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 N 2 is the ultimate source of biological nitrogen Nitrogen fixation: biosynthetic process of the reduction of N 2 to NH 3 (ammonia) Higher organisms are unable to fix nitrogen. Some bacteria and archaea can fix nitrogen.

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

The amount of N 2 fixed by nitrogen-fixing microorganisms is about 60% of Earth's newly fixed nitrogen. 25% is fixed by industrial processes (fertilizer factories) Lightning and ultraviolet radiation fix 15%

Nitrogen-fixing bacteria possess nitrogenase complex which can reduce N 2 to ammonia The nitrogenase complex consists of two proteins:  reductase, which provides electrons  nitrogenase, which uses these electrons to reduce N 2 to NH 3. The transfer of electrons from the reductase to the nitrogenase is coupled to the hydrolysis of ATP.

Nitrogenase reaction: N H e ATP  2 NH 3 + H ADP + 16 P i The high-potential electrons come from protein ferredoxin, generated by photosynthesis or oxidative processes. 16 molecules of ATP are hydrolyzed for each molecule of N 2 reduced. 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.

Nitrosomonas Ammonia in the presence of water becomes NH 4 + which can be used by plants NH 4 + can be rapidly oxidized by soil bacteria Nitrosomonas and Nitrobacter to NO 2 - and NO 3 - (nitrification) NO 2 - and NO 3 - are used by higher plants Another soil bacteria can reverse the nitrification process and convert NO 2 - and NO 3 - 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 N 2 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 (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.