Ammonia Assimilation and Recycling

The glutamate synthase or GS-GOGAT cycle

For many years it was thought that bacteria and higher plants assimilate ammonia into glutamate via
the GDH pathway, as in certain fungi and yeasts. However, in bacteria it became clear in 1970 that an
alternative pathway of ammonia assimilation [involving glutamine synthetase (GS) [EC 6.3.1.2] and an
NADPH-dependent glutamine:2-oxoglutarate amidotransferase (GOGAT) [EC 1.4.1.13] or glutamate
synthase, must be operating when ammonia is present in the growth medium at low levels (Tempest
et al, 1970). Thus, N-starvation leads to derepression and activation of GS (with a high affinity for NH 3)
and derepression of GOGAT, and repression of GDH (with a relatively low affinity for NH 3) (Tempest et
al, 1970). High ammonia availability leads to repression and deactivation of GS and induction of GDH
(Tempest et al, 1970).
GDH
NH3 + 2-oxoglutarate + NADPH + H+ <---> glutamate + NADP+
GS-GOGAT
NH3 + glutamate + ATP ---> glutamine + ADP + Pi
glutamine + 2-oxoglutarate + NADPH + H+ ---> 2 glutamate + NADP+
Both the GDH and GS-GOGAT pathways produce 1 mole of glutamate from 1 mole each of NH 3, 2oxoglutarate and NADPH. But note that the GS-GOGAT pathway is energetically more costly than the
GDH pathway, consuming 1 ATP.
Escherichia coli is now known to have two primary pathways for glutamate synthesis (Hellig, 1994;
1998). The GS-GOGAT pathway is essential for glutamate synthesis at low ammonium concentrations
and for regulation of the glutamine pool, and is used when the cell is not under energy limitation
(Hellig, 1994; 1998). The GDH pathway is used in glutamate synthesis when the cell is limited for
energy (and carbon; i.e. glucose-limited growth) but ammonium and phosphate are present in excess
(Hellig, 1994; 1998). Synechocystis sp. strain PCC 6803 utilizes the GS-GOGAT pathway as the
primary pathway of ammonia assimilation, but the presence of GDH appears to offer a selective
advantage for the cyanobacterium under nonexponential growth conditions (Chavez et al, 1999).
These dual pathways may be common to bacteria, cyanobacteria, algae, yeasts and fungi (Huth and
Liebs, 1988).
Re-examination of ammonia assimilation in yeasts and fungi now reveals the operation of alternative
pathways of glutamate synthesis, independent of NADPH-GDH:

Mutants of Aspergillus nidulans lacking NADP-GDH activity grow more poorly than wild-type
strains on ammonium as a sole nitrogen source (Macheda et al, 1999). The leaky growth of
these mutants is indicative of an alternative pathway of ammonium assimilation and
glutamate biosynthesis (Macheda et al, 1999). A. nidulans mutants disrupted in the gltA
encoding GOGAT, were found to be dispensable for growth on ammonium in the presence of
NADP-GDH (Macheda et al, 1999). However, a strain carrying the gltA inactivation together
with an NADP-GDH structural gene mutation (gdhA) was unable to grow on ammonium or on
nitrogen sources metabolized via ammonium (Macheda et al, 1999).
Schizosaccharomyces pombe mutants lacking either NADPH-GDH or GOGAT are still able to
grow on ammonium as sole nitrogen source (Perysinakis et al, 1995). Complete lack of
growth on ammonium as sole N source is seen only in double mutants lacking both NADPHGDH and GOGAT (Perysinakis et al, 1995).
In contrast to Candida utilis (Sims and Folkes, 1964), analysis of 15N-ammonium assimilation
in actively growing mycelium of Agaricus bisporus indicates participation of the GS-GOGAT
pathway, and no participation of NADP-GDH (Baars et al, 1996). 13NH3 tracer studies indicate
that the GS-GOGAT pathway is the major route of ammonium assimilation in Candida
albicans and also in nitrogen-starved cultures of Saccharomyces cerevisiae and Candida
tropicalis (Holmes et al, 1989; 1991).

