Information

Is glutamate always involved in the deamination and amination of the other amino acids?


For example, are there pathways for the deamination of phenylalanine that simply produce ammonia or pathways for it to be synthesized from phenylpyruvate with ammonia being utilized to form the amine group?

Preferably, I want to know how it is with human metabolism mainly.


For most amino acids, the removal of the α-amino group involves α-ketoglutarate and glutamate. The amino group is first transferred to a-ketoglutarate by transaminases, and the resulting glutamate is then deaminated (via glutamate dehydrogenase) to yield ammonia.

The same is true for amination. Glutamate and glutamine are the two major amino group-donors. Most ketoacids are converted to their respective amino acids by transamination involving glutamate or glutamine. Glutamine can be synthesized by amination of glutamate with ammonia without transamination (via a synthetase enzyme) and glutamate can be aminated with ammonia too.

Exceptions do exist, of course. For example, Not all transaminations involve glutamate/glutamine (as this user has replied), and serine and threonine can be directly deaminated (via dehydratase enzymes, as opposed to the dehydrogenase used for glutamate).


Glutamic acid

Glutamic acid (symbol Glu or E [4] the ionic form is known as glutamate) is an α-amino acid that is used by almost all living beings in the biosynthesis of proteins. It is non-essential in humans, meaning that the body can synthesize it. It is also an excitatory neurotransmitter, in fact the most abundant one, in the vertebrate nervous system. It serves as the precursor for the synthesis of the inhibitory gamma-aminobutyric acid (GABA) in GABA-ergic neurons.

  • l isomer: 56-86-0 Y
  • racemate: 617-65-2 Y
  • d isomer: 6893-26-1 Y
  • l isomer: CHEBI:16015 Y
  • racemate: CHEBI:18237
  • d isomer: CHEBI:15966
  • l isomer: ChEMBL575060 Y
  • l isomer: 591 Y
  • l isomer: C00025 N
  • d isomer: C00217
  • l isomer: 3KX376GY7L Y
  • racemate: 61LJO5I15S Y
  • d isomer: Q479989WEA Y
InChI=1S/C5H9NO4/c6-3(5(9)10)1-2-4(7)8/h3H,1-2,6H2,(H,7,8)(H,9,10) Y Key: WHUUTDBJXJRKMK-UHFFFAOYSA-N Y

It has a formula C
5 H
9 NO
4 . Glutamic acid exists in three optically isomeric forms the dextrorotatory L -form is usually obtained by hydrolysis of gluten or from the waste waters of beet-sugar manufacture or by fermentation. [5] Its molecular structure could be idealized as HOOC−CH( NH
2 )−( CH
2 )2−COOH, with two carboxyl groups −COOH and one amino group − NH
2 . However, in the solid state and mildly acidic water solutions, the molecule assumes an electrically neutral zwitterion structure − OOC−CH( NH +
3 )−( CH
2 )2−COOH. It is encoded by the codons GAA or GAG.

The acid can lose one proton from its second carboxyl group to form the conjugate base, the singly-negative anion glutamate − OOC−CH( NH +
3 )−( CH
2 )2−COO − . This form of the compound is prevalent in neutral solutions. The glutamate neurotransmitter plays the principal role in neural activation. [6] This anion creates the savory umami flavor of foods and is found in glutamate flavorings such as MSG. In Europe it is classified as food additive E620. In highly alkaline solutions the doubly negative anion − OOC−CH( NH
2 )−( CH
2 )2−COO − prevails. The radical corresponding to glutamate is called glutamyl.


1. Introduction

Glutamate is the most abundant of the common protein-coded amino acids in the brain [1]. Table 1 lists the concentration of the more abundant amino acids in cat, rat, and human brain. In addition to high concentrations of glutamate, the brain contains mM concentrations of glutamine, aspartate, and GABA ( Table 1 ). Taurine is included in this table because, although it is not a protein-coded amino acid, its concentration in brain is high. Taurine plays important roles in the brain as an osmolyte (volume regulation), calcium homeostasis, and regulation of neurotransmission, e.g., [2,3,4]. Glutathione (GSH: a tripeptide, γ-glutamylcysteinylglycine) is also included in this table because it is synthesized from glutamate. In the brain, GSH is a major redox buffer, water soluble antioxidant (along with ascorbate), enzyme cofactor and participant in detoxification processes, especially in astrocytes, e.g., [5]. Glutamate and, to a lesser extent, aspartate are the major excitatory neurotransmitters in the brain, whereas GABA is the main inhibitory neurotransmitter. Therefore, these amino acids must be maintained at very low concentrations in the extracellular fluid compartments of the brain. For example, the concentrations of glutamate and aspartate in human cerebrospinal fluid (CSF) are

8 and 0.2 µM, respectively [1]. The concentration of GABA in human CSF is 𢙀.1 µM [6]. Interestingly, the concentration of glutamine in human CSF is remarkably high (

50 µM) and greater than that of all the other common amino acids combined [1]. In point of fact, the concentration of glutamine in human CSF is 㹐 times greater than that of glutamate [1]. This high concentration of glutamine is a reflection of the release of glutamine from astrocytes to the extracellular fluid as a means of maintaining nitrogen balance and as part of the glutamate-glutamine cycle hereinafter simply referred to as the glutamine cycle (Section 6).

