PROTEIN PROTEIN METABOLISM: METABOLISM:

PROTEIN PROTEIN TURNOVER; TURNOVER;

GENERAL WAYS OF GENERAL WAYS OF AMINO ACIDS AMINO ACIDS METABOLISM METABOLISM

Proteins function in the organism.

All enzymes are proteins. Storing amino acids as nutrients and as building blocks for the growing organism. Transport function (proteins transport fatty acids, bilirubin, ions, hormones, some drugs etc.). Proteins are essential elements in contractile and motile systems (actin, myosin). Protective or defensive function (fibrinogen, antibodies). Some hormones are proteins (insulin, somatotropin). Structural function (collagen, elastin).

PROTEIN TURNOVER

How can a cell distinguish proteins that are meant for degradation?

Protein turnover — the degradation and resynthesis of proteins

Half-lives of proteins – from several minutes to many years

Structural proteins – usually stable (lens protein crystallin lives during the whole life of the organism)Regulatory proteins - short lived (altering the amounts of these proteins can rapidly change the rate of metabolic processes)

Ubiquitin - is the tag that marks proteins for destruction ("black spot" - the signal for death)

Ubiquitin - a small (8.5-kd) protein present in all eukaryotic cells

Structure: extended carboxyl terminus (glycine) that is linked to other proteins; lysine residues for linking additional ubiquitin molecules

Ubiquitin covalently binds to -amino group of lysine residue on a protein destined to be degraded.

Isopeptide bond is formed.

E1 - ubiquitin-activating enzyme (attachment of ubiquitin to a sulfhydryl group of E1; ATP-driven reaction)

E2 - ubiquitin-conjugating enzyme (ubiquitin is shuttled to a sulfhydryl group of E2)

E3 - ubiquitin-protein ligase (transfer of ubiquitin from E2 to -amino group on the target protein)

Mechanism of the binding of ubiquitin to target protein

Attachment of a single molecule of ubiquitin - weak signal for degradation.

Chains of ubiquitin are generated.

Linkage – between -amino group of lysine residue of one ubiquitin to the terminal carboxylate of another.

Chains of ubiquitin molecules are more effective in signaling degradation.

What determines ubiquitination of the protein?

1. The half-life of a protein is determined by its amino-terminal residue (N-terminal rule). E3 enzymes are the readers of N-terminal residues.

2. Cyclin destruction boxes - specific amino acid sequences (proline, glutamic acid, serine, and threonine –PEST)

What is the executioner of the protein death?

A large protease complex proteasome or the 26S proteasome digests the ubiquitinated proteins.

26S proteasome - ATP-driven multisubunit protease.

26S proteasome consists of two components: 20S - catalytic subunit 19S - regulatory subunit

Digestion of the Ubiquitin-Tagged Proteins

20S subunit resembles a barrel is constructed from 28 polipeptide chains which are arranged in four rings (two and two ) active sites are located in rings on the interior of the barrel degrades proteins to peptides (seven-nine residues)

made up of 20 polipeptide chains

controls the access to interior of 20S barrel

binds to both ends of the 20S proteasome core

binds to polyubiquitin chains and cleaves them off

possesses ATPase activity

unfold the substrate

induce conformational changes in the 20S proteasome (the substrate can be passed into the center of the complex)

19S subunit

Overview of Amino Acid Catabolism:Interorgan Relationships

Overview of Amino Acid Catabolism:Interorgan Relationships

• Liver– Synthesis of liver and plasma proteins– Catabolism of amino acids

• Gluconeogenesis• Ketogenesis• Branched chain amino acids (BCAA) not catabolized• Urea synthesis

– Amino acids released into general circulation• Enriched in BCAA (2-3X)

Overview of Amino Acid Catabolism:Interorgan Relationships

• Skeletal Muscle– Muscle protein synthesis– Catabolism of BCAA

• Amino groups transported away as alanine and glutamine (50% of AA released)

– Alanine to liver for gluconeogenesis– Glutamine to kidneys

• Kidney– Glutamine metabolized to a-KG + NH4

• a-KG for gluconeogenesis• NH4 excreted or used for urea cycle (arginine synthesis)

– Important buffer from acidosis

GENERAL WAYS OF AMINO ACIDS METABOLISM

The fates of amino acids: 1) for protein synthesis;

2) for synthesis of other nitrogen containing compounds (creatine, purines, choline, pyrimidine);

3) as the source of energy;

4) for the gluconeogenesis.

