What is the fate of amino acids released on protein digestion or turnover? The first call is for use as building blocks for biosynthetic reactions. However, any not needed as building blocks are degraded to compounds able to enter the metabolic mainstream. The amino group is first removed, and then the remaining carbon skeleton is metabolized to glucose, one of several citric acid cycle intermediates, or to acetyl CoA. The major site of amino acid degradation in mammals is the liver, although muscles readily degrade the branched-chain amino acids (Leu, Ile, and Val). The fate of the α-amino group will be considered first, followed by that of the carbon skeleton (Section 23.5).

The α-amino group of many amino acids is transferred to α-ketoglutarate to form glutamate, which is then oxidatively deaminated to yield ammonium ion (NH₄⁺).

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Aminotransferases catalyze the transfer of an α-amino group from an α-amino acid to an α-ketoacid. These enzymes, also called transaminases, generally funnel α-amino groups from a variety of amino acids to α-ketoglutarate for conversion into NH₄⁺.

Aspartate aminotransferase, one of the most important of these enzymes, catalyzes the transfer of the amino group of aspartate to α-ketoglutarate.

Alanine aminotransferase catalyzes the transfer of the amino group of alanine to α-ketoglutarate.

Transamination reactions are reversible and can thus be used to synthesize amino acids from α-ketoacids, as we shall see in Chapter 24.

The nitrogen atom in glutamate is converted into free ammonium ion by oxidative deamination, a reaction catalyzed by glutamate dehydrogenase. This enzyme is unusual in being able to utilize either NAD⁺ or NADP⁺, at least in some species. The reaction proceeds by dehydrogenation of the C—N bond, followed by hydrolysis of the resulting ketimine.

This reaction equilibrium constant is close to 1 in the liver, so the direction of the reaction is determined by the concentrations of reactants and products. Normally, the reaction is driven forward by the rapid removal of ammonium ion. Glutamate dehydrogenase, essentially a liver-specific enzyme, is located in mitochondria, as are some of the other enzymes required for the production of urea. This compartmentalization sequesters free ammonium ion, which is toxic.

In mammals, but not in other organisms, glutamate dehydrogenase is allosterically inhibited by GTP and stimulated by ADP. These nucleotides exert their regulatory effects in a unique manner. An abortive complex is formed on the enzyme when a product is replaced by substrate before the reaction is complete. For instance, the enzyme bound to glutamate and NAD(P)H is an abortive complex. GTP facilitates the formation of such complexes while ADP destabilizes the complexes.

The sum of the reactions catalyzed by aminotransferases and glutamate dehydrogenase is

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In most terrestrial vertebrates, NH₄⁺ is converted into urea, which is excreted.

All aminotransferases contain the prosthetic group pyridoxal phosphate (PLP), which is derived from pyridoxine (vitamin B₆). Pyridoxal phosphate includes a pyridine ring that is slightly basic to which is attached an OH group that is slightly acidic. Thus, pyridoxal phosphate derivatives can form a stable tautomeric form in which the pyridine nitrogen atom is protonated and, hence, positively charged, while the OH group loses a proton and hence is negatively charged, forming a phenolate.

The most important functional group on PLP is the aldehyde. This group forms covalent Schiff-base intermediates with amino acid substrates. Indeed, even in the absence of substrate, the aldehyde group of PLP usually forms a Schiff-base linkage with the ε-amino group of a specific lysine residue at the enzyme’s active site. A new Schiff-base linkage is formed on addition of an amino acid substrate.

The α-amino group of the amino acid substrate displaces the ε-amino group of the active-site lysine residue. In other words, an internal aldimine becomes an external aldimine. The amino acid–PLP Schiff base that is formed remains tightly bound to the enzyme by multiple noncovalent interactions. The Schiff-base linkage often accepts a proton at the N, with the positive charge stabilized by interaction with the negatively charged phenolate group of PLP.

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The Schiff base between the amino acid substrate and PLP, the external aldimine, loses a proton from the α-carbon atom of the amino acid to form a quinonoid intermediate (Figure 23.9). Reprotonation of this intermediate at the aldehyde carbon atom yields a ketimine. The ketimine is then hydrolyzed to an α-ketoacid and pyridoxamine phosphate (PMP). These steps constitute half of the transamination reaction.

The second half takes place by the reverse of the preceding pathway. A second α-ketoacid reacts with the enzyme–pyridoxamine phosphate complex (E-PMP) to yield a second amino acid and regenerate the enzyme–pyridoxal phosphate complex (E-PLP).