Table 1

Approximate Concentration (µmol/g Wet weight) of Glutathione and the Most Abundant Amino Acids in the Brain.

CatRatHuman
Glutamate7.90 (9.88)11.6 (14.5)6.00 (7.50)
Taurine2.30 (2.88)6.60 (8.25)0.93 (1.16)
Glutamine2.80 (3.50)4.50 (5.63)5.80 (7.25)
Aspartate1.70 (2.13)2.60 (3.25)0.96 (1.20)
γ-Aminobutyrate1.40 (1.75)2.30 (2.88)0.42 (0.53)
Glycine0.78 (0.98)0.68 (0.85)0.40 (0.50)
Alanine0.48 (0.60)0.65 (0.81)0.25 (0.31)
Serine0.48 (0.60)0.98 (1.23)0.44 (0.55)
Glutathione0.49 (0.61)2.60 (3.25)0.20 (0.25)

Adapted from [1]. Values in parenthesis are concentrations (mM) assuming a water content of 80%.

The concentration of glutamate in synaptosomal vesicles is very high [7], perhaps as high as 100 mM or greater (ref. [8] and references cited therein), representing 15%�% of the total glutamate pool in synaptosomes, consistent with high levels in the nerve endings [7]. The very high concentration of glutamate in the cytosol and glutamate-containing vesicles requires strict homeostatic mechanisms for the following reason. Glutamate is the major excitatory neurotransmitter, yet levels of glutamate in the extracellular fluid must be kept low (𼄀 µM) to avoid excitotoxicity. In fact, the concentration of glutamate in the ambient extracellular fluid of the brain is normally 0.5𠄵 µM [8]. This remarkable glutamate concentration gradient between the extracellular fluid and nerve cell cytosol is accomplished by powerful uptake systems for glutamate in neurons, astrocytes and synaptosomal vesicles. (For a recent review see [9]).

Exclusion of most blood-borne glutamate at the blood-brain barrier (BBB) and a net removal of glutamine from the brain (see below) indicate that the cerebral pools of glutamate are largely produced within the brain. Thus, the tricarboxylic acid (TCA) cycle must be an important source of 5-carbon units for the synthesis of the glutamate backbone in the brain. The nitrogen of the glutamate pool in the brain is mostly supplied by aminotransferase reactions. Here, we review the central importance of aminotransferases in maintaining nitrogen homeostasis in the brain, with special emphasis on glutamate/α-ketoglutarate-linked aminotransferases. We highlight the important role of glutamate as a precursor for other important metabolites including GSH. We also emphasize the special role of glutamate in the glutamine- and glutamine-GABA cycles of the brain. The glutamate utilized in these pathways is maintained at a remarkably constant level. For example, during hyperammonemia there is a marked increase in the rate of conversion of cerebral glutamate to glutamine, yet although there is some depletion in glutamate the decrease is not stoichiometric with the concomitant large increase in glutamine concentration. There is also no change in the concentration of cerebral α-ketoglutarate or a modest increase. Thus, there is a considerable net increase in the concentration of 5-carbon units (i.e., glutamate plus glutamine plus α-ketoglutarate) resulting from an ammonia-induced stimulation of anaplerosis. An especially prominent anaplerotic enzyme stimulated by hyperammonemia is pyruvate decarboxylase. The main pathways of glutamate metabolism in normoammonemia and hyperammonemia are depicted diagrammatically in Figure 1 .

Schematic of the major pathways by which cerebral glutamate levels are maintained during normoammonemia (top panel) and hyperammonemia (bottom panel). Relative changes in pool size of cerebral metabolites (α-ketoglutarate, ammonia, glutamate, and glutamine) between nomoammonemic and hyperammonemic brain are indicated by differences in font size. Enzymes: 1, glutamate dehydrogenase (GDH) 2, α-ketoglutarate/glutamate-linked aminotransferases (notably aspartate aminotransferase (AspAT) and branched-chain aminotransferases) 3, glutamine synthetase 4, glutaminase. Note that despite the ammonia-stimulated increase in glutamine (Gln), glutamate (Glu) levels are only modestly depleted. This is in large part due to hyperammonemia-induced increase in the activity of anaplerotic enzymes including pyruvate carboxylase. Note also that for simplicity not all reactants are shown in the enzyme-catalyzed reactions.

Note that throughout the text we use the term ammonia to refer to the sum of ammonium (NH4 + ) ions and ammonia free base (NH3). Since the pKa of ammonia is

9.2 at physiological pH values (7.2𠄷.4) only

1% of ammonia will be in the form of NH3.


The Kinetic Mechanism of Phenylalanine Hydroxylase: Intrinsic Binding and Rate Constants from Single Turnover Experiments &dagger

A possible mechanism is shown below.

"1) oxidation of the pterin cofactor to form the reactive hydroxylating intermediate, followed by 2) insertion of oxygen into the amino acid substrate (20). Supporting this view is the observation that pterin oxidation can become uncoupled from amino acid oxidation, either when nonphysiological amino acids are used as substrates (11, 25) or in a variety of TyrH active-site mutants "

As in the case with the the conversion of dihydrofolate back to tetrahydrofolate (FH4) by dihydrofolate reductase, the 4a-OH-BH4 is conveted to dihydrobiopterin and then to tetrahydrobiopterin by dihyrobiopterin reductase.

