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 nonessential amino acids
Uses : 1.Protein synthesis 2.Nitrogen and carbon source of general and special product biosynthesis 3.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 is N2, an extremely stable compound: the N N 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 This is followed by the 6-electron reduction of nitrite to ammonium: NO2- + 8 H+ + 6 e- NH4+ + 2 H2O

(2)

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 Excretion

Digestion.

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
pepsin

pancreas to intestinal small intestine wall

Trypsin Chymotrypsin carboxypeptidase A carboxypeptidase B dipeptidases

elastase

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
1

Biosynthesis 2 3

Protein

a AMINO ACIDS c
Degradatio n (required) (ketogenic)
Urea
acetoacetate acetyl CoA

b c
Purines Pyrimidines Porphyrins

Nitrogen

Carbon skeletons

Used for energy

(glucogenic) pyruvate -ketoglutarate succinyl-CoA fumarate oxaloacetate

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 glucosealanine cycle Gluconeogenesis (in liver) Oxidised in Krebs Cycle
From: Summerlin LR (1981) Chemistry for the Life Sciences. New York: Random House p 563.

Carbon skeletons used for:

Amino groups used for

Synthesis of nonprotein nitrogen compounds disposed of via Urea Cycle

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 -ketoglutarate, producing GLUTAMATE 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
oxaloacetate

X ASPARTATE

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 -keto acid Used to synthesize amino acids as needed

Some essential amino acids

Not lysine or threonine

Must have appropriate -keto acid in diet Pyridoxal phosphate (PLP)

Requires vitamin B6 in coenzyme form

Catalyzed by amino transferases

Amino Acid

Transamination
-Keto Acid PLP amino Amino transferase

amino -Keto Acid -Keto Acid

-Keto Acid

-Amino Acid

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

+ NH3

O O

NH3+ HO C C H2 H C

O O

C C H2 H

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

LIVER
Amino acids CO2 NH3 Urea Blood keto acids

KIDNEY

Urea

Urine

Deamination

Removal of amino group from an amino acid with no transfer Produces ammonia and -keto acid

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

Pyridoxal phosphate (PLP) required (B6)

Deamination
amino Dehydratase PLP

-Keto Acid

-Keto Acid
H2O

+ NH4

Use of Keto Acids for Energy

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

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

Corn SBM

CP % 8 48

ME Mcal/kg 3.42 3.38

ME Mcal/ton 3,102 3,066

Cost $/ton 71.4 150.0

Cost /Mcal 2.30 4.89

Disposal of NH3

NH3 is very toxic and must be detoxified and excreted from the body

Fish: Mammals: Birds:

NH3 Urea Uric acid

Synthesis of uric acid

Same pathway as for purines Detoxifies NH3 to urea Synthesizes arginine

Synthesis of ureathe urea cycle

UREA

Urea Cycle

Urea Cycle

Overall reaction
2 NH3 + CO2

O || H2NCNH2 + H2O

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

Urea excreted in urine

UREA CYCLE

mitochondri a cytosol

Function: detoxification of ammonia (prevents hyperammonemia)

O H 2N C NH2

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

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
49

Blood Urea Nitrogen

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

50

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 retardation Condition 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

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

Phenylalanine important in synthesis of melanin

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)