Nitrogen Metabolism
Amino acid: Disposal of nitrogenIntroduction
Amino acid pool
Protein Turnover: Rate of turnover
Protein degradation
Chemical signals for protein degradation
Digestion of dietary protein
Absorption of amino acids and dipeptides
Transport of amino acids into cells
Fat and carbohydrates are stored in the body as triglycerides in adipose tissues and carbohydrates as glycogen in muscles and liver, while amino acids are not stored by the body for further use.
Any amino acids in excess of the biosynthetic need of the cell are rapidly degraded.
The degradation of amino acids forming ammonia and the corresponding α-keto acid.
A portionof free ammonia is excreted in the urin but most is used in the synthesis of uria, which is quantitatively the most important route for disposing of nitrogen from the body.
The carbon skeletons of the α-keto acids are converted to common intermediate of energy producing, metabolic pathways.
Thesecompounds can be metabolized to CO2 and water, glucose, fatty acids, or keton bodies by central pathways of metabolism.
Amino acid pool:
Free amino acids are present throughout the body, for example, in cells, blood, and the extracellular fluids. Let us imagine that these amino acids belonged to a single entity, called. the amino acid pool. This pool supplied by three sources :Amino acids provided by the degradation of body proteins.
Amino acids derived from dietary protein.
Synthesis of nonessential amino acids from simple intermediates of metabolism.
The amino acid pool depleted by three routes:
Synthesis of body protein.
Amino acid consumed as precursors of essential nitrogen-containing small molecules.
Conversion of AA to glycogen, free fatty acids or CO2.
AA. acid pool is small comprised of 90-100g of AA., compared with protein in the body (about 12 Kg in a 70 Kg man).
In a healthy, well fed individuals, the input to AA. Pool is balanced by the output, this mean that the amount of AA. Contained in the pool is constant. The AA. Pool is said to be in a steady state.
Protein Turnover: In a healthy adults:
The total amount of protein in the body remains constant, because the rate of protein synthesis is just sufficient to replace the protein that is degraded.
This process, called protein turnover, and lead to hydrolysis and resynthesis of 300-400 g of body protein each day.
The rat of protein turnover varies widely for individual protein:
Short-lived proteins: regulatory proteins and misfolded proteins are rapidly degraded, having half-lives measured in minutes or hours.
Long- live proteins: Constitute the majority of proteins in the cell having half-lives of days to weeks.
Structural proteins: Collagen are metabolically stable, having half-lives of months or years.
Degradation of protein: Two major enzyme systems responsible for degradation damgad or unneeded proteins.
Ubiquitin-proteasome mechanism: a. energy dependent.
b. mainly degrade endogenous proteins (proteins that were synthesized within the cell.)
2.Degradative enzyme of lysosomes (acid hydrolysis):
a. non-energy dependent.b. mainly degraded extracellular proteins (such as plasma proteins that are taken into the cell by endocytosis, and cell- surface membrane proteins that are used in receptor-mediated endocytosis.
a. Ubiquitin-proteasomeproteolytic pathway:
Proteins selected for degradation by this mechanism
Ubiquitination: linkage of ubiquitin with target substrate (protein)take place in three step enzyme catalyzed process: The targetprotein first covalently attached to ubiquitin, the linkage of the α-carboxyl glycine of ubiquitin to a lysine∈-amino group on protein.
The consecutive of addition of ubiquitin moieties generates a polyubiquitin chain.
Protein that tagged with ubiquitin are then recognized by a large barrel shaped macromolecular proteolytic complex called a proteasome which cuts the target protein into fragments that are then further degraded to amino acids, which enter the amino acid pool.
The ubiquitins are recycled. The selective degradation of protein by the ubiquitin-proteosom complex require ATP that is, it is energy-dependent.
Chemical signals for protein degradation:
Protein degradation is not random because protein have different half-lives. But rather is influenced by some structural aspect of protein. For example, some proteins that have been chemically altered by oxidation or tagged with ubiquitin are preferentially degraded.The half-live of protein is influenced by the nature of the N-terminal residue:
Proteins that have serine as N-terminal amino acid are long-lived with half-life more than 20 hours.
Proteins with aspartate as the N-terminal amino acid have half-life of only three minutes.
Proteins rich in sequence containing: proline, glutamate, serine, and threonine (PEST) are rapidly degraded and exhibit short intracellular half- lives.
