Removal of Nitrogen From Amino Acids

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Chapter: Biochemistry : Amino Acids: Disposal of Nitrogen

The presence of the α-amino group keeps amino acids safely locked away from oxidative breakdown. Removing the α-amino group is essential for producing energy from any amino acid and is an obligatory step in the catabolism of all amino acids.


REMOVAL OF NITROGEN FROM AMINO ACIDS

The presence of the α-amino group keeps amino acids safely locked away from oxidative breakdown. Removing the α-amino group is essential for producing energy from any amino acid and is an obligatory step in the catabolism of all amino acids. Once removed, this nitrogen can be incorporated into other compounds or excreted as urea, with the carbon skeletons being metabolized. This section describes transamination and oxidative deamination, reactions that ultimately provide ammonia and aspartate, the two sources of urea nitrogen.

 

A. Transamination: the funneling of amino groups to glutamate

The first step in the catabolism of most amino acids is the transfer of their α-amino group to α-ketoglutarate (Figure 19.7), producing an α-keto acid (derived from the original amino acid) and glutamate. α-Ketoglutarate plays a pivotal role in amino acid metabolism by accepting the amino groups from most amino acids, thereby becoming glutamate. Glutamate produced by transamination can be oxidatively deaminated (see below) 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 (also called transaminases). These enzymes are found in the cytosol and mitochondria of cells throughout the body. 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.]


Figure 19.7 Aminotransferase reaction using α-ketoglutarate as the amino group acceptor.

 

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. Two important aminotransferase reactions are catalyzed by alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as shown in Figure 19.8).


Figure 19.8 Reactions catalyzed during amino acid catabolism. A. Alanine aminotransferase (ALT). B. Aspartate aminotransferase (AST). PLP = pyridoxal phosphate.

 

a. Alanine aminotransferase: 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. The reaction is readily reversible. However, during amino acid catabolism, this enzyme (like most aminotransferases) functions in the direction of glutamate synthesis. [Note: Glutamate, in effect, acts as a “collector” of nitrogen from most amino acids.]

 

b. Aspartate aminotransferase: AST is an exception to the rule that aminotransferases funnel amino groups to form glutamate. 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. Like other transaminations, the AST reaction is reversible.

 

2. Mechanism of action of aminotransferases: All aminotransferases require the coenzyme pyridoxal phosphate (a derivative of vitamin B6), 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. Figure 19.9 shows these two component reactions for the reaction catalyzed by AST.


Figure 19.9 Cyclic interconversion of pyridoxal phosphate and pyridoxamine phosphate during the aspartate aminotransferase reaction.P = phosphate group.

 

3. Equilibrium of transamination reactions: For most transamination reactions, the equilibrium constant is near 1. This allows 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 of nonessential amino acids 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: Aminotrans-ferases are normally intracellular enzymes, with the low levels found in the plasma representing the release of cellular contents during normal cell turnover. Elevated plasma levels of aminotransferases indicate 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 measurements of AST and ALT (so-called “liver function tests”) are often useful in determining the course of liver damage. Figure 19.10 shows the early release of ALT into the serum, following ingestion of a liver toxin. [Note: Elevated serum bilirubin results from hepatocellular damage that decreases the hepatic conjugation and excretion of bilirubin.]


Figure 19.10 Pattern of serum ALT and bilirubin in the plasma, following poisoning with the toxic mushroom Amanita phalloides.

 

b. Nonhepatic disease: Aminotransferases may be elevated in nonhepatic diseases such as those that cause damage to cardiac or skeletal muscle. However, these disorders can usually be distinguished clinically from liver disease.

 

B. Oxidative deamination of amino acids

In contrast to transamination reactions that transfer amino groups, oxidative deamination reactions result 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 pathways of energy metabolism and ammonia, which is a source of nitrogen in hepatic urea synthesis. [Note: Ammonia exists primarily as ammonium (NH4+) in aqueous solution, but it is the un-ionized form (NH3) that crosses membranes.]


Figure 19.11 Oxidative deamination by glutamate dehydrogenase. [Note: The enzyme is unusual in that it uses both NAD+ (nicotinamide adenine dinucleotide) and NADPH (nicotinamide adenine dinucleotide phosphate).]

 

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 19.11). Therefore, the sequential action of transamination (resulting in the transfer of amino groups from most amino acids to 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, a mitochondrial enzyme, is unusual in that it can use either nicotinamide adenine dinucleotide (NAD+) or its phosphorylated reduced form (NADPH) 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, as shown in Figure 19.12A), and NADPH is used in reductive amination (the simultaneous gain of ammonia coupled with the reduction of the carbon skeleton, as shown in Figure 19.12B).


Figure 19.12 Combined actions of aminotransferase and glutamate dehydrogenase reactions. [Note: Reductive amination occurs only when ammonia (NH3) level is high.] NAD(H) = nicotinamide adenine dinucleotide; NADP(H) = nicotinamide adenine dinucleotide phosphate.

 

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). High ammonia levels are required to drive the reaction to glutamate synthesis.

 

c. Allosteric regulators: Guanosine triphosphate is an allosteric inhibitor of glutamate dehydrogenase, whereas adenosine diphosphate (ADP) is an activator. Therefore, 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 (DAO) is a flavin adenine dinucleotide– dependent peroxisomal enzyme that catalyzes the oxidative deamination of these amino acid isomers, thereby producing α-keto acids, ammonia, and hydrogen peroxide. The α-keto acids can enter the general pathways of amino acid metabolism and be reaminated to L-isomers or catabolized for energy. [Note: DAO degrades D-serine, the isomeric form of serine that modulates N-methyl-D-aspartate (NMDA)-type glutamate receptors. Increased DAO activity has been linked to increased susceptibility to schizophrenia.] L-amino acid oxidases are known, but their physiologic significance is unclear.

 

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. Both are important in, but not exclusive to, skeletal muscle. The first 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 ammonia is converted to urea. The second transport mechanism involves the formation of alanine by the transamination of pyruvate produced from both aerobic glycolysis and metabolism of the succinyl coenzyme A (CoA) generated by the catabolism of the branched-chain amino acids isoleucine and valine. Alanine is transported by the blood to the liver, where it is converted to pyruvate, again by transamination. The pyruvate is used to synthesize glucose, which can enter the blood and be used by muscle, a pathway called the glucose–alanine cycle.


Figure 19.13 Transport of ammonia (NH3) from muscle to the liver. ADP = adenosine diphosphate; Pi = inorganic phosphate; CoA = coenzyme A.


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