The sum of these partial reactions is

The mitochondrial enzyme aspartate aminotransferase provides an especially well studied example of PLP as a coenzyme for transamination reactions (Figure 23.10). X-ray crystallographic studies provided detailed views of how PLP and substrates are bound and confirmed much of the proposed catalytic mechanism. Each of the identical 45-kDa subunits of this dimer consists of a large domain and a small one. PLP is bound to the large domain, in a pocket near the domain interface. In the absence of substrate, the aldehyde group of PLP is in a Schiff-base linkage with lysine 258, as expected. Adjacent to the coenzyme’s binding site is a conserved arginine residue that interacts with the α-carboxylate group of the amino acid substrate, helping to orient the substrate appropriately in the active site. A base is necessary to remove a proton from the α-carbon group of the amino acid and to transfer it to the aldehyde carbon atom of PLP (Figure 23.9, steps 1 and 2). The lysine amino group that was initially in Schiff-base linkage with PLP appears to serve as the proton donor and acceptor.

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The presence of alanine and aspartate aminotransferase in the blood is an indication of liver damage. Liver damage can occur for a number of reasons, including viral hepatitis, long-term excessive alcohol consumption, and reaction to drugs such as acetaminophen (Section 36.1). Under these conditions, liver cell membranes are damaged and some cellular proteins, including the aminotransferases, leak into the blood. Normal blood values for alanine and aspartate aminotransferase activity are 5–30 units/l and 40–125 units/l, respectively. Depending on the extent of liver damage, the values will reach 200–300 units/l.

Transamination is just one of a wide range of amino acid transformations that are catalyzed by PLP enzymes. The other reactions catalyzed by PLP enzymes at the α-carbon atom of amino acids are decarboxylations, deaminations, racemizations, and aldol cleavages (Figure 23.11). In addition, PLP enzymes catalyze elimination and replacement reactions at the β-carbon atom (e.g., tryptophan synthetase in the synthesis of tryptophan) and the γ-carbon atom (e.g., cystathionine β-synthase in the synthesis of cysteine) of amino acid substrates. Three common features of PLP catalysis underlie these diverse reactions.

How does an enzyme selectively break a particular one of three bonds at the α-carbon atom of an amino acid substrate? An important principle is that the bond being broken must be perpendicular to the π orbitals of the electron sink (Figure 23.12). An aminotransferase, for example, binds the amino acid substrate so that the C_α—H bond is perpendicular to the PLP ring (Figure 23.13). In serine hydroxymethyltransferase, the enzyme that converts serine into glycine, the N—C_α bond is rotated so that the C_α—C_β bond is most nearly perpendicular to the plane of the PLP ring, favoring its cleavage. This means of choosing one of several possible catalytic outcomes is called stereoelectronic control.

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Many of the PLP enzymes that catalyze amino acid transformations, such as serine hydroxymethyltransferase, have a similar structure and are clearly related by divergent evolution. Others, such as tryptophan synthetase, have quite different overall structures. Nonetheless, the active sites of these enzymes are remarkably similar to that of aspartate aminotransferase, revealing the effects of convergent evolution.

The α-amino groups of serine and threonine can be directly converted into NH₄⁺ without first being transferred to α-ketoglutarate. These direct deaminations are catalyzed by serine dehydratase and threonine dehydratase, in which PLP is the prosthetic group.

These enzymes are called dehydratases because dehydration precedes deamination. Serine loses a hydrogen ion from its α-carbon atom and a hydroxide ion group from its β-carbon atom to yield aminoacrylate. This unstable compound reacts with H₂O to give pyruvate and NH₄⁺. Thus, the presence of a hydroxyl group attached to the β-carbon atom in each of these amino acids permits the direct deamination.

Although most amino acid degradation takes place in the liver, other tissues can degrade amino acids. For instance, muscle uses branched-chain amino acids as a source of fuel during prolonged exercise and fasting. How is the nitrogen processed in these other tissues? As in the liver, the first step is the removal of the nitrogen from the amino acid. However, muscle lacks the enzymes of the urea cycle, and so the nitrogen must be released in a nontoxic form that can be absorbed by the liver and converted into urea.

Nitrogen is transported from muscle to the liver in two principal transport forms. Glutamate is formed by transamination reactions, but the nitrogen is then transferred to pyruvate to form alanine, which is released into the blood (Figure 23.14). The liver takes up the alanine and converts it back into pyruvate by transamination. The pyruvate can be used for gluconeogenesis and the amino group eventually appears as urea. This transport is referred to as the glucose–alanine cycle. It is reminiscent of the Cori cycle discussed earlier (Figure 16.33). However, in contrast with the Cori cycle, pyruvate is not reduced to lactate by NADH, and thus more high-energy electrons are available for oxidative phosphorylation in the muscle.

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Glutamine is also a key transport form of nitrogen. Glutamine synthetase catalyzes the synthesis of glutamine from glutamate and NH₄⁺ in an ATP-dependent reaction:

The nitrogens of glutamine can be converted into urea in the liver.