December 2, 2005

AMINO ACID METABOLISM I,II,III

Lecturer:

Eileen M. Lafer

Reading:

Stryer Edition 5: Chapters 23 and 24

OBJECTIVES:

1.

2.

Understand the fates and sources of the amino acids in general terms.

Understand the general features of lysosomal protein degradation, including what types of proteins are degraded in lysosomes.

3.

Understand how "controlled proteolysis" is effected in cells. This includes an understanding of the mechanism of selection and attachment of ubiquitin to target proteins, as well as where and how ubiquitinated proteins are degraded.

4.

Understand the common pathways for the removal of the group from an a -amino acid during amino acid catabolism.

a -amino 5. Understand how the fate of the ammonium ions generated during amino acid degradation differs in the liver versus the peripheral tissues.

6.

Understand the fundamentals of the urea cycle and how it is regulated.

Understand how defective urea cycle enzymes can lead to disease, and how those diseases are treated.

7.

Understand which a -amino acid carbon skeletons feed into which major metabolic intermediates during amino acid catabolism.

8.

Know which amino acids are solely ketogenic, solely glucogenic, and both ketogenic and glucogenic.

9.

Know which steps in amino acid degradation lead to the following diseases: methyl-malonic acedemia, homocystinuria, maple syrup disease, phenylketonuria, tyrosinemia I, II and III, and alkaptonuria. For each disease, you should know the name of the defective enzyme, the reaction catalyzed by the enzyme, and the pathway in which the enzyme functions. 10.

humans.

Know which amino acids are essential and which are non-essential in 11.

Know in humans, which major metabolic intermediates are able to serve as carbon skeletons for the biosynthesis of which amino acids.

12.

13.

Know which amino acids can act as neurotransmitters.

Know which amino acids can be used for the synthesis of which neurotransmitters.

14.

Understand in general terms the biosynthesis of spermine, spermidine, creatine and phosphocreatine.

SOURCES AND FATES OF AMINO ACIDS IN THE BODY

PROTEIN CATABOLISM Proteolysis

of

dietary

proteins in the

stomach

and lumen of the

small intestine

releases free amino acids into the bloodstream.

Proteolysis

of proteins that move through the

endocytic pathway

takes place in the

lysosomes

of all cells.

Controlled proteolysis

of

ubiquitin-tagged

intracellular proteins takes place in the

proteasomes

of all cells.

AMINO ACID POOL

LYSOSOMAL DEGRADATION

1. Lysosomes degrade proteins taken up by

endocytosis

, or proteins that traffic within the endocytic pathway.

2.

Lysosomes contain ~50 hydrolytic enzymes (proteases). Their

pH optima is acidic

.

3.

The

pH

of the lysosome is

~5

.

4.

In

well-nourished

cells, lysosomal protein degradation is

non-selective

.

5.

In

starving

cells, there is a

selective

pathway that preferentially degrades cytosolic proteins containing the pentapeptide KFERQ (Lys-Phe-Glu-Arg-Gln).

CONTROLLED PROTEOLYSIS

1.

Ubiquitin

tags proteins for destruction.

2.

The

proteasome

digests the ubiquitin tagged proteins.

3.

Protein degradation can be used to

regulate

biological function.

UBIQUITIN

76 aa polypeptide.

The Mark of Death C-terminal gly attaches to the e -amino groups of several lys on a protein destined for degradation.

Additional ubiquitin molecules can be added to Lys48.

UBIQUITIN CONJUGATION E1=Ubiquitin-Activating Enzyme E2=Ubiquitin-Conjugating Enzyme E3=Ubiquitin-Protein Ligase

UBIQUITIN IS ATTACHED TO THE e

-AMINO GROUP

OF LYSINE RESIDUES ON TARGET PROTEINS

CLINICAL CORRELATION: Human papilloma virus (HPV) encodes a protein that activates a specific E3 enzyme. The enzyme ubiquitinates the tumor suppressor p53 and other proteins that control DNA repair, which are then destroyed. The activation of this E3 enzyme is observed in more than 90% of all cervical carcinomas.

