CHAPTER 19 Amino Acid Oxidation

Production of Urea

– How proteins are digested in animals– How amino acids are degraded in animals– How urea is made and excreted

Key topics:

Oxidation of Amino Acids is a Significant Energy-Yielding Pathway in Carnivores

• Not all organisms use amino acids as the source of energy

• About 90% of energy needs of carnivores can be met by amino acids immediately after a meal

• Only a small fraction of energy needs of herbivores are met by amino acids

• Microorganisms scavenge amino acids from their environment for fuel

Sources and Uses of Amino Acids

Sources1.Proteins in the diet provide both essential and non-essential amino acids in contrast to microorganisms that for the most part synthesize their own.2.Turnover of endogenous proteins3.de novo biosynthesis (non-essential amino acids)

Uses1.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)

Metabolic Circumstances of Amino Acid Oxidation

Amino acids undergo oxidative catabolism under three circumstances:

– Protein amino-acid residues from normal turnover are recycled to generate energy and molecular components

– Dietary amino acids that exceed body’s protein synthesis needs are degraded

– Proteins in the body are broken down to supply amino acids for catabolism when carbohydrates are in short supply (starvation, diabetes mellitus),

Protein Turnover and Nitrogen Balance

Protein Degradation:

• Endogenous proteins degrade continuously- Damaged- Mis-folded- Un-needed

• Dietary protein intake - mostly degraded

Nitrogen Balance - expresses the patient’s current status - are they gaining or losing net Nitrogen?

Dietary Proteins are Enzymatically Hydrolyzed

• Pepsin cuts protein into peptides in the stomach• Trypsin and chymotrypsin cut proteins and larger

peptides into smaller peptides in the small intestine

• Aminopeptidase and carboxypeptidases A and B degrade peptides into amino acids in the small intestine

stomach pancreas to small intestine

intestinal wall

pepsin Trypsin

Chymotrypsin

carboxypeptidase A

carboxypeptidase B

elastase

dipeptidases

• (a) gastrin -> secretion of HCl by parietal cells and pepsin by chief cells

• (b) exocrine cells synthesize zymogens

– zymogen granules fuse with plasma membrane

– zymogens released into the lumen of the collecting duct

– collecting ducts -> pancreatic duct -> small intestine.

• (c) Amino acids -> villi -> capillaries

Enzymatic Degradation of

Dietary Proteins

Overview of Amino

Acid Catabolism

OVERVIEW OF AMINO ACID METABOLISM

ENVIRONMENT ORGANISM

Ingested protein

Bio- synthesis Protein

AMINO ACIDS

Nitrogen Carbon

skeletons

Urea

Degradation (required)

1 2 3

a

b

PurinesPyrimidinesPorphyrins

c c

Used for energy

pyruvateα-ketoglutaratesuccinyl-CoAfumarateoxaloacetate

acetoacetateacetyl CoA

(glucogenic)(ketogenic)

Degradation of amino acids to one of seven common metabolic intermediates.

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.

Transamination is a reaction between an amino acid and a keto-acid in which the amino group is transferred from the donor amino acid onto the acceptor keto-acid.

TRANSAMINATION

Enzymatic Transamination

• Typically, -ketoglutarate accepts amino groups

• L-Glutamine acts as a temporary storage of nitrogen

• L-Glutamine can donate the amino group when needed for amino acid biosynthesis

• All aminotransferases rely on the pyridoxal phosphate (PLP) cofactor

Structure of Pyridoxal Phosphate and Pyridoxamine Phosphate

• Intermediate, enzyme-bound carrier of amino groups

• Aldehyde form can

react reversibly with

amino groups

• Aminated form can

react reversibly with

carbonyl groups

A small number of amino acids undergo oxidative or non-oxidative deamination

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Urea Formation

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

• Defects in urea cycle enzymes• Severe neurological defects in neonates

Treatment of deficiency of Urea Cycle enzymes (depends on which enzyme is deficient):

limiting protein intake to the amount barely adequate to supply amino acids for growth, while adding to the diet the -keto acid analogs of essential amino acids.

Liver transplantation has also been used, since liver is the organ that carries out Urea Cycle.