Here is the full pathway for the conversion of Phe and Tyr to acetoacetate and fumarate

henylalanine normally has only two fates: incorporation into polypeptide chains, and hydroxylation to tyrosine via the tetrahydrobiopterin-requiring phenylalanine hydroxylase (PAH) reaction. Thus, phenylalanine catabolism always ensues in the pathway of tyrosine biosynthesis followed by tyrosine catabolism. Tyrosine is equally important for protein biosynthesis as well as an intermediate in the biosynthesis of the catecholamines: dopamine, norepinephrine and epinephrine (see Amino Acid Derivatives).

The pathway of tyrosine degradation involves conversion to fumarate and acetoacetate, allowing phenylalanine and tyrosine to be classified as both glucogenic and ketogenic. The catabolism of tyrosine involves five reactions, four of which have been shown to associated with inborn errors in metabolism and three of these result in clinically significant disorders. The first reaction of tyrosine catabolism involves the nuclear genome encoded mitochondrial enzyme tyrosine aminotransferase and generates the corresponding ketoacid, p-hydroxyphenylpyruvic acid.

Like most aminotransferase reaction, tyrosine aminotransferase utilizes 2-oxoglutarate (&alpha-ketoglutarate) as the amino acceptor with the consequent generation of glutamate. Tyrosine aminotransferase is encoded by the TAT gene on chromosome 16q22.2 which is composed of 12 exons that generate a protein of 454 amino acids. The second reaction of tyrosine catabolism is catalyzed by 4-hydroxyphenylpyruvate dioxygenase which is encoded by the HPD gene located on chromosome 12q24.31 which is composed of 17 exons that generate two alternatively spliced mRNAs encoding proteins of 393 amino acids (isoform 1) and 354 amino acids (isoform 2).

The product of the HPD reaction is homogentisic acid (homogentisate). Homogentisate is oxidized by the second dioxygenase enzyme of tyrosine catabolism, homogentisate oxidase. Homogentisate oxidase is encoded by the homogentisate 1,2-dioxygenase gene, HGD. The HGD gene is located on chromosome 3q13.33 and is composed of 16 exons that encode a protein of 445 amino acids.

Oxidation of homogentisate yields 4-maleylacetoacetate which is isomerized to 4-fumarylacetoacetate by the enzyme glutathione S-transferase zeta (&zeta) 1 which is encoded by the GSTZ1 gene. Glutathione S-transferase zeta 1 was formerly called 4-maleylacetoacetate isomerase or maleylacetoacetate cis&ndashtrans-isomerase. The GSTZ1 gene is located on chromosome 14q24.3 and is composed of 9 exons that generate four alternatively spliced mRNAs, each of which encode a distinct protein isoform.

Fumarylacetoacetate is hydrolyzed to fumarate and acetoacetate by the enzyme fumarylacetoacetate hydrolase which is encoded by the FAH gene located on chromosome 15q25.1 and is composed of 15 exons that generate a 419 amino acid protein.

The fumarate end product of tyrosine catabolism feeds directly into the TCA cycle for further oxidation. The acetoacetate is activated to acetoacetyl-CoA via the action of the mitochondrial ketone body utilization enzyme, succinyl-CoA:3-oxoacid-CoA transferase (SCOT) which is encoded by the OXCT1 (3-oxoacid-CoA transferase 1) gene. Acetoacetate can also be activated in the cytosol by the cytosolic enzyme, acetoacetyl-CoA synthetase (AACS)."

E. Leu to Acetoacetate

"The first step in each case is a transamination using a pyridoxal phosphate-dependent BCAA aminotransferase (termed a branched-chain aminotransferase, BCAT), with 2-oxoglutarate (&alpha-ketoglutarate) as amine acceptor."

F. Isoleucine to Acetyl-CoA

Conversion to &alpha-ketoglutarate: Pro, Glu, Gln, Arg,His

A. Proline and Arginine

aldehyde dehydrogenase 4 family, member A1 (ALDH4A1) or D1-pyrroline-5-carboxylate dehydrogenase, (P5CDH)

"lutamate that results from ornithine and proline catabolism can then be converted to 2-oxoglutarate (&alpha-ketoglutarate) in a transamination reaction. Therefore, ornithine and proline are both glucogenic. Since arginine is metabolized to urea and ornithine, and the resulting ornithine is a glucogenic precursor, arginine is also a glucogenic amino acid."

The overall pathway is shown below

B. Histidine

C. Glutamine and Glutamic Acid

As described in the reactions above, can be converted to &alpha-ketoglutarate through transamination reactions. Also as described in sections 18.x, gluatamine can be deaminated through the action of glutaminase to form glutamine which can likewise form &alpha-ketoglutarate, a gluconeogenic intermediate.

