The removal of the α-amino groups, the first step in the
catabolism of most of the L-amino acids, is promoted by enzymes
called aminotransferases or transaminases. In
these transamination reactions, the α-amino group is
transferred to the α-carbon atom of α-ketoglutarate, leaving behind the
corresponding α-keto acid analog of the amino acid (Fig. 17-5). There is no net
deamination (i.e., loss of amino groups) in such reactions because the
α-ketoglutarate becomes aminated as the α-amino acid is deaminated. The effect
of transamination reactions is to collect the amino groups from many different
amino acids in the form of only one, namely, L-glutamate. The
glutamate channels amino groups either into biosynthetic pathways or into a
final sequence of reactions by which nitrogenous waste products are formed and
then excreted.
Cells contain several different aminotransferases, many specific for
a-ketoglutarate as the amino group acceptor. The aminotransferases differ in
their specificity for the other substrate, the L-amino acid that
donates the amino group, and are named for the amino group donor (Fig. 17-5b).
The reactions catalyzed by the aminotransferases are freely reversible, having
an equilibrium constant of about 1.0 (ΔG°' = 0 kJ/mol).
All aminotransferases share a common prosthetic group and a common reaction
mechanism. The prosthetic group is pyridoxal phosphate (PLP),
the coenzyme form of pyridoxine or vitamin B6.
Pyridoxal phosphate was briefly introduced in Chapter 14 (p. 422) as a cofactor
in the glycogen phosphorylase reaction. Its role in that reaction, however, is
not representative of its normal coenzyme function. Its more typical functions
occur in the metabolism of molecules with amino groups.
Pyridoxal phosphate functions as an intermediate carrier of amino groups at
the active site of arninotransferases. It undergoes reversible transformations
between its aldehyde form, pyridoxal phosphate, which can accept an amino group,
and its aminated form, pyridoxamine phosphate, which can donate its amino group
to an α-keto acid (Fig. 17-6a). Pyridoxal phosphate is generally bound
covalently to the enzyme's active site through an imine (Schiff base) linkage to
the ε-amino group of a Lys residue (Fig. 17-6b).
Pyridoxal phosphate is involved in a variety of reactions at the α and β
carbons of amino acids. Reactions at the a carbon (Fig. 17-7) include
racemizations (interconverting L- and D-amino
acids) and decarboxylations, as well as transaminations. Pyridoxal phosphate
plays the same chemical role in each of these reactions. One of the bonds to the
a carbon is broken, removing either a proton or a carboxyl group and leaving
behind a free electron pair on the carbon (a carbanion). This intermediate is
very unstable and normally would not form at a significant rate. Pyridoxal
phosphate provides a highly conjugated structure (an electron sink) that permits
delocalization of the negative charge, stabilizing the carbanion
Aminotransferases are classic examples of enzymes catalyzing bimolecular
ping-pong reactions (see Fig. 8-13b). In such reactions the first substrate must
leave the active site before the second substrate can bind. Thus the incoming
amino acid binds to the active site, donates its amino group to pyridoxal
phosphate, and departs in the form of an a-keto acid. Then the incoming
a-keto acid is bound, accepts the amino group from pyridoxamine
phosphate, and departs in the form of an amino acid.
The measurement of alanine aminotransferase and aspartate aminotransferase
levels in blood serum is an important diagnostic procedure in medicine, used as
an indicator of heart damage and to monitor recovery from the damage
Ammonia Is Formed from Glutamate
We have seen that, in the liver, amino groups are removed from many of the
a-amino acids by transamination with a-ketoglutarate to form L-glutamate. How
are amino groups removed from glutamate to prepare them for excretion?
|
Glutamate is transported from the cytosol to the mitochondria,
where it undergoes oxidative deamination catalyzed by
L-glutamate dehydrogenase (Mr
330,000). This enzyme, which is present only in the mitochondrial matrix,
requires NAD+ (or NADP+) as
the acceptor of the reducing equivalents (Fig. 17-8). The combined action of the
aminotransferases and glutamate dehydrogenase is referred to as
transdeamination. A few amino acids bypass the transdeamination pathway and
undergo direct oxidative deamination. The fate of the NH4 produced by either of
these processes is discussed in detail later.
As might be expected from its central role in amino group metabolism,
glutamate dehydrogenase is a complex allosteric enzyme. The enzyme molecule
consists of six identical subunits. It is influenced by the positive modulator
ADP and by the negative modulator GTP, a product of the succinyl-CoA synthetase
reaction in the citric acid cycle (p. 456). Whenever a hepatocyte needs fuel for
the citric acid cycle, glutamate dehydrogenase activity increases, making
α-ketoglutarate available for the citric acid cycle and releasing NH4 for
excretion. On the other hand, whenever GTP accumulates in the mitochondria as a
result of high citric acid cycle activity, oxidative deamination of glutamate is
inhibited.