Dialysis

Increase ammonia excretion: Na benzoate, Na phenylbutyrate, L-arginine, L-citrulline

Postulated mechanisms for toxicity of high [ammonia]:

1. High [NH3] would drive Glutamine Synthase:

glutamate + ATP + NH3 glutamine + ADP + Pi

This would deplete glutamate – a neurotransmitter & precursor for synthesis of the neurotransmitter GABA.

2. Depletion of glutamate & high ammonia level would drive Glutamate Dehydrogenase reaction to reverse: glutamate + NAD(P)+ -ketoglutarate + NAD(P)H + NH4

+

The resulting depletion of -ketoglutarate, an essential Krebs Cycle intermediate, could impair energy metabolism in the brain.

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GABA Formation

NH3+

-O2CCH 2CH2CHCO 2-

NH3+

-O2CCH 2CH2CH2

Glutamate Gamma-aminobutyrate(GABA)

GABA is an important inhibitory neurotransmitter in the brainDrugs (e.g., benzodiazepines) that enhance the effectsof GABA are useful in treating epilepsy

Glutamatedecarboxylase

CO2

• Glutamate is the precursor of free GABA in GABAergic terminals and comes from two different sources (Kreb's cycle in glia cells and glutamine in nerve terminals). Next, the enzyme glutamic acid decarboxylase (GAD) forms GABA from glutamate. After being released into the synapses, GABA is inactivated by reuptake mediated by GABA transporters (GATs) into presynaptic terminals or into glia cells where it is metabolized by GABA-transaminase (GABA-T).

N balance = NN balance = Ninin - N - Noutout

1 Major dietary source of N is Protein (>95%), since the diet has very few free amino acids (AA)

2 AA are used for Protein Synthesis & N containing compounds

3 AA in excess are degraded (used for energy)

N is disposed of in urea (80%), ammonia, uric acid or creatinine in urine with small amounts in fecal matter (undigested)

Nitrogen Acquisition

• Nitrogen Fixation

• Nitrate Assimilation

• Ammonium Assimilation

N2 is converted to metabolically useful forms (is "fixed") only by a few species of prokaryotes, called Diazotrophs.Diazotrophs 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 ( catalyzed by nitrate reductase)

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

NO2- + 8 H+ + 6 e- → NH4

+ + 2 H2O ( catalyzed by nitrite reductase)

*NH3/NH4+ can be incorporated into the amino acids glutamate

by glutamate dehydrogenase (and glutamine by glutamine synthetase.

Nitrate Assimilation(Green plants, some fungi and bacteria)

NO3– + NADH + H+ NO2

– + H2O + NAD+

Nitrate Reductase

NO2– + 8H+ + 6e– NH4

+ + 2H2ONitrite Reductase

Ammonium Assimilation(Carbamoyl Phosphate Synthetase)

H2N C

O

OP

2ATP 2ADP + Pi

NH3 + HCO3–

(Biosynthetic Glutamate Dehydrogenase)and/or

(Glutamine Synthetase)

NH3 Glutamate

NH3

Glutamine

No animals are capable of either N-fixation or nitrate assimilation, so animals 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.

Nitrogen balance = nitrogen ingested - nitrogen excreted

(primarily as protein) (primarily as urea)

Nitrogen balance = 0 (nitrogen equilibrium)

protein synthesis = protein degradation

Positive nitrogen balance

protein synthesis > protein degradation

Negative nitrogen balance

protein synthesis < protein degradation

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

Fates of Nitrogen in Organisms• Plants conserve almost all the nitrogen• Many aquatic vertebrates release ammonia to their environment

– Passive diffusion from epithelial cells– Active transport via gills

• Many terrestrial vertebrates and sharks excrete nitrogen in the form of urea– Urea is far less toxic that ammonia– Urea has very high solubility

• Some animals, such as birds and reptiles excrete nitrogen as uric acid– Uric acid is rather insoluble– Excretion as paste allows to conserve water

• Humans and great apes excrete both urea (from amino acids) and uric acid (from purines)

Excretory Forms of Nitrogen

The Amino Group is

Removed From All Amino Acids

First

Glutamate can Donate Ammonia to Pyruvate to

Make Alanine

• Vigorously working muscles operate nearly anaerobically and rely on glycolysis for energy