Conversion to succinyl-CoA: Met, Ile, Thr, Val

We just saw that two branched chain amino acids, Leu and Ile, are converted to acetyl-CoA and hence are ketogenic (E and F above). Another branched chain hydrophobic amino acid, Val, and also Leu again, can be converted to succinyl-CoA which can be converted to &alpha-ketoglutarate in the Kreb's cycle in net fashion and hence are glucogenic amino acids. We saw in the introduction to amino acids that produce acetyl-CoA that threonine and isoleucine, two branched chains amino acids, also form proprionyl-CoA which goes on to succinyl CoA. So, let's consider Val, another branched chain amino acid, before we consider Met, both of which have 3 Cs in their side chains.

Methionine

The principal fates of the essential amino acid methionine are incorporation into polypeptide chains, and use in the production of cysteine and &alpha-ketobutyrate via the reaction pathway involving the synthesis of SAM and cysteine as described above. The transulfuration reactions that produce cysteine from homocysteine and serine also produce &alpha-ketobutyrate, the latter being converted first to propionyl-CoA and then via a 3-step process to succinyl-CoA.

In the catabolism of methionine the &alpha-ketobutyrate is converted to propionyl-CoA. The propionyl-CoA is converted, via a mitochondrially-localized three reaction ATP-dependent pathway, to succinyl-CoA. The succinyl-CoA can then enter the TCA cycle for further oxidation. The enzymes required for this conversion are propionyl-CoA carboxylase, methylmalonyl-CoA epimerase, and methylmalonyl-CoA mutase, respectively. Propionyl-CoA carboxylase is called an ABC enzyme due to the requirements for ATP, Biotin, and CO2 for the reaction. The clinical significance of methylmalonyl-CoA mutase in this pathway is that it is one of only two enzymes that requires a vitamin B12-derived co-factor for activity. The other B12-requiring enzyme is methionine synthase (see the Cysteine Synthesis section above). This propionyl-CoA conversion pathway is also required for the metabolism of the amino acids valine, isoleucine, and threonine and fatty acids with an odd number of carbon atoms. For this reason this three-step reaction pathway is often remembered by the mnemonic as the VOMIT pathway, where V stands for valine, O for odd-chain fatty acids, M for methionine, I for isoleucine, and T for threonin

above from med chem

Methionine metabolism in mammals happens within two pathways, a methionine cycle and a transsulfuration sequence. These pathways have three common reactions with both pathways including the transformation of methionine to S-adenosylmethionine (SAM), the use of SAM in many different transmethylation reactions resulting in a methylated product plus S-adenosylhomocysteine, and the conversion of S-adenosylhomocysteine to produce the compounds homocysteine and adenosine. The reactions mentioned above not only produce cysteine, they also create a-ketobutyrate. This compound is then converted to succinyl-CoA through a three step process after being converted to propionyl-CoA. If the amino acids cysteine and methionine are available in enough quantity, the pathway will accumulate SAM and this will in turn encourage the production of cysteine and a-ketobutyrate, which are both glucogenic, through cystathionine synthase. When there is a lack of methionine, there is a decrease in the production of SAM, which limits cystathionine synthase activity.

MEt to SAM give Met product + SAHC which produces homcys and adenosie also alpha keto butyrate which then proprionyl and to succinyll coa. If enough cys and met acumulate SAM lead to Cys and alpha keto

Special: Branched Chains

. The three branched-chain amino acids, isoleucine, leucine, and valine enter the catabolic pathway via the action of the same two enzymes. The initial deamination of all three amino acids is catalyzed by one of two branched-chain amino acid transaminases (BCATc or BCATm). The resulting &alpha-ketoacids are then oxidatively decarboxylated via the action of the enzyme complex, branched-chain ketoacid dehydrogenase (BCKD). The BCKD reaction generates the CoA derivatives of the decarboxylated ketoacids while also generating the reduced electron carrier, NADH. After these first two reactions the remainder of the catabolic pathways for the three amino acids diverges. The third reaction of branched-chain amino acid catabolism involves a dehydrogenation step that involve three distinct enzymes, one for each of the CoA derivatives generated via the BCKD reaction. This latter dehydrogenation step also yields additional reduced electron carrier as FADH2. The third reaction of isoleucine catabolism involves the enzyme short/branched-chain acyl-CoA dehydrogenase (SBCAD). The SBCAD enzyme is encoded by the ACADSB gene. The third reaction of leucine catabolism involves the enzyme isovaleryl-CoA dehydrogenase (IVD). The third reaction of valine catabolism involves the enzyme isobutyryl-CoA dehydrogenase (IBD). The IBD enzyme is encoded by the acyl-CoA dehydrogenase family, member 8 (ACAD8) gene. These CoA dehydrogenases belong to the same family of enzymes involved in the process of mitochondrial fatty acid oxidation.


Selenocysteine

A cysteine analog commonly referred to as the 21st amino acid, selenocysteine (Figure 6.163) is an unusual amino acid occasionally found in proteins. Although it is rare, selenocysteine has been found in proteins in bacteria, archaea and eukaryotes.

In contrast to amino acids such as phosphoserine, hydroxyproline, or acetyl-lysine, which arise as a result of post-translational modifications, selenocysteine is actually built into growing peptide chains in ribosomes during the process of translation.