Digestion of dietary proteins:
In the stomach, hydrochloric acid denatures dietary protein making them more susceptible to proteases. Pepsin, an enzyme secreted in zymogen form by the serous cells of the stomach, release peptides and few free amino acids from dietary protein.
In small intestine, proteases released by pancreas as zymogen become active. Each has different specificity for the amino acid
R- groups adjacent to the susceptible peptide bond.
Examples of these enzymes are trypsin, chymotrypsin, elastase, and carboxypeptidase A and B.
The resulting oligopeptides are cleaved by aminopeptidase found on the luminal surface of the intestine.
Free amino acids and dipeptides are then absorbed by the intestinal epithelial cells.
Absorption of amino acids and dipeptides:
Free amino acids and dipeptides are taken up the epithelial cells. There, the dipeptides are hydrolyzed in the cytosol to amino acids before being released into the portal system. Thus, only free AAs are found in the portal vain after a meal containing protein.
These AAs are either metabolized by the liver or released into the general circulation.
Transport of amino acids into cells:
The concentration of free AA in the extracellular fluids is significantly lower than that within the cells of the body.
This concentration gradient maintained by active transport systems which are required for movement of AA from extracellular space into cells.This transport systems driven by hydrolysis of ATP.
At least seven different transport systems are known that have overlapping specificities for different amino acids.
The small intestine and proximal tubule of the kidney have common transport systems for AA uptake, therefore, a defect in any one in these systems results in an inability to absorb particular AA into thegut and into the kidney tubules.
For example, one system is responsible for the uptake of cysteine and dibasic AAs, ornithine, arginine, and lysine (COAL).
In the inherited disorder cystinuria, this carrier system is defective, and all four AAs appear in urine.
Cystinuria occurs at frequency of 1 in 7,000 individuals, and is the most common inherited disease, and common genetic error of AA transport, characterized by the precipitation of cystin to form kidney stones(calculi), which can block urinary tract.
Oral hydration is an important part of treatment for this disorder.
Note: Defect in transport of tryptophan(and other nutral AAs can result in Hartnup disorder and pellagra- like
dermatologic and nurologicsymptoms.
Nitrogen balance:
In an adult healthy individual maintaining constant weight, the amount of intake of N in food (mainly as dietary proteins) will be balanced by an excretion of an equal amount of N in urine (in form of urea mainly, uric acid, creatinine/and creatin, and amino acids contribute to a minor extent) and in faeces (mainly as unabsorbed N). The individual is then said to be in "nitrogen balance" or "nitrogenous equilibrium".
A subject in nitrogenous equilibrium is said to be in nitrogen balance i.e. intake of N equalizes the output.
A subject whose intake of N is greater than the output e.g. in growth, is said to have a +ve nitrogen balance . In the growing period and also during convalescence from illness or when anabolic hormones are given, the body puts on weight and nitrogen intake will be more than N-output, since some of the N is retained as tissue proteins.
A subject whose intake of N is less than the output of N, (e.g. in losing weight), is said to have a –ve nitrogen balance. In old age and during illness and starvation weight is lost and results in –ev nitrogen balance.
During starvation when protein is not available from dietary sources, it is the liver, which loses the largest proportions of its proteins compared to other protein such as, proteins of kidney and blood which come next in degree of lability.
To establish N-balance, certain minimum amounts of proteins or equivalent specific type of amino acids must be provided to replace the inevitable losses from the dynamic equilibrium and metabolic utilization of amino acids. The specific type of amino acids called "essential amino acids" which must be provided in the diet simultaneously together and they cannot be synthesized in the body.
It is impossible to maintain N-equilibrium on diets which are deficient in anyone or more of these essential amino acids, no matter how much protein is consumed.
Exclusion of any one of these amino acids leads to –ve N- balance manifesting as loss of weigt, fatigue, loss of appetite and nervous irritability. When missing essential amino acid is supplemented, perfect health is promptly restored.
For normal adult, the minimum amount of each EAA which must be supplied/day, when all other AAs are present has been sat as 0.3 to 1.0 gm of the natural L-form.
The requirement for EAA varies, for example during growth, pregnancy or lactation, a higher intake (as mixed protein) is required.
Removal of Nitrogen from Amino Acids:
α
R—CH—COOH
│
NH2
In mammalian tissues, α–NH2 group of amino acids, derived either from the diet or breakdown of tissue proteins, ultimately is converted first to NH3 and then to urea and is excreted in urine.