• Glycolysis yields pyruvate that muscles cannot metabolize aerobically; if not eliminated lactic acid will build up

• This pyruvate can be converted to alanine for transport into liver

Ammonia in Transported in the

Bloodstream Safely as Glutamate

• Un-needed glutamine is processed in intestines, kidneys and liver

Excess Glutamate is Metabolized in the Mitochondria of Hepatocytes

The Glutamate Dehydrogenase

Reaction

• Two-electron oxidation of glutamate followed by hydrolysis

• Net process is oxidative deamination of glutamate

• Occurs in mitochondrial matrix in mammals

• Can use either NAD+ or NADP+ as electron acceptor

Nitrogen from

Carbamoyl Phosphate Enters the

Urea Cycle

• Urea is produced in the Urea is produced in the liverliver• From the liver, it is transported in the blood to the From the liver, it is transported in the blood to the kidneyskidneys for for

excretion in urine excretion in urine

Urea is composed of:Urea is composed of:

Two nitrogen atomsTwo nitrogen atoms• First nitrogen atom is from free ammoniafree ammonia• Second nitrogen atom is from aspartateaspartate

Carbon & oxygen atoms are from COCarbon & oxygen atoms are from CO22

Urea Cycle

The Reactions in the Urea Cycle

• 1 ornithine + carbamoyl phosphate => citrulline – (entry of the first amino group). – citrulline passes into the cytosol.

• 2a citrulline + ATP => citrullyl-AMP + PPi• 2b citrullyl-AMP + Aspartate => argininosuccinate + AMP

– (entry of the second amino group).

• 3 argininosuccinate => arginine + fumarate– fumarate enters the citric acid cycle.

• 4 arginine => urea + ornithine– Ornithine passes to the mitochondria to continue the cycle

Urea Cycle N-2

from Aspartate

Ammonia is Re-captured via Synthesis of Carbamoyl Phosphate

• This is the first nitrogen-acquiring reaction

Entry of Aspartate into the Urea Cycle

• This is the second nitrogen-acquiring reaction

Aspartate –Argininosuccinate Shunt Links Urea Cycle and Citric Acid Cycle

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.

Fate of UreaFate of Urea

Urea Urea (synthesized in the liver) (synthesized in the liver)

BloodBlood

KidneyKidney intestine

Urine cleaved by bacterial urease

AmmoniaAmmonia CO2

In stool Reabsorbed in blood

1- Urea Urea in the liverin the liver

• is quantitatively the most important most important disposal route for ammonia

• Urea is formed in the liver liver from ammonia (urea cycle)

• UreaUrea travels in the blood from the liver to the kidneyskidneys where it is filtered to appear in urineurine

Disposal of Ammonia

2- GGlutamine lutamine in in most peripheral tissues most peripheral tissues especiallyespecially brain, Skeletal Muscles brain, Skeletal Muscles

& liver& liver

• In most peripheral tissues, glutamate binds with ammoniaammonia by action of glutamine synthase glutamine synthase

• in the brainbrain, it is the major mechanism of removal of ammonia from the brain

• This structure provides a nontoxic storage & transport form of ammonia nontoxic storage & transport form of ammonia • Glutamine is transported to blood to other organs esp. liver & kidneys• In the liver & Kidney, glutamine is converted to ammonia & glutamate

by the enzyme glutaminaseglutaminase.

Disposal of Ammonia

3- Alanine Alanine in skeletal musclesin skeletal muscles

• AmmoniaAmmonia + Pyruvate form alanine alanine in skeletal muscles• Alanine is transported in blood to liver• In liver, alanine is converted to pyruvate & ammoniaammonia • Pyruvate can be converted to glucoseglucose (by gluconeogenesis)• GlucoseGlucose can enter the blood to be used by skeletal muscles

(GLUCOSE - ALANINE PATHWAY)(GLUCOSE - ALANINE PATHWAY)

Disposal of Ammonia

Alanine Alanine in Skeletal Musclesin Skeletal Muscles

GlutamineGlutaminein Most Tissuesin Most Tissues

Esp. brain & KidneysEsp. brain & Kidneys

UreaUreain Liverin Liver

Disposal of Ammonia

Not All Amino Acids can be Synthesized in Humans

• These amino acids must be obtained as dietary protein

• Consumption of a variety of foods (including vegetarian only diets) well supplies all the essential amino acids

Essential amino acids

Mammalian cells lack enzymes to synthesize their carbon skeletons (-keto acids).