A

SINGLE

UBIQUITIN MOLECULE IS A

POOR SIGNAL

FOR DEGRADATION.

CHAINS

OF 4 OR MORE UBIQUITIN MOLECULES ARE VERY

STRONG SIGNALS

FOR DEGRADATION.

WHAT DETERMINES WHETHER A PROTEIN IS UBIQUITINATED? The substrate specificity of each E3.

1.

The

N-terminal rule:

the chemical nature of the amino-terminal amino acid.

2.

Cyclin destructive boxes:

specific amino acid sequences that mark cell-cycle proteins for destruction.

3.

PEST sequences:

proteins rich in proline, glutamic acid, serine and threonine.

THE 26S PROTEASOME DIGESTS THE UBIQUITIN TAGGED PROTEINS

19S regulatory subunit 20S proteasome (catalytic activity) 19S regulatory subunit The Executioner

THE 20S PROTEASOME

1. 700kD, 28 homologous subunits: 14 of type a and 14 of type b.

2. Subunits are arranged in 4 rings of 7 subunits each to form a

sealed barrel

. 7 7 7 7

PROTEOLYTIC ACTIVITY RESIDES IN THE N-TERMINALTHREONINE RESIDUES OF THE BETA SUBUNITS

7 7 7 7

ACCESS TO THE 20S PROTEASOME IS CONTROLLED BY THE 19S CAPS The 19S regulatory subunits bind to polyubiquitin chains.

SOURCES AND FATES OF AMINO ACIDS IN THE BODY

PROTEIN DEGRADATION CAN REGULATE BIOLOGICAL PROCESSES Dynamically alter the stablity of regulatory proteins.

SOURCES AND FATES OF AMINO ACIDS IN THE BODY

AMINO ACID DEGRADATION

1.

Any amino acids generated by protein catabolism that are

not needed

as building blocks for new biomolecular synthetic reactions are

degraded

to carbon skeletons in the liver.

2.

The first step in amino acid degradation is the

removal of nitrogen.

a

-AMINO GROUPS ARE CONVERTED INTO AMMONIUM IONS BY OXIDATIVE DEAMINATION OF GLUTAMATE

The a

-amino group

of the a -amino acid is

transferred

to a -ketoglutarate to form glutamate, which is

oxidatively deaminated

to yield

ammonium ion

.

1. THE TRANSAMINATION REACTION: Aminotransferases

(also called transaminases) catalyze the

transfer of an

a

-amino group

from an a -amino acid to an a -keto acid.

These enzymes generally utilize a -ketoglutarate as the acceptor.

The enzymes are named after their amino acid substrates, i.e.

aspartate transaminase

catalyzes the transfer of the a -amino group of

aspartate

to a ketoglutarate, yielding oxaloacetate plus glutamate.

2. THE OXIDATIVE DEAMINATION REACTION:

The nitrogen atom that is transferred to a -ketoglutarate in the transamination reaction is

converted into free ammonium ion by oxidative deamination.

This reaction is catalyzed by

glutamate dehydrogenase.

This reaction takes place in the mitochondria, and is driven by the

consumption of ammonia.

Dehydrogenation Hydrolysis

The

sum

of the aminotransferase and glutamate dehydrogenase reactions yield ammonium ion: aminotransferase dehydrogenase urea cycle

AMINO ACID 1 ALPHA KETOACID 2

excreted

ALPHA KETOACID 1 AMINO ACID 2

ALL AMINOTRANSFERASES CONTAIN THE PROSTHETIC GROUP PYRIDOXAL PHOSPHATE (PLP) PLP is derived from Pyridoxine (Vitamin B6)

PLP TAUTOMERS:

The phenolic hydroxyl group is slightly acidic, favoring deprotonation.

The pyridine ring is slightly basic, which favors protonation of the pyrimidine N.

PHENOLATE

PLP FORMS SCHIFF BASE INTERMEDIATES IN AMINOTRANSFERASES AMINO ACID 1

(schiff-base linkage with the enzyme) (schiff-base linkage with the substrate) The

positively

charged schiff-base linkages are stabilized by the

negatively

charged phenolate group.