α–NH2 group—— NH3——urea (excreted in urin )
* The presence of α–amino group keeps AA safely locked away from oxidative breakdown.
* Removing of α–amino group is essential for producing energy from amino acids, and obligatory step in the catabolism of all AAs.
* Once removed this nitrogen can be incorporated into other compounds or excreted with the carbon skeletons being metabolized.
* Formation of NH3 and urea can be discussed under the following heads:
1. Transamination
2. Deamination: a. Oxidative deamination
b. Non-oxidative deamination
3. Transdeamination
4. NH3 transport, and
5. Formation of urea and
* Verebrates other than mammals share all features of the above scheme except urea formation.
* Urea is the characteristic end-product of amino acid N-catabolism in human beings and ureotelic organism..
* Urea synthesis is replaced,
- by uric acid formation in uricotelic organism, e. g reptiles and birds,
- by NH3 in ammonotelic organism, e.g. bony fish.
1. Transamination: Is a reversible reaction in which α–NH2 group of one AA is transferred to a α–keto–acid resulting in formation of a new amino acid and new keto acid.
COOH COOH COOH COOH
| | | |
CH–NH2 + C=O ======= C=O + CH–NH2
| | | |
R1 R2 R1 R2
1 2 1 2
α–AA α–keto acid New keto- New amino
(donor) (rescipient) acid acid
Donor amino acid (1) thus becomes a new ketoacid (1) after losing the
α–NH2, and the recipient keto acid (2) becomes a new amino acid (2) after receving the NH2 group.
A. The funneling of amino group to glutamate:
The first step in the catabolism of most amino acids is the transfer of their α-amino group to α-ketoglutarate .The products are an α-keto acid (derived from the original amino acid) and glutamate, which can be oxidativelydeaminated, or used as an amino group donor in the synthesis of nonessential amino acids.
This transfer of amino groups from one carbon skeleton to another is catalyzed by a family of enzymes called aminotransferases (formerly called transaminases). These enzymes are found in the cytosol and mitochondria of cells throughout the body—especially those of the liver, kidney, intestine, and muscle.
All amino acids, with the exception of lysine and threonine, participate in transamination at some point in their catabolism. [Note: These two amino acids lose their α-amino groups by deamination.
1. Substrate specificity of aminotransferases:
* Each aminotransferase is specific for one or, at most, a few amino group
donors
* Aminotransferases are named after the specific amino group donor,
because the acceptor of the amino group is almost always α-
ketoglutarate.
* The two most important aminotransferase reactions are catalyzed by
Alanine aminotransferase (ALT):
*Formerly called glutamate-pyruvate transaminase, ALT is present in many tissues.
* The enzyme catalyzes the transfer of the amino group of alanine to α-ketoglutarate, resulting in the formation of pyruvate and glutamate.
b. Aspartate aminotransferase (AST):
* AST formerly called glutamate-oxaloacetate transaminase.
* During amino acid catabolism, AST transfers amino groups from glutamate to oxaloacetate, forming aspartate, which is used as a source of nitrogen in the urea cycle. [Note: The AST reaction is also reversible.]
Mechanism of action of aminotransferases:
* All aminotransferases require the coenzyme pyridoxal phosphate (a derivative of vitamin B), which is covalently linked to the ε-amino group of a specific lysine residue at the active site of the enzyme.
* Aminotransferases act by transferring the amino group of an amino acid to the pyridoxal part of the coenzyme to generate pyridoxamine phosphate. The pyridoxamine form of the coenzyme then reacts with an α-keto acid to form an amino acid, at the same time regenerating the original aldehyde form of the coenzyme.
3. Equilibrium of transamination reactions:
* For most transamination reactions, the equilibrium constant is near one, allowing the reaction to function in both amino acid degradation through removal of α-amino groups (for example, after consumption of a protein-rich meal) and biosynthesis through addition of amino groups to the carbon skeletons of α-keto acids (for example, when the supply of amino acids from the diet is not adequate to meet the synthetic needs of cells).
4. Diagnostic value of plasma aminotransferases:
* Aminotransferases are normally intracellular enzymes, with the low levels found in the plasma.
* The presence of elevated plasma levels of aminotransferases indicates damage to cells rich in these enzymes. For example, physical trauma or a disease process can cause cell lysis, resulting in release of intracellular enzymes into the blood.
