Protein Metabolism

A.Sources and Uses of Amino Acids

B. Nutritional (Dietary) Requirements for Amino Acids

C. Digestion, Adsorption and Excretion

D. Nitrogen Balance

E. Endogenous Amino acid Degradation and Urea Cycle

F. Hepatic Glutamine Metabolism

G.Biosynthesis and Utilization of Various Amino Acids in Specialized Biosynthetic Pathways

A. Sources and Uses of Amino Acids

Sources :Proteins in the diet provide both essential and non-essential amino acids

Uses :1.Protein synthesis2.Nitrogen and carbon source of general and special product biosynthesis3.Energy source a.glucogenic(those that can be used for the synthesis of glucose) b.ketogenic(those whose metabolism leads to ketone bodies)

Amino acid roles:

1) protein monomeric units (primary purpose)2) energy metabolites (about 10% of energy)3) precursors of many biologically important nitrogen containing compounds such as: a) HEME b) physiologically active AMINES [(nor)epinephrine, dopamine, GABA (g-aminobutyric acid), serotonin, histamine] c) GLUTATHIONE e) NUCLEOTIDES f) nucleotide COENZYMES

Amino acid metabolism is intimately intertwined with nitrogen acquisition.

All organisms need bioavailable sources of nitrogen for proteins and nucleic acids.

The major form of nitrogen in the atmosphere isN2, an extremely stable compound: the N ΞN triple bond has a bond energy of 945kJ/mol.

N2 is converted to metabolically useful forms (is "fixed") only by a few species of prokaryotes, called diazatrophs.Diazatrophs of the genus Rhizobium live symbiotically in the root nodules of legumes, where they convert N2 to NH3 (ammonia) in a process called

NITROGEN FIXATION:

NITROGENASE

N2 + 8 H+ + 8 e- + 16 ATP + 16 H2O → 2 NH3 + H2 + 16 ADP + 16 Pi

* But, less than 1% of N entering the biosphere comes from N fixation.

Another oxidized form of nitrogen, NO3 - (nitrate ion) is also found in the soils and oceans. It is converted to NH4 + through... NITRATE ASSIMILATION:

* The reduction of NO3- to NH4 + (ammonium ion) occurs in green plants, various fungi, and certain bacteria in a two-step pathway:(1) The 2-electron reduction of nitrate to nitrite: NO3

- + 2 H+ + 2 e- → NO2- + H2O

(2) This is followed by the 6-electron reduction of nitrite to ammonium:

NO2- + 8 H+ + 6 e- → NH4

+ + 2 H2O

No animals are capable of either N-fixation or nitrate assimilation, so they (we!) are totally dependent on plants and microorganisms for the synthesis of organic nitrogenous compounds, such as amino acids and proteins, to provide this essential nutrient.

C.Digestion, Adsorption and ExcretionDigestion.

Dietary proteins can not be absorbed directly from the intestine. They must be hydrolyzed by a group of proteases and peptidases to amino acids, dipeptides and tripeptides.

The first major site of digestion is in the stomach by the action of pepsin, then by the action of pancreatic enzymes(e.g.trypsin and chymotrypsin) and intestinal peptidases functioning in the intestine then complete the hydrolytic process.

Absorption.The amino acids and small peptides are transported into the intestinal cells by a family of amino acid specific transports many of which require Na+.

stomach pancreas to small intestine

intestinal wall

pepsin Trypsin

Chymotrypsin

carboxypeptidase A

carboxypeptidase B

elastase

dipeptidases

Excretion.

Nitrogen is also constantly being lost from the body in a variety of different forms.

The principal excretory nitrogen product in mammals is urea (about 12-20 g urea nitrogen/day).

Other excretory products include ammonium ion, uric acid and creatinine. The amounts of each of these is dependent of the metabolic state of the individual: -

Ureotelic-excrete urea (mammals) Uricotelic-excrete uric acid (birds, insects) Ammonotelic-excrete ammonium ion (fish)

Some animals switch from one form to another during development (more about that later).