TRANSAMINATION MECHANISM

1. The schiff base loses a proton from the a -carbon of the amino acid to become a

quinonoid

intermediate.

2. Reprontonation of the quinonoid at the aldehyde carbon yields a

ketimine

intermediate.

3. The ketimine is then hydrolyzed to an a

-ketoacid

and

PMP

.

ALPHA KETOACID 1 1 2 3

ONCE THE AMINO GROUP HAS BEEN TRANSFERRED TO PMP, PMP TRANSFERS THE AMINO GROUP TO ANOTHER ALPHA-KETOACID BY REVERSING THE REACTION SCHEME WE JUST DISCUSSED (FOLLOW THE RED ARROWS):

ALPHA KETOACID 2 (ALPHA KETOGLUTARATE) 1 3 4 AMINO ACID 2 (GLUTAMATE) 2

ASPARTATE AMINOTRANSFERASE

Active site Arg386 helps orient substrates by binding to their a -carboxylate groups.

PLP is bound to active site Lys268 by a Schiff-base linkage.

MECHANISM OF THE AMINOTRANSFERASE REACTION:

MOVIE:

SERINE AND THREONINE CAN BE DIRECTLY DEAMINATED

1.

The nitrogen atoms of

MOST

amino acids are transferred to a ketoglutarate.

2.

The a -amino groups of serine and threonine can be directly converted into ammonium ion by the action of

dehydratases.

Threonine

a

-ketobutyrate + NH 4 +

PERIPHERAL TISSUES TRANSPORT NITROGEN TO THE LIVER BY THE ALANINE CYCLE OR AS GLUTAMINE

If amino acids are produced in tissues that

lack the urea cycle

, they need a mechanism to release nitrogen in a form that can be absorbed by the liver and converted into urea.

EXAMPLE:

Muscle uses amino acids as fuel during prolonged exercise and fasting.

THE ALANINE CYCLE

1. In peripheral tissues,the a -amino groups of the amino acids are transferred to glutamate by a

transamination

reaction, as in the liver.

2. However, rather than oxidatively deaminating glutamate to form ammonium ion, the a -amino group is transferred to pyruvate to form

alanine.

3. The liver takes up the alanine, and converts it back to pyruvate by another

transamination

reaction.

4. The pyruvate can be used for gluconeogenesis, and the amino group eventually ends up as

urea

by the usual pathway.

a -ketoglutarate a -ketoglutarate glutamate pyruvate

NITROGEN CAN ALSO BE TRANSPORTED AS GLUTAMINE Glutamine Synthetase: NH 4 + + glutamate + ATP glutamine + ADP + P i

SOURCES AND FATES OF AMINO ACIDS IN THE BODY

IN LIVER THE AMMONIUM IONS GENERATED DURING AMINO ACID DEGRADATION FEED INTO THE UREA CYCLE

Urea cycle: importance

• NH 4 + is a product of the breakdown of amino acids.

• NH 4 + is required by cells for synthesis of nitrogen containing compounds.

• Excess NH 4 + is very toxic. Normal levels in human blood are: [NH 4 + ] < 70  M.

• Excess NH 4 + is converted to urea via the urea cycle and excreted. The urea cycle accounts of ~80% of the excreted nitrogen.

Urea cycle: location and source of atoms

• Urea synthesis takes place mostly in the liver.

• One N atom of urea comes from Asp (blue).

• One N atom comes from NH 4 + (green).

• One C atom comes from CO 2 (red).

• Ornithine acts as a carrier of various atoms in the process of synthesizing urea.

Urea cycle reactions: carbamoyl phosphate synthetase

• Catalyzes formation of carbamoyl phosphate from H 2 O, 2 ATPs, CO 2 and NH is required for activity.

3 + .

• The positive heterotropic activator, N-acetylglutamate, • Brings one C atom and one N atom into the urea cycle as a carbamoyl group.

• Catalyzes the critical step in removing NH 4 + blood.

from the

Urea cycle reactions: carbamoyl phosphate synthetase

• The reaction is made irreversible by cleaving two ATP molecules to two ADP. • One molecule of phosphate is released while the second phosphate ends up as part of carbamoyl phosphate.