Isoleucine, leucine, & valine

Lysine

Threonine

Tryptophan

Phenylalanine (Tyr can be made from Phe.)

Methionine (Cys can be made from Met.)

Histidine (Essential for infants.)

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V Valine

F Phenylalanine

W Tryptophan

I Isoleucine

T Threonine

H Histidine

M Methionine

L Leucine

K Lysine

•One way to remember the 9 essential amino acids is with the mnemonic VF WITH MLK (Very Full With Milk):

Fates of carbon

skeleton of amino acids

Fate of Individual Amino Acids

• Seven to acetyl-CoA– Leu, Ile, Thr, Lys, Phe, Tyr, Trp

• Six to pyruvate– Ala, Cys, Gly, Ser, Thr, Trp

• Five to -ketoglutarate– Arg, Glu, Gln, His, Pro

• Four to succinyl-CoA– Ile, Met, Thr, Val

• Two to fumarate– Phe, Tyr

• Two to oxaloacetate – Asp, Asn

Glucogenic vs ketogenic amino acids

• Glucogenic amino acids (are degraded to pyruvate or citric acid cycle intermediates) - can supply gluconeogenesis pathway

• Ketogenic amino acids (are degraded to acetyl CoA or acetoacetyl CoA) - can contribute to synthesis of fatty acids or ketone bodies

• Some amino acids are both glucogenic and ketogenic

Summary of Amino Acid Catabolism

6 Amino Acids -> Pyruvate

Ala, Gly, Ser,

Cys,Trp,Thr.

7 AAs -> Acetyl CoA [W,K,F,Y, L]

I, M, T, V-> Succinyl-CoA

Albinism – genetically determined lack or deficit of enzyme tyrosinase

Tyrosinase in melanocytes oxidases tyrosine to DOPA and DOPA-chinone

tyrosinase

Phenylalanine

Tyrosine Tyroxine

MelaninDOPA

Dopamine

Norepinephrine

Epinephrine

The pathways for the biosynthesis of amino acids are diverse

Common feature: carbon skeletons come from intermediates of glycolysis, pentose phosphate pathway, citric acid cycle.

All amino acids are grouped into families according to the intermediates that they are made from

Summary

• Amino acids from protein are an important energy source

in carnivorous animals

• Catabolism of amino acids involves transfer of the amino

group via PLP-dependent aminotransferase to a donor

such as -ketoglutarate to yield L-glutamine

• L-glutamine can be used to synthesize new amino acids,

or it can dispose of excess nitrogen as ammonia

• In most mammals, toxic ammonia is quickly recaptured

into carbamoyl phosphate and passed into the urea cycle

Sample question

• The site of amino acid catabolism is the:A. StomachB. Small intestineC. Large intestineD. Liver

Sample question

• The first step in the catabolism of most amino acids is

• A. Removal of carboxylate groups• B. Enzymatic hydrolysis of peptide bonds• C. Removal of the amino group• D. Zymogen cleavage

Sample question

Which of the following is true of urea?  • A. more toxic to human cells than ammonia • B. the primary nitrogenous waste products of

humans. • C. insoluble in water • D. the primary nitrogenous waste product of

most aquatic invertebrates

Sample question

A glucogenic amino acid is one which is degraded to

• A. keto-sugars• B. either acetyl CoA or acetoacetyl CoA• C. pyruvate or citric acid cycle

intermediates• D. none of the above

Sample question

Transamination is the process where

• A. carboxyl group is transferred from amino acid

• B. α-amino group is removed from the amino acid

• C. polymerization of amino acid takes place• D. none of the above

Sample question

Transamination is the transfer of an amino

• A. acid to a carboxylic acid plus ammonia• B. group from an amino acid to a keto acid• C. acid to a keto acid plus ammonia• D. group from an amino acid to a

carboxylic acid