PROTEIN METABOLISM: NITROGEN CYCLE; DIGESTION OF PROTEINS Red meat is an important dietary source of protein nitrogen

The Nitrogen Cycle and Nitrogen Fixation Nitrogen is needed for amino acids, nucleotides, etc Atmospheric N2 is the ultimate source of biological nitrogen Nitrogen fixation: biosynthetic process of the reduction of N2 to NH3 (ammonia) Higher organisms are unable to fix nitrogen. Some bacteria and archaea can fix nitrogen.

Nodules of Rhizobium bacteria Symbiotic Rhizobium bacteria invade the roots of leguminous plants and form root nodules. Nodules of Rhizobium bacteria Rhizobium bacteria fix nitrogen supplying both the bacteria and the plants. Archaea

The amount of N2 fixed by nitrogen-fixing microorganisms is about 60% of Earth's newly fixed nitrogen. Lightning and ultraviolet radiation fix 15% 25% is fixed by industrial processes (fertilizer factories)

Nitrogen-fixing bacteria possess nitrogenase complex which can reduce N2 to ammonia The nitrogenase complex consists of two proteins: reductase, which provides electrons nitrogenase, which uses these electrons to reduce N2 to NH3. The transfer of electrons from the reductase to the nitrogenase is coupled to the hydrolysis of ATP.

Nitrogenase reaction: The high-potential electrons come from protein ferredoxin, generated by photosynthesis or oxidative processes. 16 molecules of ATP are hydrolyzed for each molecule of N2 reduced. Nitrogenase reaction: N2 + 8 H+ + 8 e- + 16 ATP  2 NH3 + H2 + 16 ADP + 16 Pi Reductase – dimer containing Fe-S clusters and ATP-binding site

The nitrogenase component is an 22 tetramer. Contains P cluster (Fe-S) and FeMo cofactor. FeMo cofactor is the site of nitrogen fixation.

NO2- and NO3- are used by higher plants Ammonia in the presence of water becomes NH4+ which can be used by plants NH4+ can be rapidly oxidized by soil bacteria Nitrosomonas and Nitrobacter to NO2- and NO3- (nitrification) Nitrosomonas NO2- and NO3- are used by higher plants Another soil bacteria can reverse the nitrification process and convert NO2- and NO3- back to nitrogen Nitrogen from plants and animals is recycled to soil (excretion of nitrogen in the form of urea or uric acid; decay of plants and animals) - nitrogen cycle

Assimilation of Ammonia Ammonia generated from N2 is assimilated into amino acids such as glutamate or glutamine

A. Ammonia Is Incorporated into Glutamate Reductive amination of a-ketoglutarate by glutamate dehydrogenase occurs in plants, animals and microorganisms This reaction establishes the stereochemistry of the -carbon atom in glutamate. Only the L isomer of glutamate is synthesized.

B. Glutamine Is a Nitrogen Carrier A second route in assimilation of ammonia is via glutamine synthetase All organisms have both enzymes: glutamate dehydrogenase and glutamine synthetase. Amino acids are used for the synthesis of proteins. Animals and humans consume proteins. Proteins undergo digestion in the stomach and intestine.

Protein digestion Digestion in Stomach Stimulated by food acetylcholine, histamine and gastrin are released onto the cells of the stomach The combination of acetylcholine, histamine and gastrin cause the release of the gastric juice. Mucin - is always secreted in the stomach HCl - pH 0.8-2.5 (secreted by parietal cells) Pepsinogen (a zymogen, secreted by the chief cells) Hydrochloric acid: Creates optimal pH for pepsin Denaturates proteins Kills most bacteria and other foreign cells

Pepsinogen (MW=40,000) is activated by the enzyme pepsin present already in the stomach and the stomach acid.  Pepsinogen cleaved off to become the enzyme pepsin (MW=33,000) and a peptide fragment to be degraded. Pepsin partially digests proteins by cleaving the peptide bond formed by aromatic amino acids: Phe, Tyr, Trp

Digestion in the Duodenum Stimulated by food secretin and cholecystokinin regulate the secretion of bicarbonate and zymogens trypsinogen, chymotrypsinogen, proelastase and procarboxypeptidase by pancreas into the duodenum Bicarbonate changes the pH to about 7 The intestinal cells secrete an enzyme called enteropeptidase that acts on trypsinogen cleaving it into trypsin