* Two aminotransferases—AST and ALT—are of particular diagnostic value when they are found in the plasma.
a. Liver disease:
* Plasma AST and ALT are elevated in nearly all liver diseases, but are particularly high in conditions that cause extensive cell necrosis, such as severe viral hepatitis, toxic injury, and prolonged circulatory collapse. *ALT is more specific than AST for liver disease, but the latter is more sensitive because the liver contains larger amounts of AST.
* Serial enzyme measurements are often useful in determining the course of liver damage.
b. Nonhepatic disease:
* ALT,and AST elevated in nonhepatic disease, such as myocardial infarction and muscle disorders.* These disorders can usually be distinguished clinically from liver disease.
B. Glutamate dehydrogenase: the oxidative deamination of amino acids
In contrast to transamination reactions that transfer amino groups, oxidative deamination by glutamate dehydrogenase results in the liberation of the amino group as free ammonia (Figure 19.11). These reactions occur primarily in the liver and kidney. They provide α-keto acids that can enter the central pathway of energy metabolism, and ammonia, which is a source of nitrogen in urea synthesis.1. Glutamate dehydrogenase: As described above, the amino groups of most amino acids are ultimately funneled to glutamate by means of transamination with α-ketoglutarate.Glutamate is unique in that it is the only amino acid that undergoes rapid oxidative deamination—a reaction catalyzed by glutamate dehydrogenase (see Figure 19.9). Therefore, the sequential action of transamination (resulting in the collection of amino groups from other amino acids onto α-ketoglutarate to produce glutamate) and the oxidative deamination of that glutamate (regenerating α-ketoglutarate) provide a pathway whereby the amino groups of most amino acids can be released as ammonia.
a. Coenzymes:Glutamate dehydrogenase is unusual in that it can use either NAD+ or NADP+ as a coenzyme (see Figure 19.11). NAD+ is used primarily in oxidative deamination (the simultaneous loss of ammonia coupled with the oxidation of the carbon skeleton (Figure 19.12A),andNADPH is used in reductive amination (the simultaneous gain of ammonia coupled with the reduction of the carbon skeleton, Figure 19.12B).
b. Direction of reactions:The direction of the reaction depends on the relative concentrations of glutamate, α-ketoglutarate, and ammonia, and the ratio of oxidized to reduced coenzymes. For example, after ingestion of a meal containing protein, glutamate levels in the liver are elevated, and the reaction proceeds in the direction of amino acid degradation and the formation of ammonia (see Figure 19.12A). [Note: the reaction can also be used to synthesize amino acids from the corresponding α-keto acids (see Figure 19.12B).]
c. Allosteric regulators:guanosine triphosphate is an allosteric inhibitor of glutamate dehydrogenase,whereasadenosine diphosphate (ADP) is an activator. Thus, when energy levels are low in the cell, amino acid degradation by glutamate dehydrogenase is high, facilitating energy production from the carbon skeletons derived from amino acids.
2. D-Amino acid oxidase: D-Amino acids are found in plants and in the cell walls of microorganisms, but are not used in the synthesis of mammalian proteins. D-Amino acids are, however, present in the diet, and are efficiently metabolized by the kidney and liver. D-Amino acid oxidase is an FAD-dependent peroxisomal enzyme that catalyzes the oxidative deamination of these amino acid isomers. The resulting α-keto acids can enter the general pathways of amino acid metabolism, and be reaminated to L-isomers, or catabolized for energy.
C. Transport of ammonia to the liver
Two mechanisms are available in humans for the transport of ammonia from the peripheral tissues to the liver for its ultimate conversion to urea.The first, found in most tissues, uses glutamine synthetase to combine ammonia with glutamate to form glutamine—a nontoxic transport form of ammonia (Figure 19.13). The glutamine is transported in the blood to the liver where it is cleaved by glutaminase to produce glutamate and free ammonia . The second transport mechanism, used primarily by muscle, involves transamination of pyruvate (the end product of aerobic glycolysis) to form alanine (see Figure 19.8). Alanine is transported by the blood to the liver, where it is converted to pyruvate, again by transamination. In the liver, the pathway of gluconeogenesis can use the pyruvate to synthesize glucose, which can enter the blood and be used by muscle—a pathway called the glucose-alanine cycle.
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