Urea cycle reactions: carbamoyl phosphate synthetase

• Carbamoyl phosphate synthetase is present at very high concentration in the mitochondrial matrix (~1 mM).

• The high enzyme concentration allows the enzyme to work well below the K m ~ 250  M for NH 4 + .

• By operating well below K m , a small increase in NH 4 + leads to a large increase in the rate of removal of NH 4 + insuring that NH 4 + remains low.

Urea cycle reactions: ornithine transcarbamoylase

• Catalyzes the formation of citrulline and P i ornithine and carbamoyl phosphate.

from • Transfer of a carbamoyl group to ornithine is facilitated by rupture of a high energy phosphoanhydride bond.

• Catalyzes introduction of one C atom and one N atom into the urea cycle from carbamoyl phosphate.

Urea cycle reactions: argininosuccinate synthetase

• Catalyzes condensation of citrulline and aspartate to form argininosuccinate.

• Catalyzes the introduction of one N atom into the urea cycle from aspartate.

Urea cycle reactions: argininosuccinase

• Cleaves argininosuccinate to arginine and fumarate.

• Completes the transfer of the amino group from aspartate to make arginine.

• Retains the carbon skeleton of aspartate (as a fumarate molecule).

Urea cycle reactions: arginase

• Catalyzes hydrolysis of arginine to ornithine and urea.

• Ornithine “cycles” back to the first step and picks up another carbamoyl group from carbamoyl phosphate.

Urea cycle: overall reaction

• PP i  2 P i quickly in a reaction catalyzed by pyrophosphotase.

• Overall, four high energy phosphate bonds are broken to synthesize each molecule of urea.

Urea cycle and the citric acid cycle

General amino acid catabolism • Fumarate production connects the urea cycle and the citric acid cycle (fumarate  malate  oxaloacetate).

• In the citric acid cycle fumarate is converted to oxaloacetate.

• Oxaloacetate is transaminated to aspartate.

• Aspartate carries the amino groups of other amino acids into the urea cycle.

Compartmentalization of the urea cycle

• Takes place in the liver.

• Two intracellular locations.

• Mitochondrial matrix: carbamoyl phosphate formation and citrulline synthesis.

• Cytosol: argininosuccinate formation; cleavage of argininosuccinate to arginine and fumarate; hydrolysis of arginine to ornithine and urea.

Regulation of the urea cycle

• The urea cycle removes excess NH 4 + breakdown of dietary amino acids. which comes from the • Overall control of the urea cycle is by enzyme levels, which change by as much as ten-fold depending on the diet.

• The flow of compounds through the urea cycle also depends on the concentrations of cycle intermediates.

• Several reactions convert amino acids into urea cycle intermediates.

• Arginine from the diet can be converted to ornithine.

• Glutamate can be converted to ornithine by intestinal enzymes.

Regulation of the urea cycle

• Fine control of the urea cycle is through regulation of carbamoyl phosphate synthetase.

• N-acetylglutamate is a heterotropic allosteric activator of carbamoyl phosphate synthetase. (Heterotropic means an effector molecule that is different from the substrate.)

Regulation of the urea cycle

• N-acetylglutamate acts as a signal for high amino acid concentrations.

• N-acetylglutamate is synthesized in the liver from acetyl CoA and glutamate in a reaction catalyzed by N acetylglutamate synthetase.

Regulation of the urea cycle

• The steady state concentration of N-acetylglutamate is determined by two factors.

• Concentrations of substrates: acetyl CoA and glutamate.

• Concentration of arginine, which activates N acetylglutamate synthetase.

Defective urea cycle enzymes and inherited disease

• High NH 4 + is toxic and a complete lack of any urea cycle enzyme is fatal.

• Some diseases are believed to be due to partially active defective enzymes.

• Defective enzymes cause high levels of NH 4 + in the blood.

• Sometimes a low protein diet can help. The diet decreases the amount of NH 4 + that needs to be eliminated through the urea cycle.

Defective enzymes and treatment

• Problem: argininosuccinase deficiency.

• Treatment: a low protein diet high in arginine. • Result: argininosuccinate is secreted in place of urea.