No codon specifies selenocysteine, so to incorporate it into a protein, a tRNA carrying it must bind to a codon that normally specifies STOP (UGA). This alternative reading of the UGA is dependent on formation of a special hairpin loop structure in the mRNA encoding selenoproteins.

Selenium is rather toxic, so cellular and dietary concentrations are typically exceedingly low. About 25 human proteins are known to contain the amino acid. These include five glutathione peroxidases, and three thioredoxin reductases. Iodothyronine deiodinase, a key enzyme that converts thyroxine to the active T3 form, also contains selenocysteine in its active site. All of these proteins contain a single selenocysteine.

A eukaryotic protein known as selenoprotein P, found in the blood plasma of animals, contains ten selenocysteine residues and is thought to function as an antioxidant and/or in heavy metal detoxification. Besides selenocysteine, at least two other biological forms of a seleno-amino acid are known. These include 1) selenomethionine (Figure 6.164), a naturally occurring amino acid in Brazil nuts, cereal grains, soybeans, and grassland legumes and 2) methylated forms of selenocysteine, such as Se-methylselenocysteine, are found in Astragalus, Allium, and Brassica species.

The specifics of the process of translation will be described elsewhere in the book, but to get selenocysteine into a protein, the tRNA carrying selenocysteine pairs with a stop codon (UGA) in the mRNA in the ribosome. Thus, instead of stopping translation, selenocysteine can incorporated into a growing protein and translation continues instead of stopping.

Four genes are involved in preparation of selenocysteine for incorporation into proteins. They are known as sel A, sel B, sel C, and sel D. Sel C codes for the special tRNA that carries selenocysteine. The amino acid initially put onto the selenocysteine tRNA is not selenocysteine, but rather serine. Action of sel A and sel D are necessary to convert the serine to a selenocysteine.

An intermediate in the process is selenophosphate, which is the selenium donor. It is derived from H2Se, the form in which selenium is found in the cell. The tRNA carrying selenocysteine has a slightly different structure than other tRNAs, so it requires assistance in translation. The sel B gene encodes for an EF-Tu-like protein that helps incorporate the selenocysteine into the protein during translation.

Using UGA codons to incorporate selenocysteine into proteins could wreak havoc if done routinely, as UGA, in fact, almost always functions as a stop codon and is only rarely used to code for selenocysteine. Fortunately, there is a mechanism to ensure that the reading of a UGA codon as selenocysteine occurs only when the mRNA encodes a selenoprotein.

Unusual structures in mRNAs

The mRNAs for selenocysteine-containing proteins form unusual mRNA structures around the UGA codon that make the ribosome &ldquomiss&rdquo it as a stop codon and permit the tRNA with selenocysteine to be incorporated instead.

Like selenocysteine, pyrrolysine is a rare, unusual, genetically encoded amino acid found in some cells. Proteins containing it are enzymes involved in methane metabolism and so far have been found only methanogenic archaeans and one species of bacterium. The amino acid is found in the active site of the enzymes containing it. It is sometimes referred to as the 22nd amino acid.

Synthesis of the amino acid biologically begins with two lysines. One is converted to (3R)-3-Methyl-D-ornithine, which is attached to the second lysine. After elimination of an amine group, cyclization, and dehydration, L-pyrrolysine is produced. Pyrrolysine is attached to an unusual tRNA (pylT gene product) by action of the aminoacyl tRNA synthetase encoded by the pylS gene. This unusual tRNA can pair with the UAG stop codon during translation and allow for incorporation of pyrrolysine into the growing polypeptide chain during translation in a manner similar to incorporation of selenocysteine.

The urea cycle holds the distinction of being the first metabolic cycle discovered - in 1932, five years before the citric acid cycle. It is an important metabolic pathway for balancing nitrogen in the bodies of animals and it takes place primarily in the liver and kidney.

Organisms, like humans, that excrete urea are called ureotelic. Those that excrete uric acid (birds, for example) are called uricotelic and those that excrete ammonia (fish) are ammonotelic. Ammonia, of course, is generated by metabolism of amines and is toxic, so managing levels of it is critical for any organism. Excretion of ammonia by fish is one reason that an aquarium periodically requires cleaning and replacement of water.

Liver failure can lead to accumulation of nitrogenous waste and exacerbates the problem. As shown in Figure 1.166, the cycle contains five reactions, with each turn of the cycle producing a molecule of urea. Of the five reactions, three occur in the cytoplasm and two take place in the mitochondrion. (The reaction making carbamoyl phosphate, catalyzed by carbamoyl phosphate synthetase is not shown in the figure.)

Though the cycle doesn&rsquot really have a starting point, a common place to begin discussion is with the molecule of ornithine. As discussed elsewhere in this book, ornithine intersects the metabolic pathways of arginine and proline.

Ornithine is found in the cytoplasm and is transported into the mitochondrion by the ornithine-citrulline antiport of the inner mitochonrial membrane. In the matrix of the mitochondrion, two reactions occur relevant to the cycle. The first is formation of carbamoyl phosphate from bicarbonate, ammonia, and ATP catalyzed by carbamoyl phosphate synthetase I.

Carbamoyl phosphate then combines with ornithine in a reaction catalyzed by ornithine transcarbamoylase to make citrulline.

