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.