Defective enzymes and treatment

• Problem: carbamoyl phosphate synthetase or ornithine transcarbamoylase deficiency.

• Result: glycine and glutamine build up.

• Treatment: feed benzoate to remove Gly as hippurate.

• Treatment: feed phenylacetate to remove Gln as phenylacetylglutamine.

NH

4 +

toxicity and excess glutamine

• NH 4 + toxicity may be due to formation of excess glutamine.

• High glutamine levels are found in the cerebrospinal fluid of those with high NH 4 + .

• High glutamine and glutamate may lead to brain damage, possibly by producing osmotic effects that cause brain swelling.

• Treatment: a low protein diet high in arginine. • Result: argininosuccinate is secreted in place of urea.

Urea cycle: overall reaction

• PP i  2 P i quickly in a reaction catalyzed by pyrophosphotase.

• Overall, four high energy phosphate bonds are broken to synthesize each molecule of urea.

Hyperammonemia: Why is NH 4 + toxic?

a

-ketoglutarate + NH 4 + <---> Glutamate + NH 4 + <---> Glutamine Excess Glutamate and Glutamine lead to osmotic effects, i.e. brain swelling; Exitatory Amino Acid Neurotoxicity

SOURCES AND FATES OF AMINO ACIDS IN THE BODY

FATES OF THE CARBON SKELETONS OF THE AMINO ACIDS

1.

The strategy of amino acid degradation is to transform the carbon skeletons into

major metabolic intermediates

that can be converted into glucose, or oxidized by the citric acid cycle.

2.

The carbon skeletons of a diverse set of 20 amino acids are funneled into only 7 molecules:

pyruvate, acetyl CoA, acetoacetyl CoA,

a

-ketoglutarate, succinyl CoA, fumarate and oxaloacetate.

PYRUVATE AS AN ENTRY POINT INTO METABOLISM 3-carbon

amino acids ala, ser, cys enter via pyruvate.

OXALOACETATE AS AN ENTRY POINT INTO METABOLISM

The skeletons of

4-carbon

amino acids enter at oxaloacetate.

a

-KETOGLUTARATE AS AN ENTRY POINT INTO METABOLISM

The skeletons of several

5-carbon

amino acids enter the TCA cycle at a -ketoglutarate. All first transfer their amino groups to glutamate, which is then oxidatively deaminated by glutamate dehydrogenase to yield a -ketoglutarate.

SUCCINYL COENZYME A AS AN ENTRY POINT INTO METABOLISM FOR SEVERAL NON-POLAR AMINO ACIDS

Threonine (in humans)

CLINICAL CORRELATION:

methyl-malonic acedemia methylmalonyl CoA mutase is defective

CLINICAL CORRELATION:

homocystinuria Scoliosis, muscle weakness, mental retardation, thin blond hair cystathione b -synthase is defective

Glucogenic Amino Acids:

amino acids (aa) that are converted to metabolites that can be converted to

glucose.

TCA cycle intermediates and pyruvate can be converted to phosphoenolpyruvate and then glucose.

Ketogenic Amino Acids:

aa that give rise to

ketone

bodies or fatty acids.

Only leucine and lysine are solely ketogenic.

Isoleucine,phenylalanine, tryptophan and tyrosine are both ketogenic and glucogenic.

Remaining 14 amino acids are solely glucogenic.

WHEN THE BRANCHED CHAIN AMINO ACIDS VALINE, ISOLEUCINE, AND LEUCINE ARE DEGRADED IN EXTRA-HEPATIC TISSUES THEY SHARE TWO COMMON ENZYMES: branched-chain aminotransferase branched-chain

a

-ketoacid dehydrogenase complex

While much of the catabolism of amino acids takes place in the liver, the branched chain amino acids are oxidized as primary fuels in muscle, adipose, kidney, and brain tissues.

CLINICAL CORRELATION: maple syrup disease;

Defective branched-chain a -keto acid dehydrogenase complex; urine has odor of maple syrup, mental and physical retardation UNLESS patients are placed on a diet low in valine, isoleucine and leucine early in life.

