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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 (HCO3-) –
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 & pyrimidinebases, 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.
Stoker 2014, Figure 26-3 p955
Possible fates for amino acid degradation products
Amino Acid Degradation
• AA degradation takes place in the liver in 2 stages:
• Removal of the –NH2 group
• Degradation of the remaining carbon skeleton
• Removal of the –NH2 group is a 3 step process:
• 1. Transamination
• 2. Oxidative Deamination
• 3. The Urea Cycle
Transamination
• Transfer of the –NH2 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 / AspartateStoker 2014, p958
Stoker 2014, Figure 26-4 p957
Key keto/amino acid pairs encountered in Transamination reactions
GeneralizedTransamination
Reaction
Stoker 2014, p956
Transamination
• Catalyzed by enzyme Transaminase / Aminotransferase.
• Transamination involves several steps & requires pyridoxalphosphate (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 –NH2 group from a variety of AAs onto just 1 AA = glutamate, which acts a –NH2 donor for further processing of –NH2 group.
• Glutamate is further processed via 2nd transamination with oxaloacetate forming aspartate or via oxidative deamination forming ammonium ion (NH4
+) – both are –NH2 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 –NH2 group from Glutamate in the form of ammonium ion (NH4
+) & 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
• A series of biochemical reactions, in which urea is produced from NH4
+ & Aspartate as nitrogen sources.
• The NH4+ 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 odouror 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.
Urea
3 AA intermediates involved in
the Urea cycle:
ArginineOrnithineCitruline
Carbamoyl Phosphate
• The fuel for the Urea cycle.
• 1 molecule of carbamoyl phosphate is produced from NH4+,
CO2, H2O & 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 1st 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 Ornithineto from Citruline, releasing Pi.
• 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
Stoker 2014, Figure 26-8 p967
The connection between Urea Cycle & Citric Acid Cycle
Amino Acid Carbon Skeletons
• The removal of –NH2 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.
Stoker 2014, Figure 26-9 p970
Glucogenic & KetogenicAmino Acids
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 O2, NADPH & phenylalanine hydroxylase – lack of this enzyme causes the metabolic disease Phenylkenonuria (PKU).
Stoker 2014, Figure 26-10 p972
Phenylketonuria (PKU)
• 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.
Timberlake 2014,
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, 7th edn,
Brooks/Cole, Cengage Learning, Belmont, CA.
• Stoker, HS 2004, General, Organic and Biological Chemistry, 3rd edn, Houghton Mifflin, Boston, MA.
• Timberlake, KC 2014, General, organic, and biological chemistry: structures of life, 4th edn, Pearson, Boston, MA.
• Alberts, B, Johnson, A, Lewis, J, Raff, M, Roberts, K & Walter P 2008, Molecular biology of the cell, 5th edn, Garland Science, New York.
• Berg, JM, Tymoczko, JL & Stryer, L 2012, Biochemistry, 7th 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, 14th edn, John Wiley & Sons, Hoboken, NJ.
• Tortora, GJ & Grabowski, SR 2003, Principles of Anatomy and Physiology, 10th edn, John Wiley & Sons, New York, NY.