PROTEIN PROTEIN METABOLISM: METABOLISM:

NITROGEN NITROGEN CYCLE; CYCLE;

DIGESTION OF DIGESTION OF PROTEINS 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.

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 N2 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 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:

N2 + 8 H+ + 8 e- + 16 ATP

2 NH3 + H2 + 16 ADP + 16 Pi

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.

Reductase – dimer containing Fe-S clusters and ATP-binding site

The nitrogenase component is an 22 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 NH4

+ which can be used by plants

NH4+ can be rapidly oxidized

by soil bacteria Nitrosomonas and Nitrobacter to NO2

- and NO3-

(nitrification)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.

The nitrogen cycle

Nitrogen balance is a comparison between Nitrogen intake (in the form of dietary protein) and Nitrogen loss (as undigested protein in feces, NPN as urea, ammonia, creatinine & uric acid in urine, sweat & saliva & losses by hair, nail, skin).

NB is important in defining1.overall protein metabolism of an individual2.nutritional nitrogen requirement.

Nitrogen Balance (NB):

Nitrogenous balance Positive nitrogenous balance – the amount of nitrogen entered the organism is more than amount of nitrogen removed from the organism. It occurs in young growing organism, during recovering after severe diseases, at the using of anabolic medicines pregnancy, lactation and convulascence

Negative nitrogenous balance – the amount of nitrogen removed from the organism is more than amount of nitrogen entered the organism. It occurs in senile age, destroying of malignant tumor, vast combustions, poisoning by some toxins. High loss of tissue proteins in wasting diseases like burns, hemorrhage & kidney diseases with albuminurea (High breakdown of tissue proteins ) in hyperthyroidism, fever, infection

Zero nitrogenous balance – the amount of nitrogen removed from the organism is equal to the amount of nitrogen entered the organism. It occurs in healthy adult people Normal adult: will be in nitrogen equilibrium, Losses = Intake

A deficiency of even one amino acid results in a negative nitrogen balance. In this state, more protein is degraded than synthesized.

The normal daily requirement of protein for adults is 0.8 g/Kg body wt. day-1.

• That requirement is increased in healthy conditions:during the periods of rapid growth, pregnancy, lactation and adolescence.• Protein requirement is increased in disease states:illness, major trauma and surgery.• RDA for protein should be reduced in:hepatic failure and renal failure

Protein Requirement for humans in Healthy and Disease Conditions

The Fate of Dietary Protein

The intake of dietary protein is in the range of 50-100g/day.

Digestion and absorption .Maintenance of body protein stores.Net protein synthesis. Synthesis of non-protein compoundsOxidative deamination

BV is : a measure for the ability of dietary protein to provide the essential amino acids required for tissue protein maintenance.

•Proteins of animal sources (meat, milk, eggs) have high BV because they contain all the essential amino acids.•Proteins from plant sources (wheat, corn, beans) have low BV thus combination of more than one plant protein is required (a vegetarian diet) to increase its BV.

Biological Value for Protein (BV)

PROTEINS in the BODY

• Amino Acid Pool – amino acids that are available throughout the body (tissues and fluids) for use when needed.

• Protein Turnover – of the ~ 300 grams of protein synthesized by the body each day, 200 grams are made from recycled amino acids.

Chemical composition of digestive juices.

Gastric juice contains water, enzymes, hydrochloric acid, mineral salts and other compounds. About 2,5 l of gastric juice is secreted per day.

The role of hydrochloric acid in digestion. Denaturate proteins (denaturated proteins easier undergo digestion by pepsin than native proteins). Stimulates the activity of pepsin. Hydrochloric acid has bactericidial properties. Stimulates the peristalsis. Regulate the enzymatic function of pancreas.

Protein digestion

Protein digestionDigestion 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

Proteolytic enzymes and their activation.

Three enzymes are in gastric juice: pepsin, gastricsin and rennin. All these enzymes cleave proteins or peptides.

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

Optimal pH for gastricsin is 2,0-3,0. The ratio between gastricsin and pepsin in gastric juice is 1:5,5. This ratio can be changed in some pathological states.

Rennin also possesses a proteolytic activity and causes a rapid coagulation of ingested casein. But this enzyme plays important role only in children because the optimal pH for it is 5-6.

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

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Enteropeptidase secreted by the mucosa of duodenum initiatesthe activation of the pancreatic proenzymes

Proteolytic enzymes exhibit the preference for particular types of peptide bonds

Proteinases preferentially attacks the bond after:

Pepsin aromatic (Phe, Tyr) and acidic AA (Glu, Asp)

Trypsin basic AA (Arg, Lys)

Chymotrypsin hydrophobic (Phe, Tyr, Trp, Leu) and acidic AA (Glu, Asp)

Elastase AA with a small side chain (Gly, Ala, Ser)

Peptidases:

Carboxypeptidase A nearly all AA (not Arg and Lys)

Carboxypeptidase B basic AA (Arg, Lys)

aminopeptidase nearly all AA

Prolidase proline

Dipeptidase only dipeptides

The splitting of elastin in an intestine is catalyzed by elastase and collagen is decomposed by collagenase.

Digestion of protein takes place not only in the intestinal cavity but also on the surface of mucosa cells.

Mechanism of amino acid absorbtion.

This explanation is called the sodium cotransport theory for amino acid transport; it is also called secondary active transport of amino acid.Absorption of amino acids through the intestine mucosa can occur far more rapidly than protein can be digested in the lumen of the intestine. Since most protein digestion occurs in the upper small intestine most protein absorption occurs in the duodenum and jejunum.

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.