The citrulline is transported out to the cytoplasm by the ornithine-citrulline antiport mentioned above. In the cytoplasm, citrulline combines with L-aspartate using energy of ATP to make citrullyl-AMP (an intermediate) followed by argininosuccinate. The reaction is catalyzed by argininosuccinate synthase.

Next, fumarate is split from argininosuccinate by argininosuccinate lyase to form arginine.

Water is used by arginase to cleave arginine into urea and ornithine, completing the cycle.

Urea is less toxic than ammonia and is released in the urine. Some organisms make uric acid for the same reason.

It is worth noting that aspartic acid, ammonia, and bicarbonate enter the cycle and fumarate and urea are produced by it. Points to take away include 1) ammonia is converted to urea using bicarbonate and the amine from aspartate 2) aspartate is converted to fumarate which releases more energy than if aspartate were converted to oxaloacetate, since conversion of fumarate to malate to oxaloacetate in the citric acid cycle generates an NADH, but direct conversion of aspartate to oxaloacetate does not and 3) glutamate and aspartate are acting as shuttles to funnel ammonia into the cycle. Glutamate, as will be seen below, is a scavenger of ammonia.

The urea cycle is controlled both allosterically and by substrate concentration. The cycle requires N-acetylglutamate (NAG) for allosteric activation of carbamoyl phosphate synthetase I. The enzyme that catalyzes synthesis of NAG, NAG synthetase, is activated by arginine and glutamate. Thus, an indicator of high amine levels, arginine, and an important shuttler of amine groups, glutamate, stimulates the enzyme that activates the cycle.

The reaction catalyzed by NAG synthetase is

At the substrate level, all of the other enzymes of the urea cycle are controlled by the concentrations of substrates they act upon. Only at high concentrations are the enzymes fully utilized.

Complete deficiency of any urea cycle enzyme is fatal at birth, but mutations resulting in reduced expression of enzymes can have mixed effects. Since the enzymes are usually not limiting for these reactions, increasing substrate can often overcome reduced enzyme amounts to a point by simply fully activating enzymes present in reduced quantities.

However, if the deficiencies are sufficient, ammonium can accumulate and this can be quite problematic, especially in the brain, where mental deficiencies or lethargy can result. Reduction of ammonium concentration relies on the glutamate dehydrogenase reaction (named for the reverse reaction).

Additional ammonia can be taken up by glutamate in the glutamine synthetase reaction.

The result of these reactions is that &alpha-ketoglutarate and glutamate concentrations will be reduced and the concentration of glutamine will increase. For the brain, this is a yin/yang situation. Removal of ammonia is good, but reduction of &alpha-ketoglutarate concentration means less energy can be generated by the citric acid cycle. Further, glutamate is, itself, an important neurotransmitter and a precursor of another neurotransmitter - &gamma-aminobutyric acid (GABA).

From an energy perspective, the urea cycle can be said to break even or generate a small amount of energy, if one includes the energy produced in releasing ammonia from glutamate (one NADH). There are two NADHs produced (including the one for converting fumarate to oxaloacetate), which give 4-6 ATPs, depending on how efficiently the cell performs electron transport and oxidative phosphorylation.

The cycle takes in 3 ATPs and produces 2 ADPs and one AMP. Since AMP is equivalent to 2 ATP, the cycle uses 4 ATP. Thus, the cycle either breaks even in the worst case or generates 2 ATPs in the best case.

Amino acid catabolism

Amino acids are divided according to the pathways involved in their degradation. There are three general categories. Ones that yield intermediates in the glycolysis pathway are called glucogenic and those that yield intermediates of acetyl-CoA or acetoacetate are called ketogenic. Those that involve both are called glucogenic and ketogenic. These are shown in Figures 6.167 and 6.168.

As seen in the two figures, amino acids largely produce breakdown products related to intermediates of the citric acid cycle or glycolysis, but this isn&rsquot the complete picture. Some amino acids, like tryptophan, phenylalanine, and tyrosine yield hormones or neurotransmitters on further metabolism (as noted earlier). Others like cysteine and methionine must dispose of their sulfur and all of the amino acids must rid themselves of nitrogen, which can happen via the urea cycle, transamination, or both.

Tyrosine catabolism

Breakdown of tyrosine (Figure 6.169) is a five step process that yields acetoacetate and fumarate. Enzymes involved include 1) tyrosine transaminase 2) p-hydroxylphenylpyruvate dioxygenase 3) homogentisate dioxygenase 4) maleylacetoacetate cis-trans-isomerase and 5) 4-fumaryl acetoacetate hydrolase.

Breakdown of leucine is a multi-step process ultimately yielding the ketone body acetoacetate and acetyl-CoA. Branched chain amino acids (BCAAs - valine, leucine, and isoleucine) rely on Branched Chain AminoTransferase (BCAT) followed by Branched Chain &alpha-ketoacid dehydrogenase (BCKD) for catabolism.

Breakdown of isoleucine yields intermediates that are both ketogenic and glucogenic. These include acetyl-CoA and propionyl-CoA.

Breakdown of valine is a multi-step process ultimately yielding propionyl-CoA.