OXYGENASES ARE REQUIRED FOR THE DEGRADATION OF AROMATIC AMINO ACIDS

Monooxygenase- one atom of O 2 in the product and one in H 2 0.

appears

CLINICAL CORRELATION:

MUTATIONS IN THE GENE ENCODING PHENYLALANINE HYDROXYLASE CAUSE PHENYLKETONURIA More than 200 mutations have been identified. Mutations effecting the

active site

, the

biopterin binding site

, and

other regions of the protein

are indicated as colored spheres.

AROMATIC AMINO ACID METABOLISM Clinical Correlation:

Phenylketonuria-phenylalanine accumulates in body fluids, if untreated, severe mental retardation. Treatment-low phenylalanine diet.

Tyrosinemias-if untreated, weakness, self-mutilation, liver damage, mental retardation. Treatment?

Alkaptonuria homogentisate accumulates in the urine, and is excreted, which turns dark on standing due to the oxidation of homogentisate.

Harmless.

TRYPTOPHAN DEGRADATION

Nearly all cleavages of aromatic rings in biological systems are catalyzed by dioxygenases.

SUMMARY OF DEFECTS IN AMINO ACID CATABOLISM THAT CONTRIBUTE TO HUMAN DISEASE

FATES OF THE CARBON SKELETONS OF THE AMINO ACIDS

SOURCES AND FATES OF AMINO ACIDS IN THE BODY

AMINO ACID BIOSYNTHESIS

1. The nitrogen in amino acids, purines, pyrimidines and other biomolecules

ultimately

comes from atmospheric N 2 .

2. This process begins with the reduction of N 2 to NH 3. This process is called

NITROGEN FIXATION

, and is carried out by some bacteria. 3. Since nitrogen fixation does not take place in higher organisms, the source of nitrogen for amino acid biosynthesis in humans are the

metabolites of dietary nitrogen

.

AMINO ACIDS ARE MADE FROM METABOLITES OF THE MAJOR METABOLIC PATHWAYS

MOST MICROORGANISMS CAN SYNTHESIZE ALL 20 AMINO ACIDS HUMANS CAN ONLY SYNTHESIZE 11 AMINO ACIDS

The essential amino acids cannot be made by humans and must be obtained in the diet.

1. The synthesis of many of the amino acids is a simple

reversal of their degradation

, utilizing a

transamination

reaction.

2. Amino acids skeletons end up as

major metabolic intermediates

during

degradation

. Likewise, amino acids are also

biosynthesized

from

major metabolic intermediates.

SOURCES AND FATES OF AMINO ACIDS IN THE BODY

BIOSYNTHETIC FATES OF THE AMINO ACIDS:

1. Protein synthesis (Dr. Lee) 2. Nucleic acid synthesis (Dr. Lee) 3. Heme synthesis (Dr. Luduena) 4. Thyroid hormone synthesis (Dr. Adamo) 5. Neurotransmitter synthesis 6. Spermine and spermidine 7. Creatine and phosphocreatine

Several amino acids can function as neurotransmitters without any chemical modification:

Glutamate Glycine Aspartate

MOST OF THE SMALL-MOLECULE NEUROTRANSMITTERS ARE AMINO ACIDS OR THEIR DERIVATIVES

* Tyrosine Tyrosine Glutamate Tyrosine Tryptophan Histidine * (not directly from an amino acid, from acetyl CoA + choline)

CATHECHOLAMINE BIOSYNTHESIS

GABA BIOSYNTHESIS

HISTAMINE BIOSYNTHESIS

SEROTONIN BIOSYNTHESIS

BIOSYNTHESIS OF SPERMINE AND SPERMIDINE

BIOSYNTHESIS OF CREATINE AND PHOSPHOCREATINE

BIOSYNTHETIC FATES OF THE AMINO ACIDS:

1. Protein synthesis 2. Nucleic acid synthesis 3. Heme synthesis 4. Thyroid hormone synthesis 5. Neurotransmitter synthesis 6. Spermine and spermidine 7. Creatine and phosphocreatine

SOURCES AND FATES OF AMINO ACIDS IN THE BODY