The yeast Saccharomyces cerevisiae synthesizes glutamate through the action of either NADPglutamate dehydrogenase (NADP-GDH), encoded by GDH1 (under conditions of ammonia excess),
or through the combined action of glutamine synthetase (GS) and glutamate synthase (GOGAT),
encoded by GLN1 and GLT1 (under conditions of ammonia limitation) (Avendano et al, 1997).
Dynamic modeling indicates that the GS-GOGAT pathway plays a more important physiological role in
yeast than is generally assumed (van Riel et al, 1998). However, a double mutant of S. cerevisiae
lacking NADP-GDH and GOGAT activities was able to grow on ammonium as the sole nitrogen
source and thus to synthesize glutamate through a third pathway (Avendano et al, 1997). A computer
search for similarities between the GDH1 nucleotide sequence and the complete yeast genome led to
the discovery that GDH1 showed high identity to an open reading frame (GDH3) on chromosome I
(Avendano et al, 1997). Triple mutants impaired in GDH1, GLT1, and GDH3 are strict glutamate
auxotrophs, indicating that GDH3 plays a significant physiological role, providing glutamate when
GDH1 and GLT1 are impaired (Avendano et al, 1997). This appears to be the first example of a
microorganism possessing three pathways for glutamate biosynthesis (Avendano et al, 1997).
Following the discovery of glutamate synthase (GOGAT) in bacteria, a similar activity was sought in
plants. A ferredoxin-dependent glutamate synthase [EC 1.4.7.1] was discovered in photosynthetic
tissues of higher plants in 1974 (Lea and Miflin, 1974), and an NADH-dependent "glutamate
synthetase" in non-photosynthetic plant tissues in the same year (Fowler et al, 1974).

Evidence in favor of the operation of the GS-GOGAT cycle as the primary pathway of ammonia
assimilation in higher plants has been reviewed by several authors (e.g. Miflin and Lea, 1980; Lea et
al, 1992). This evidence includes:

almost complete inhibition of 15NH4+ assimilation by the glutamine synthetase (GS) inhibitor,
methionine sulfoximine (MSX) (Miflin and Lea, 1980; Lea et al, 1992).
quantitative analysis of 15NH4+ in Lemna minor is consistent with incorporation of 15N primarily
into glutamine-amide, followed by transfer to glutamate and the amino-group of glutamine via
the action of GOGAT and GS, respectively, provided that it is assumed that the GS-GOGAT
cycle is compartmentilized in the chloroplast, and that a second site of glutamine synthesis
occurs in the cytoplasm (Rhodes et al, 1980).

the maize gdh1-null mutant exhibits about 5% of the total GDH enzyme activity of wildtype
plants. Although this mutant exhibits a slightly reduced total rate of 15NH4+ assimilation, when
methionine sulfoximine (MSX), a potent inhibitor of GS is supplied, this completely blocks
15
NH4+ assimilation in both the mutant and wildtype roots and shoots (Magalhaes et al, 1990).
The contribution of GDH to net ammonia assimilation is small in comparison to that catalyzed
by the GS-GOGAT cycle (Rhodes et al, 1989; Magalhaes et al, 1990).

mutants of Arabidopsis and barley defective in GS or GOGAT exhibit markedly impaired

ammonia assimilation, especially under photorespiratory conditions (Lea et al, 1992).

Enzyme kinetic considerations also suggest a role for the GS-GOGAT pathway in ammonia
assimilation at low tissue/cell ammonia concentrations. GS has a much higher affinity for ammonia
than GDH and is viewed as a scavenger of ammonia in bacteria (Tempest et al, 1970) and in plants
(see e.g. Miflin and Lea, 1980; Lea et al, 1992).
The major role of GDH in tissue cultured cells is the oxidation of glutamate to provide sufficient carbon
skeletons for effective functioning of the TCA cycle (Robinson et al, 1991).
In wildtype Arapidopsis, GDH1 mRNA accumulates to high levels in dark-adapted or sucrose-starved
plants; light or sucrose treatment each repress GDH1 mRNA accumulation. These results suggest
that the GDH1 gene product functions in the direction of glutamate catabolism under carbon-limiting
conditions (Melo-Oliveira et al, 1996). Low levels of GDH1 mRNA present in leaves of light-grown
plants can be induced by exogenously supplied ammonia (Melo-Oliveira et al, 1996). Under such
conditions of carbon and ammonia excess, GDH1 may function in the direction of glutamate
biosynthesis (Melo-Oliveira et al, 1996). The recessive Arabidopsis glutamate dehydrogenasedeficient mutant allele gdh1-1 cosegregates with the GDH1. The gdh1-1 mutant displays a conditional
phenotype; seedling growth is specifically retarded on media containing exogenously supplied
inorganic nitrogen, suggesting that GDH1 plays a nonredundant role in ammonia assimilation under
conditions of inorganic nitrogen excess (Melo-Oliveira et al, 1996). This is consistent with the fact that
the levels of mRNA for GDH1 and chloroplastic glutamine synthetase (GS2) are reciprocally regulated
by light (Melo-Oliveira et al, 1996).

