3: Arginosuccinate Cleavage Argininosuccinate is cleaved to Arginine (standard AA) & Fumarate (CAC intermediate). Catalyzed by Argininosuccinate lyase.
Step 4: Hydrolysis of Arginine Produces Urea & regenerates Ornithine transported back into the mitochondria to participate in the Urea cycle again. Catalyzed by Arginase.
Stoker 2014, Figure 26-6 p963
Urea Cycle Net Reaction The equivalent of a total 4 ATP molecules are expended in the Urea cycle. 2 ATP molecules are used to produce Carbamoyl phosphate. The equivalent of 2ATP molecules is consumed in Step 2 of the Urea cycle, when ATP is hydrolyzed to AMP.
Stoker 2014, p967
The connection between Urea Cycle & Citric Acid Cycle Stoker 2014, Figure 26-8 p967
Amino Acid Carbon Skeletons The removal of NH 2 group from an AA in transamination & oxidative deamination produce an α-keto acid that contain the carbon skeleton from the original AA. Each of 20 AAs have a different carbon skeleton (CS) each CS undergoes a different degradation pathway, eventually forming 7 degradation products. The 7 degradation products formed are: Pyruvate Acetyl CoA Aacetoacetyl CoA, α-ketoglutarate, Succinyl CoA, Fumarate & Oxaloacetate are all intermediates of the CAC.
Amino Acid Carbon Skeletons Glucogenic Amino Acids The AAs that are converted to CAC intermediates can be used to produce glucose via Gluconeogenesis. Ketogenic Amino Acids The AAs the are converted to Acetyl CoA or Acetoacetyl CoA can be used to produce ketone bodies. AA that are degraded to Pyruvate are either Glucogenic or Ketogenic, as pyruvate can be metabolized into Oxaloacetate (glucogenic) or Acetyl CoA (ketogenic). Purely Ketogenic AAs = Leu & Lys.
Glucogenic & Ketogenic Amino Acids Stoker 2014, Figure 26-9 p970
Amino Acid Biosynthesis Different species synthesize AAs in different ways. In microorganisms: Non-essential AA can be produced in 1-3 steps. Essential AA biosynthetic pathways require 7-10 steps. Most bacteria & plants can synthesize all the AA via the biochemical pathways not present in humans. In humans: Non-essential AAs can be made in the body from other compounds. Essential AAs the human body can not synthesize them & they have to by supplied in the diet!
Stoker 2014, Table 26-2 p954
Amino Acid Biosynthesis Non-essential AA in humans are synthesized from: Glycolysis Intermediates 3-Phosphoglycerate & Pyruvate CAC Intermediates Oxaloacetate & α-ketoglutarate The essential AA Phenylalanine produces Tyrosine via oxidation with molecular O 2, NADPH & phenylalanine hydroxylase lack of this enzyme causes the metabolic disease Phenylkenonuria (PKU).
Stoker 2014, Figure 26-10 p972
Phenylketonuria (PKU) Timberlake 2014, The genetic disorder, in which the gene that codes for the enzyme phenylalanine hydroxylase is defective therefore Phenylalanine forms Phenylpyruvate (transamination), which is converted to Phenylacetate (decarboxylation). High levels of Phenylacetate cause severe mental retardation. A diet low in phenylalanine and high in tyrosine is recommended.
Amino Acid Biosynthesis 3 non-essential AA (Alanine, Aspartate & Glutamate) are biosynthesized by transamination of the appropriate α-keto acid.
B Vitamins & Protein Metabolism Many B vitamins function as coenzymes in protein metabolism without these the body would be unable to undertake the various degradation & biosynthesis pathways of amino acids. B vitamins involved in protein metabolism: Niacin as NAD + & NADH in oxidative deamination Pyridoxine as PLP in transamination reactions All 8 B viamins involved in degradation & biosynthesis of AAs
Stoker 2013, Figure 26-15 p982
Stoker 2014, p980
Readings & Resources Stoker, HS 2014, General, Organic and Biological Chemistry, 7 th edn, Brooks/Cole, Cengage Learning, Belmont, CA. Stoker, HS 2004, General, Organic and Biological Chemistry, 3 rd edn, Houghton Mifflin, Boston, MA. Timberlake, KC 2014, General, organic, and biological chemistry: structures of life, 4 th edn, Pearson, Boston, MA. Alberts, B, Johnson, A, Lewis, J, Raff, M, Roberts, K & Walter P 2008, Molecular biology of the cell, 5 th edn, Garland Science, New York. Berg, JM, Tymoczko, JL & Stryer, L 2012, Biochemistry, 7 th edn, W.H. Freeman, New York. Dominiczak, MH 2007, Flesh and bones of metabolism, Elsevier Mosby, Edinburgh. Tortora, GJ & Derrickson, B 2014, Principles of Anatomy and Physiology, 14 th edn, John Wiley & Sons, Hoboken, NJ. Tortora, GJ & Grabowski, SR 2003, Principles of Anatomy and Physiology, 10 th edn, John Wiley & Sons, New York, NY.