Nitrogen balance Protein content of adult body

remains remarkably constant Protein constitutes 10-15% of diet

Equivalent amount of amino acids must be lost each day

OVERVIEW OF AMINO ACID METABOLISM

ENVIRONMENT ORGANISM

Ingested protein

Bio- synthesis Protein

AMINO ACIDS

Nitrogen Carbonskeletons

Urea

Degradation (required)

1 2 3

a

b

PurinesPyrimidinesPorphyrins

c c

Used for energy

pyruvateα-ketoglutaratesuccinyl-CoAfumarateoxaloacetate

acetoacetateacetyl CoA

(glucogenic)

(ketogenic)

Mammals cannot select the specific amino acids they have in their diet, therefore they take in some amino acids in excess of their needs.

The mechanisms of utilization of those consumed in excess for the purpose of synthesizing the deficient ones is part of the dynamic metabolism of nitrogen metabolism.

Of course the essential amino acids cannot be made de novo in the animal cell.

Amino acids, which are not utilized for protein synthesis or other pathways of nitrogen utilization, are not excreted in any large amounts but are deaminated in one of several ways.

The carbon skeletons are oxidatively degraded for the production of energy or stored as carbohyrdrate.

Ammonia produced is re-utilized or new amino acid synthesis or converted to urea for excretion.

E.Endogenous Amino Acid Degradation and Urea Cycle

1.Transamination(aminotransferases, transaminases)

2.Deamination a.Oxidative (NAD an FAD dependent) b. Non-oxidative

3.Ammonia Assimilation a.Glutamate dehydrogenate b.Glutamine synthetase c.Carbamoyl phosphate synthetaseI

4.Urea Cycle

Fate of amino acids If not required for protein

synthesis amino groups removed For most amino acids occurs

primarily in liver For (leucine, isoleucine, valine)

occurs primarily in skeletal muscle amino groups transferred to alanine

and taken to liver for disposal via glucose-alanine cycle

Carbon skeletons used for: Gluconeogenesis (in liver) Oxidised in Krebs Cycle

Amino groups used for Synthesis of nonprotein nitrogen

compounds disposed of via Urea Cycle

From: Summerlin LR (1981) Chemistry for the Life Sciences. New York: Random House p 563.

Amino acid metabolism Metabolism of amino acids differs,

but 3 common reactions: Transamination Deamination Formation of urea

Typical first transamination reaction:

The usual AA acceptor is α-ketoglutarate, producingGLUTAMATE and the new a-keto acid.

What is produced if oxaloacetate is the acceptor?

What is produced if oxaloacetate is the acceptor?It is one C shorter than a-ketoglutarate, so produces…..

X

X

ASPARTATE

oxaloacetate

The second typical step in AA deamination involves transfer of the amino group from GLU to oxaloacetate, yeilding a-ketoglutarate and ASP:

glutamate-aspartate aminotransferase

TRANSAMINATION

Biosynthesis of Nonessential Amino Acids Transamination reactions

Allow extensive interconversion between nonessential amino acids

Requires vitamin B6 as a coenzyme

Transamination Transfer of amino group from an amino

acid to an α-keto acid Used to synthesize amino acids as needed

Some essential amino acids Not lysine or threonine

Must have appropriate α-keto acid in diet Requires vitamin B6 in coenzyme form

Pyridoxal phosphate (PLP) Catalyzed by amino transferases

Transaminationα-Keto Acid

aminoAmin

o Acid

α-Keto Acid

α-Keto Acid

α-Keto Acid

amino

α-Amino Acid

PLPAmino transferase

Transamination

Amino Acid Interrelationships Methionine can be converted to Cys

If too little Cys in diet, Met is converted to Cys and Met becomes deficient

Up to 50% of Cys ‘requirement’ met through Met Phe can be converted to Tyr

Requirement is typically stated for Phe + Tyr

CCH

O

O–

NH3+

CH2

CCH

O

O–

NH3+

CH2

HO

Protein Catabolism Occurs when

Dietary protein exceeds protein requirements of body

Normal situation in true carnivores Abnormal in omnivores and herbivores

Composition of absorbed amino acids is unbalanced

Gluconeogenesis is increased

Protein Catabolism Some net catabolism of body

proteins occurs at all times Expressed as urinary nitrogen

excretion Use the carbon backbone for energy,

excrete the nitrogen as urea

Urinary Nitrogen Excretion

Urine

KIDNEY

LIVER

Urea

Urea

CO2

Amino acids keto acidsNH3

Blood

Deamination Removal of amino group from an

amino acid with no transfer Produces ammonia and α-keto acid

Ammonia removed by urea cycle α-keto acid is metabolized via several

potential pathways Pyridoxal phosphate (PLP) required

(B6)

