PROTEIN METABOLISM: PROTEIN TURNOVER; GENERAL WAYS OF AMINO ACIDS 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 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) How can a cell distinguish proteins that are meant for degradation?

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.

Mechanism of the binding of ubiquitin to target protein 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)

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)

Digestion of the Ubiquitin-Tagged Proteins 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

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)

19S subunit 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)

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 2-oxoglutarate amino acid aminotransferase pyridoxal phosphate 2-oxo acid glutamate

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 deamination L-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

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). Ping-pong kinetic mechanism 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 of aspartate transaminase aspartate + -ketoglutarate  oxaloacetate + glutamate

Enzyme: decarboxylases Coenzyme – pyrydoxalphosphate Decarboxylation of amino acids Decarboxylation – removal of carbon dioxide from amino acid with formation of amines. amine Usually amines have high physiological activity (hormones, neurotransmitters etc). Enzyme: decarboxylases Coenzyme – pyrydoxalphosphate

Significance of amino acid decarboxylation 1. Formation of physiologically active compounds GABA – mediator of nervous system glutamate gamma-aminobutyric acid (GABA) histamine histidine 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  Serotonin Vasokonstrictor Takes part in regulation of arterial pressure, body temperature, respiration, kidney filtration, mediator of nervous system 3) Tyrosine  Dopamine It 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 Enzymes of microorganisms (in colon; dead organisms) decarboxylate amino acids with the formation of diamines. ornithine putrescine lysine cadaverine