Decarboxylation of Amino Acids

When a carboxyl group is cleaved from an amino acid, 1 amine and CO2 are released as side products. The reaction is catalyzed by the enzyme decarboxylase using PLP as a partner. The resulting amines fulfill important functions in the body which is why they are called biogenous amines.

A well-known representative is histamine which is formed hrough decarboxylation from the basic amino acid histidine. The responsible enzyme is accordingly called histidine decarboxylase. Histamine is an important mediator and plays a vital role in, e.g., immediate hypersensitivity reactions. Further well-known biogenous amines relevant for metabolism are, e.g., GABA (gamma-aminobutyric acid from glutamine acid) and dopamine (from 3,4-dihydroxyphenylalanine).


What is glutamate signalling doing in plants?

An evolutionary perspective

The existence of a large family of plant GLR genes, together with the presence of glutamate-activated channel activity in roots and shoots, strongly suggests that glutamate signalling in plants is a reality. When glutamate signalling was thought of as something strictly associated with neurons and nervous systems, the notion of its existence in plants seemed paradoxical ( Lam et al., 1998). However, in the past decade, it has emerged that glutamate signalling in animals is not restricted to the nervous system, and indeed that it also occurs in primitive metazoans that lack a nervous system.

Examples where functional glutamate receptors (mGluRs and iGluRs) have been detected in non-neuronal mammalian tissues include bone cells, the pineal gland, and the pancreas ( Gill and Pulido, 2001 Hinoi et al., 2004 Moriyama and Yamamoto, 2004). The same tissues also express glutamate/aspartate transporters (GLASTs), which are required in synapses for signal termination, and vesicular glutamate transporters (VGLUTs), which are responsible for the active transport and storage of L -glutamate in synaptic vesicles. These findings have led to the conclusion that glutamatergic signalling is a general and ubiquitous system for intercellular communication in animals ( Hinoi et al., 2004 Moriyama and Yamamoto, 2004). Amongst the diverse functions ascribed to glutamate signalling in non-neuronal cells are included the regulation of insulin secretion ( Storto et al., 2006), the control of cellular differentiation in osteoblasts ( Hinoi et al., 2003), and the modulation of tumour cell proliferation ( Kalariti et al., 2005).

That glutamate signalling has early evolutionary origins, before the divergence of plants and animals, was an idea first proposed by Chiu et al. (1999). Support for this idea has come from studies on the marine sponge Geodia cydonium and the cellular slime mould Dictyostelium. Isolated Geodia cells were shown to be responsive to glutamate and to agonists and anatagonists of mGluRs, and a gene related to the mGluR/GABAB-type receptors was identified ( Perovic et al., 1999). Marine sponges are regarded as belonging to the most primitive metazoan phylum ( Muller, 2001), indicating that sophisticated glutamate signalling systems existed in the earliest metazoans. The slime mould Dictyostelium is an even more primitive unicellular eukaryote that appears to have split from the animal–fungal lineage after the divergence of plants and animals ( Eichinger et al., 2005). The formation of spores on the fruiting body of this social amoeba is induced by a GABA signal, and glutamate acts as a competitive inhibitor of this process ( Anjard and Loomis, 2006). Dictyostelium possesses a mGluR-like gene (DdmGluPR or GrlE) ( Taniura et al., 2006), and disrupting this gene abolishes the responses to GABA and glutamate, suggesting that both amino acids can bind to the DdmGluPR receptor ( Anjard and Loomis, 2006).

Homologues of iGluRs/GLRs even exist in cyanobacteria (GluR0) where they function as glutamate-gated K + channels ( Chen et al., 1999). Although these receptors lack the extended C-terminal and N-terminal domains that characterize the eukaryotic members of the family ( Fig. 2), it is likely that iGluRs suitable for intercellular communication evolved from an ancestral GluR0-type channel ( Oswald, 2004).

In summary, it appears that both iGluR/GLR-type and mGluR/GABAB-type receptors were already present in the progenitors of the Viridiplantae and the Metazoa. Since multicellularity is thought to have arisen independently in the evolution of the major eukaryotic kingdoms (Viridiplantae, Fungi, and Metazoa) ( Schopf, 1993), it seems that glutamate/GABA signalling mechanisms may have been a feature of the most primitive unicellular life forms. It is possible that the earliest function of glutamate/GABA signalling in single-celled organisms was in triggering chemotactic responses. Chemotaxis is recognized as an important adaptive mechanism which enhances an organism's ability to exploit nutrient patches ( Fenchel, 2002), and glutamate has been identified as a chemotactic signal in a number of modern day unicellular organisms, both prokaryotic ( Brown and Berg, 1974 Barbour et al., 1991) and eukaryotic ( Lee et al., 1999 Van Houten et al., 2000). Glutamate also acts as a cue that initiates a range of feeding-related responses in a variety of marine metazoans ( Bellis et al., 1991 Trott et al., 1997 Daniel et al., 2001 Kidawa, 2005). In some instances there is preliminary evidence suggesting that chemodetection of environmental glutamate may involve iGluR-type receptors ( Bellis et al., 1991 Murphy and Hadfield, 1997 Van Houten et al., 2000). In this context, it is intriguing that glutamate, acting through iGluR-type receptors, has a role as a guidance cue for neuronal migration during embryogenesis ( Behar et al., 1999 Manent et al., 2005 Matsugami et al., 2006 McGowan, 2006), perhaps echoing an evolutionarily ancient role as a chemoattractant.