Deaminationam

ino

α-Keto AcidDehydratasePLP H2O

α-Keto Acid + NH4

Keto acids can Enter the TCA cycle and be broken down to

CO2 and H2O with release of energy Be used for gluconeogenesis

Some, not all amino acids In liver (and kidney)

Lipogenesis (fatty acid biosynthesis) Ketogenesis

Ketone bodies (acetoacetate, acetyl-CoA) Used as energy source in various tissues

Use of Keto Acids for Energy

Ketogenic Amino Acids Leucine and isoleucine

Converted to acetoacetate or acetyl CoA in liver

Fuel for other tissues

Use of Amino Acids for Energy Not economical

Energy feeds are less expensive (per kcal) than protein feeds

Item CP ME ME Cost Cost% Mcal/kg Mcal/ton $/ton ¢/Mcal

Corn 8 3.42 3,102 71.4 2.30SBM 48 3.38 3,066 150.0 4.89

Disposal of NH3 NH3 is very toxic and must be

detoxified and excreted from the body Fish: NH3 Mammals: Urea Birds: Uric acid

Synthesis of uric acid Same pathway as for purines

Synthesis of urea—the urea cycle Detoxifies NH3 to urea Synthesizes arginine

UREA

Urea Cycle

Urea CycleO||

2 NH3 + CO2 H2N–C–NH2 + H2O

Overall reaction

• Energy required (3 ATP)• Urea diffuses from liver cells to body fluids• Excreted by the kidneys

Urea cycle Ammonia is toxic

Readily ionises to ammonium ion NH4

+

NH4+ converted to urea

in liver (urea cycle) Urea contains 2 x NH2

One from NH4+

One from aspartate

Urea excreted in urine

From: Stryer, LS (1988) Biochemistry (3rd Ed). New York: WH Freeman & Co. p500

UREA CYCLE

mitochondriacytosol

Function: detoxification of ammonia (prevents

hyperammonemia)

Most terrestrial land animals convert excess nitrogen to urea, prior to excreting it. Urea is less toxic than ammonia.

H 2 N C

O

N H 2

u r e a

49

Urea Formation Occurs primarily in liver; excreted by kidney Principal method for removing ammonia Hyperammonemia:

Defects in urea cycle enzymes (CPS, OTC, etc.) Severe neurological defects in neonates Treatment:

Stop protein intake Dialysis Increase ammonia excretion: Na benzoate, Na

phenylbutyrate, L-arginine, L-citrulline

50

Blood Urea Nitrogen Normal range: 7-18 mg./Dl Elevated in renal insufficiency Decreased in hepatic failure

Hereditary deficiency of any of the Urea Cycle enzymes leads to hyperammonemia - elevated [ammonia] in blood. Total lack of any Urea Cycle enzyme is lethal. Elevated ammonia is toxic, especially to the brain. If not treated immediately after birth, severe mental retardation results.

Conditionally Essential Amino Acids

Amino acids that can become essential in certain physiologic conditions Example: taurine in cats Example: proline in young pigs Example: tyrosine becomes essential in

people with “phenylketonuria (PKU)” PKU; 1 in 15,000 babies Hydroxylation of phenylalanine normally forms

tyrosine Tyrosine important in adrenaline, noradrenaline,

thyroxine and melanin synthesis

Phenylketonuria (PKU) Normal situation:

Phenylalanine (essential aa)

Tyrosine (nonessential aa)

In PKU: Phenylalanine builds up

Can cause mental retardationCondition inherited from parents

(genetic)Phenylalanine (essential aa)

Tyrosine (nonessential aa)

PKU Symptoms VERY FEW symptoms if diagnosed early and diet strictly

regulated IF NOT:

Lighter skin, hair and eyes than siblings Phenylalanine important in synthesis of melanin

Delayed mental and social skills Head size significantly below normal Hyperactivity Jerking movements of the arms or legs Mental retardation (severe if not diagnosed and treated

early) Seizures Skin rashes Tremors Unusual positioning of hands

PKU Treatment EXTREMELY low phenylalanine intake

Diet for life Special low-phenylalanine infant formula

Used for life Low or no milk, eggs No aspartame (NutraSweet) Fish oil supplements

Hard to get enough essential fatty acids on low phenylalanine diet

Iron supplements

Boisen et al. (2000)