Trypsin converts chymotrypsinogen into chymotrypsin, procarboxypeptidase into carboxypeptidase and proelastase into elastase, and trypsinogen into more trypsin. Trypsin which cleaves peptide bonds between basic amino acids Lys and Arg Chymotrypsin cleaves the bonds between aromatic amino acids Phe, Tyr and Trp Carboxypeptidase which cleaves one amino acid at a time from the carboxyl side Aminopeptidase is secreted by the small intestine and cleaves off the N-terminal amino acids one at a time

Most proteins are completely digested to free amino acids Amino acids and sometimes short oligopeptides are absorbed by the secondary active transport Amino acids are transported via the blood to the cells of the body.

The ways of entry and using of amino acids in tissue The sources of amino acids: 1) absorption in the intestine; 2) protein decomposition; 3) synthesis from the carbohydrates and lipids. Using 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.

PROTEIN METABOLISM: PROTEIN TURNOVER; GENERAL WAYS OF AMINO ACIDS METABOLISM

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)

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.

Deamination of amino acids 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

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.

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

PROTEIN METABOLISM: UTILIZATION OF AMMONIA IONS; UREA CYCLE The basic features of nitrogen metabolism were elucidated initially in pigeons

AMMONIA METABOLISM The ways of ammonia formation 1. Oxidative deamination of amino acids 2. Deamination of physiologically active amines and nitrogenous bases. 3. Absorption of ammonia from intestine (degradation of proteins by intestinal microorganisms results in the ammonia formation). 4. Hydrolytic deamination of AMP in the brain (enzyme – adenosine deaminase)

Ammonia is a toxic substance to plants and animals (especially for brain) Normal concentration: 25-40 mol/l (0.4-0.7 mg/l) Ammonia must be removed from the organism Terrestrial vertebrates synthesize urea (excreted by the kidneys) - ureotelic organisms Birds, reptiles synthesize uric acid Urea formation takes place in the liver

Peripheral Tissues Transport Nitrogen to the Liver Two ways of nitrogen transport from peripheral tissues (muscle) to the liver: Glutamate is not deaminated in peripheral tissues 1. Alanine cycle. Glutamate is formed by transamination reactions

Nitrogen is then transferred to pyruvate to form alanine, which is released into the blood. The liver takes up the alanine and converts it back into pyruvate by transamination. The glutamate formed in the liver is deaminated and ammonia is utilized in urea cycle.

2. Nitrogen can be transported as glutamine. Glutamine synthetase catalyzes the synthesis of glutamine from glutamate and NH4+ in an ATP-dependent reaction:

THE UREA CYCLE Urea cycle - a cyclic pathway of urea synthesis first postulated by H.Krebs The sources of nitrogen atoms in urea molecule: aspartate; NH4+. Carbon atom comes from CO2.

The free ammonia is coupling with carbon dioxide to form carbamoyl phosphate Two molecules of ATP are required Reaction takes place in the matrix of liver mitochondria Enzyme: carbamoyl phosphate synthetase (20 % of the protein of mitochondrial matrix)

Enzyme: ornithine carbamoyltransferase Carbamoyl phosphate donates carbamoyl group to ornithine The product - citruilline Enzyme: ornithine carbamoyltransferase Reaction takes place in the mitochondrial matrix Citrulline leaves the matrix and passes to the cytosol

In the cytosol citrulline in the presence of ATP reacts with aspartate to form argininosuccinate Enzyme: argininosuccinate synthetase

Argininosuccinate is cleaved to free arginine and fumarate Enzyme: argininosuccinate lyase The fumarate enters the tricarboxylic acid cycle

Arginine is hydrolyzed to generate urea and ornithine Enzyme: arginase (present only in liver of ureotelic animals) Ornithine is transported back into the mitochondrion to begin another cycle Urea is excreted (about 40 g per day)

The urea cycle

The Linkage between Urea Cycle, Citric Acid Cycle and Transamination of Oxaloacetate Fumarate formed in urea cycle enters citric acid cycle and is converted to oxaloacetate. Fates of oxaloacetate: transamination to aspartate, conversion into glucose, condensation with acetyl CoA to form citrate, conversion into pyruvate.