Central role of glutamate:

Four of the amino acids: glutamate, aspartate, alanine and glutamine are
present in mammalian cells at much higher concentrations than the other
16. All four have major metabolic functions in addition to their roles in
proteins, but glutamate occupies the prime position.

Glutamate and aspartate function as excitatory neurotransmitters in the

central nervous system, and glutamate is partly responsible for the flavour
of food. (It is the mono sodium glutamate listed on processed food labels.)
However, glutamate also occupies a special position in amino acid
breakdown, and most of the nitrogen from dietary protein is ultimately
excreted from the body via the glutamate pool.
Glutamate is special because it is chemically related to 2-oxoglutarate ( =
-ketoglutarate) which is a key intermediate in the Krebs cycle. Glutamate
can be reversibly converted into 2-oxoglutarate by transaminases or by
glutamate dehydrogenase. In addition, glutamate can be reversibly
converted into glutamine, an important nitrogen donor, and the most
common free amino acid in human blood plasma.

Transamination reactions:
Most common amino acids can be converted into the corresponding keto
acid by transamination. This reaction swops the amino group from one
amino acid to a different keto acid, thereby generating a new pairing of
amino acid and keto acid. There is no overall loss or gain of nitrogen from
the system - it is simply a question of "robbing Peter to pay Paul".

Transamination reactions are readily reversible, and the equilibrium

constant is close to 1. One of the two substrate pairs is usually glutamate
and its corresponding keto acid -oxoglutarate. All transaminases require
pyridoxal phosphate or pyridoxamine phosphate (both derived from
vitamin b6) as an essential cofactor.
The reaction mechanism is shown on the next page. The substrates bind to
the enzyme active centre one at a time, and the function of the pyridoxal
phosphate is to act as a temporary store of amino groups until the next
substrate comes along. In the process the pyridoxal phosphate is converted
into pyridoxamine phosphate, and then back again. Enzymologists call this
a "ping pong" mechanism, and it leads to a characteristic pattern of parallel
lines in a double reciprocal plot of 1/V versus 1/S 1 at various S2
concentrations.
The condensation between the alpha amino group and the aromatic
aldehyde to form a "Schiff base" makes the alpha carbon atom chemically
reactive, so the isomerisation of the Schiff base takes place very easily. In
practice the pyridoxal form of the coenzyme condenses with the epsilon
amino group of a lysine residue in the enzyme protein when no amino acid
is bound, and the free aldehyde form of the coenzyme has only a transitory
existence. Many of the enyzmes that metabolise amino acids require

pyridoxal phosphate as a cofactor. Unexpectedly, this compound also

serves in a completely different manner in the active centre of glycogen
phosphorylase.

Glutamate:oxaloacetate transaminase [GOT]

This enzyme is also known as aspartate aminotransferase and is one of the
most active enzymes in the cell. It exists in mitochondrial and cytosolic
variants, and the detailed iso-enzyme pattern is tissue-specific. It escapes
in large amounts from dead or dying tissues and enters the bloodstream, so
GOT is often measured in blood samples for medical diagnostic purposes.

The metabolic importance of this enzyme is that it brings about a free

exchange of amino groups between glutamate (which is the most common
amino acid) and aspartate which is a second major amino acid pool.
Glutamate and aspartate are each required for separate but essential steps
in the urea cycle, which is responsible for ammonia detoxication and
nitrogen excretion. The free movement of nitrogen between the glutamate
and aspartate pools is an important balancing process that is vital for
normal cellular metabolism. This reaction is close to equilibrium in both
the cytosol and the mitochondrial compartments. It forms an integral part
of the malate - asparate shuttle for the "transport" of NADH across the
inner mitochondrial membrane.

Glutamate:pyruvate transaminase [GPT]

This very active enzyme is also known as alanine aminotransferase and
exists in mitochondrial and cytosolic variants. The detailed iso-enzyme
pattern is tissue-specific. It escapes in large amounts from dead or dying
tissues and GPT may be measured in blood samples for diagnostic
purposes.

Alanine is the principal amino acid released from muscle tissue during
starvation. It is an important substrate for hepatic gluconeogenesis, and
alanine transamination is required for the proper maintenance of fasting
blood glucose concentrations.
Pyridoxine is an example of a vitamin which is required for the synthesis
of a coenzyme. Other examples which you have met in this course are
listed in the table below.

vitamin
pyridoxine (vit b6)

cofactor

example enzymes

pyridoxal phosphate transaminases

niacin (nicotinamide) NAD+/ NADP+

malate dehydrogenase

riboflavin (vit b2)

FAD

succinate dehydrogenase

thiamine (vit b1)

TPP

pyruvate dehydrogenase

pantothenate

Coenzyme A

fatty acid metabolism

Glutamate dehydrogenase [GluDH]