The general ways of amino acids degradation: Deamination Transamination Decarboxilation

The major site of amino acid degradation - the liver.

Deamination of amino acids

Deamination - elimination of amino group from amino acid with ammonia formation.

Four types of deamination: - oxidative (the most important for

higher animals), - reduction, - hydrolytic, and - intramolecular

Reduction deamination:

R-CH(NH2)-COOH + 2H+ R-CH2-COOH + NH3

amino acid fatty acid

Hydrolytic deamination:

R-CH(NH2)-COOH + H2O R-CH(OH)-COOH + NH3

amino acid hydroxyacid

Intramolecular deamination:

R-CH(NH2)-COOH R-CH-CH-COOH + NH3

amino acid unsaturated fatty acid

General scheme of oxydative transamination

CH2CH2COOH

O

CHOOC+R CH

NH2

COOH

aminokyselina 2-oxoglutarát

HOOC CH CH2CH2COOH

NH2

+R C

O

COOH

glutamát2-oxokyselina

aminotransferasapyridoxalfosfát

amino acid

2-oxo acid

2-oxoglutarate

glutamate

aminotransferase

pyridoxal phosphate

Glutamate dehydrogenase (GMD, GD, GDH)

• requires pyridine cofactor NAD(P)+

• GMD reaction is reversible: dehydrogenation with NAD+,

hydrogenation with NADPH+H+

• two steps:

• dehydrogenation of CH-NH2 to imino group C=NH

• hydrolysis of imino group to oxo group and ammonia

Oxidative deaminationL-Glutamate dehydrogenase plays a central role in amino acid deamination

In most organisms glutamate is the only amino acid that has active dehydrogenase

Present in both the cytosol and mitochondria of the liver

Transamination of amino acids

Transamination - transfer of an amino group from an -amino acid to an -keto acid (usually to -ketoglutarate)

Enzymes: aminotransferases (transaminases).

-amino acid -keto acid -keto acid -amino acid

There are different transaminases

The most common: alanine aminotransferase alanine + -ketoglutarate pyruvate + glutamate

aspartate aminotransferase aspartate + -ketoglutarate oxaloacetate + glutamate

Aminotransferases funnel -amino groups from a variety of amino acids to -ketoglutarate with glutamate formation

Glutamate can be deaminated with NH4+

release

Mechanism of transamination

All aminotransferases require the prosthetic group pyridoxal phosphate (PLP), which is derived from pyridoxine (vitamin B6).

First step: the amino group of amino acid is transferred to pyridoxal phosphate, forming pyridoxamine phosphate and releasing ketoacid.

Second step: -ketoglutarate reacts with pyridoxamine phosphate forming glutamate

Ping-pong kinetic mechanism

Ping-pong kinetic mechanism of aspartate transaminase

aspartate + -ketoglutarate oxaloacetate + glutamate

Decarboxylation – removal of carbon dioxide from amino acid with formation of amines.

Usually amines have high physiological activity (hormones, neurotransmitters etc).

amine

Enzyme: decarboxylases Coenzyme – pyrydoxalphosphate

Decarboxylation of amino acids

Significance of amino acid decarboxylation1. Formation of physiologically active

compounds

glutamate gamma-aminobutyric acid (GABA)

GABA – mediator of nervous system

histaminehistidine

Histamine – mediator of inflammation, allergic reaction.

1) A lot of histamine is formed in inflamatory place;It has vasodilator action;Mediator of inflamation, mediator of pain;Responsible for the allergy development;

Stimulate HCI secretion in stomach. -CO2

2) Tryptophan SerotoninVasokonstrictorTakes part in regulation of arterial pressure, body temperature, respiration, kidney filtration, mediator of nervous system

3) Tyrosine DopamineIt is precursor of epinephrine and norepinephrine. mediator of central nervous system

4) Glutamate -aminobutyrate (GABA)Is is ingibitory mediator of central nervous system. In medicine we use with anticonvulsion purpose (action).

2. Catabolism of amino acids during the decay of proteins

ornithine putrescine

lysine cadaverine

Enzymes of microorganisms (in colon; dead organisms) decarboxylate amino acids with the formation of diamines.