Glutamine Carries Ammonia to the Liver
Ammonia is quite toxic to animal tissues (we examine some
possible reasons for this toxicity later). In most animals excess ammonia is
converted into a nontoxic compound before export from extrahepatic tissues into
the blood and thence to the liver or kidneys. Glutamate, which is so critical to
intracellular amino group metabolism, is supplanted by
L-glutamine for this transport function. In many
tissues, including the brain, ammonia is enzymatically combined with glutamate
to yield glutamine by the action of glutamine synthetase. This
reaction requires ATP and occurs in two steps. In the first step, glutamate and
ATP react to form ADP and a γ-glutamyl phosphate intermediate, which reacts with
ammonia to produce glutamine and inorganic phosphate. We will encounter
glutamine synthetase again in Chapter 21 when we consider nitrogen metabolism in
microorganisms, where this enzyme serves as a portal for the entry of fixed
nitrogen into biological systems. Glutamine is a nontoxic, neutral compound that
can readily pass through cell membranes, whereas glutamate, which bears a net
negative charge, cannot. In most land animals glutamine is carried in the blood
to the liver. As is the case for the amino group of glutamate, the amide
nitrogen is released as ammonia only within liver mitochondria, where the enzyme
glutaminase converts glutamine to glutamate and
NH4+ .
Glutamine is a major transport form of ammonia; it is normally present in
blood in much higher concentrations than other amino acids. In addition to its
role in the transport of amino groups, glutamine serves as a source of amino
groups in a variety of biosynthetic reaction
Alanine also plays a special role in transporting amino groups to
the liver in a nontoxic form, by the glucose-alanine cycle
(Fig. 17-9). In muscle and certain other tissues that degrade amino acids for
fuel, amino groups are collected in glutamate by transamination (Fig. 17-2).
Glutamate may then be converted to glutamine for transport to the liver, or it
may transfer its α-amino group to pyruvate, a readily available product of
muscle glycolysis, by the action of alanine aminotransferase
(Fig. 17-9). The alanine, with no net charge at pH near 7, passes into the blood
and is carried to the liver. As with glutamine, excess nitrogen carried to the
liver as alanine is eventually delivered as ammonia in the mitochondria. In a
reversal of the alanine aminotransferase reaction described above, alanine
transfers its amino group to α-ketoglutarate, forming glutamate in the cytosol.
Some of this glutamate is transported into the mitochondria and acted on by
glutamate dehydrogenase, releasing
NH4+ (Fig. 17-8).
Alternatively, transamination with oxaloacetate moves amino groups from
glutamate to aspartate, another nitrogen donor in urea synthesis.
The use of alanine to transport ammonia from hard-working skeletal muscles to
the liver is another example of the intrinsic economy of living organisms.
Vigorously contracting skeletal muscles operate anaerobically, producing not
only ammonia from protein breakdown but also large amounts of pyruvate from
glycolysis. Both these products must find their way to the liver-ammonia to be
converted into urea for excretion and pyruvate to be rebuilt into glucose and
returned to the muscles. Animals thus solve two problems with one cycle: they
move the carbon atoms of pyruvate, as well as excess ammonia, from muscle to
liver as alanine. In the liver, alanine yields pyruvate, the starting material
for gluconeogenesis, and releases
NH4+ for urea synthesis. The
energetic burden of gluconeogenesis is thus imposed on the liver rather than the
muscle, so that the available ATP in the muscle can be devoted to muscle
contraction.
The catabolic production of ammonia poses a serious biochemical problem
because ammonia is very toxic. The molecular basis for this toxicity is not
entirely understood. The terminal stages of ammonia intoxication in humans are
characterized by the onset of a comatose state and other effects on the brain,
so that much of the research and speculation has focused on this tissue. The
major toxic effects of ammonia in brain probably involve changes in cellular pH
and the depletion of certain citric acid cycle intermediates.
The protonated form of ammonia (ammonium ion) is a weak acid, and the
unprotonated form is a strong base:
Most of the ammonia generated in catabolism is present as
NH4+ at neutral pH. Although
many of the reactions that produce ammonia, such as the glutamate dehydrogenase
reaction, yield NH4+ , a few
reactions, such as that of adenosine deaminase (Chapter 21), produce
NH3. Excessive amounts of
NH3 cause alkalization of cellular fluids, which has
complex effects on cellular metabolism.
Ridding the cytosol of excess ammonia involves reductive amination of
α-ketoglutarate to form glutamate by glutamate dehydrogenase (the reverse of the
reaction described earlier) and conversion of glutamate to glutamine by
glutamine synthetase. Both of these enzymes occur in high levels in the brain,
although the second probably represents the more important pathway for removal
of ammonia. The first reaction depletes cellular NADH and α-ketoglutarate
required for ATP production in the cell. The second reaction depletes ATP itself
. Overall, NH3 may interfere with the very high levels
of ATP production required to maintain brain function.
Depletion of glutamate in the glutamine synthetase reaction may have
additional effects on the brain. Glutamate, and the compound γ-aminobutyrate
(GABA) that is derived from it (Chapter 21), are both important
neurotransmitters; the sensitivity of the brain to ammonia may well reflect a
depletion of neurotransmitters as well as changes in cellular pH and ATP
metabolism.
As we close this discussion of amino group metabolism, note that we have
described several processes that deposit excess ammonia in the mitochondria of
hepatocytes (Fig. 17-2). We now turn to a discussion of the fate of that
ammonia.
|
|
|
|