This enzyme is the first committed step on the final common pathway for
mammalian nitrogen excretion, leading eventually to urea. A few of the
amino acids have specific deamination pathways, but about 75% of
ingested protein nitrogen follows the glutamate route.
Glutamate dehydrogenase in mammals is almost entirely confined to the
liver mitochondrial matrix space, where it accounts for a significant
proportion of the total protein. In contrast to the transamination reactions
which merely swop amino groups from one compound to another, GluDH
catalyses a net loss of nitrogen from the amino acid pool. The process is
therefore termed "oxidative deamination". It is the only common
dehydrogenase which is non-specific for NAD or NADP, and this may be
important for its overall regulation.

NADH / NAD and NADPH / NADP have the same standard redox
potential of -420mV when the oxidised and reduced forms are present in
equal concentrations. In practice these coenzymes have different effective
redox potentials and perform specialised functions within cells. The
NADPH / NADP pool operates almost entirely in the reduced form, but
the NADH / NAD pool is rarely more than 30% reduced. In general
NADPH is used to drive reductive biosynthetic reactions, whereas NAD is
the coenzyme for the oxidative energy-yielding pathways.

The dual coenzyme specificity is a potential source of difficulty for the

cell, since in theory this readily reversible enzyme could catalyse a futile
cycle, proceeding first in the oxidative direction with NAD, followed by a
reductive step using NADPH. The effect would be to "short circuit" the
two coenzyme pools, which normally require considerable investment in
substrates and cellular equipment to keep them separate. If this futile cycle
happens to any significant extent then it must be an advantage for the cell,
because it has persisted unchanged throughout 2,000,000,000 years of
evolutionary development.
The most likely explanation at present is that this futile cycle takes place,
but for various reasons it does not place an excessive burden on its owner.
The Km of GluDH for ammonia is quite high, and the free ammonia
concentration is kept very low by the next enzyme in the pathway,
carbamyl phosphate synthetase. This will severely reduce the rate of the
synthetic reaction, and allow the enzyme to catalyse a net glutamate
oxidation at a slow controlled rate that provides the maximum opportunity
for regulatory interference.
Regulation is plainly critical at this point, since GluDH and carbamyl
phosphate synthetase jointly control the overall rate of nitrogen excretion
and determine whether a particular individual will be in positive, neutral or
negative nitrogen balance. Control of unwanted nitrogen losses remains an
important unsolved problem after major surgery, burns or other serious
traumatic injuries.
The enzyme is modulated by adenine and guanine nucleotides, although it
is difficult to make much sense of the observed effects. GluDH has all the
hallmarks of a large multimeric allosteric enzyme, although the true nature
of the regulation remains to be identified. The situation is in some ways
similar to the parallel NAD and NADP linked oxidation pathways for
malate and isocitrate, although the competing reactions for these substrates
are separately regulated and catalysed by different proteins.

Trans-deamination
Most transaminases share a common substrate and product (glutamate and
oxoglutarate) with glutamate dehydrogenase, and this permits a combined
nitrogen excretion pathway for individual amino acids that is commonly
described as "trans-deamination".

This process underlines the central role of glutamate in the overall control
of nitrogen metabolism.

Amino Acid and Protein Metabolism

Abstract
Amino acid metabolism is one of the most important biochemical processes in
plants; similar to other topics in biochemistry, it has been affected by the
tremendous developments in science. Following are four actively studied aspects
in the field of amino acid metabolism: 1) the identification of new transporters of
amino acids and other N-forms like nitrate and ammonium; 2) the
characterization of factors controlling the metabolism in situ in distinct cells or
subcellular compartments; 3) the regulation of the multiple isoenzymes of amino
acid metabolism in the context of a single plant; and 4) the role of amino acids
as signaling molecules.
Studies on protein metabolism, on the other hand, have focused on the
processes of protein synthesis. The complex regulation of protein degradation is
today attracting more attention, particularly because proteolysis is involved in
cellular processes such as programmed cell death, circadian rhythm, and the
defense response in plants. This chapter summarizes the latest insights in the
studies of amino acid metabolism and protein degradation.
The focus of this review is to examine the regulation of amino acid metabolism
and protein processing in the context of a single plant. Particular emphasis is
given to the enzymes involved in NH4+ assimilation, which are often oligomers
located in different subcellular compartments. The mechanism operating in
oxidative protein cleavage is also discussed.
Keywords: N-form; Ammonium assimilation; Glutamate; GOGAT; Glutamine
synthetase; Nuclear magnetic resonance; Metalloprotease; Protein degradation;
Oxidative cleavage