If it is the case that glutamate signalling evolved in a primitive progenitor of plants and animals, clues to the function of glutamate signalling pathways in plants may come from a greater understanding of non-neuronal glutamate signalling in animals, and vice versa.

Glutamate signalling and root apical meristem activity

An unusual and striking effect of external glutamate on Arabidopsis root growth and branching has recently been reported ( Walch-Liu et al., 2006b). When Arabidopsis roots were exposed to low concentrations of L -glutamate there was a marked inhibition of primary root growth and an increase in root branching near the root apex. This effect on root architecture resulted from inhibition of meristematic activity at the primary root tip and an initial stimulation of lateral root outgrowth in the apical region of the primary root. Lateral roots were also glutamate sensitive, but only after they had reached 5–10 mm in length, indicating a developmentally delayed response. A similar response to glutamate was found in roots of a number of other species, including Thlaspi caerulescens, Thellungiella halophila, wild poppy, and tomato ( Walch-Liu et al., 2006a, b). Intriguingly, different Arabidopsis ecotypes displayed widely differing sensitivities to glutamate, the most sensitive being C24 and the least sensitive RLD1.

Both positive and negative effects of amino acids on plant growth have been reported by other authors ( Skinner and Street, 1953 Rognes et al., 1986 Katonoguchi et al., 1994 Barazani and Friedman, 2000), and a rapid inhibition of primary root growth by glutamate was reported by Sivaguru et al. (2003). However, the effects observed by Walch-Liu et al. (2006b) are distinctive in a number of respects. Most notably, the effects were detected at much lower concentrations than the millimolar concentrations commonly used in previous studies and were strongly genotype dependent. Furthermore, the effects were highly specific to L -glutamate, not being mimicked by related amino acids such as aspartate, glutamine, and GABA, or even by the D -stereoisomer of glutamate. The authors were able to rule out the possibility that the L -glutamate treatment was causing any kind of general nutritional or metabolic disturbance to the plant. Indeed, glutamate was only inhibitory to primary root growth if it was applied directly to the primary root tip.

The specificity of the glutamate effect, its localized nature, and its occurrence at low concentrations (≥20 μM) led to the suggestion that the root tip is probably responding to changes in the apoplastic L -glutamate concentration, and that some kind of signalling mechanism is involved ( Walch-Liu et al., 2006b). Attempts to use agonists and antagonists of mammalian iGluRs to investigate the possible involvement of AtGLR-encoded glutamate receptors proved unfruitful: the antagonists tested [DNQX, MK-801, and 2-amino-5-phosphonopentanoate (AP-5)] neither suppressed the L -glutamate effect nor had any direct effect on root growth ( Walch-Liu and Forde, 2007). Furthermore, although the agonist BMAA was inhibitory to root growth, the root phenotype it produced was very different from that seen with L -glutamate ( Walch-Liu and Forde, 2007).


Conclusion

Glutamate plays a critical role in the central metabolism of many organisms, including nitrogen assimilation, amino acid biosynthesis, and cofactor production. It is also involved in the production of secondary metabolites such as antibiotics. Whether the large intracellular pool of glutamate present in many organisms evolved to support a wide array of processes, or whether these processes evolved to make use of the large pool of glutamate is unclear. It is possible that the utilization of glutamate in these various processes is adaptation of ancient non-enzymatic processes that relied on the unique capability of glutamate and aspartate to form cyclic anhydrides. Regardless of the evolutionary history, increased understanding of the roles of glutamate could allow for the engineering of processes such as bioremediation of industrial contaminates and the production of important compounds in heterologous hosts.


Glutamate metabolism

As can be seen from Fig. 1, there is little doubt that glutamate is a central molecule in amino acid metabolism in higher plants. The α-amino group of glutamate is directly involved in both the assimilation and dissimilation of ammonia and is transferred to all other amino acids. In addition, both the carbon skeleton and α-amino group form the basis for the synthesis of γ-aminobutyric acid (GABA), arginine, and proline. It should also be noted that glutamate is the precursor for chlorophyll synthesis in developing leaves ( Yaronskaya et al., 2006). The biochemistry and molecular biology of glutamate metabolism have been reviewed previously ( Lea and Miflin, 2003 Suzuki and Knaff, 2005), and this paper provides an overview of the enzymes involved in the metabolism of glutamate as shown in Fig. 1, focusing on advances that have been made in the past few years.

Pathways of glutamate synthesis and metabolism in plants.

Pathways of glutamate synthesis and metabolism in plants.

Glutamine synthetase–glutamate synthase

The key enzyme involved in the de novo synthesis of glutamate is glutamate synthase, also known as glutamine:2-oxoglutarate amidotransferase (GOGAT). The reaction is a reductant-driven transfer of the amide amino group of glutamine to 2-oxoglutarate to yield two molecules of glutamate. The enzyme in plants is present in two distinct forms, one that uses reduced ferredoxin (Fd) as the electron donor (EC 1.4.7.1) and one that uses NADH as the electron donor (EC 1.4.1.14). The Fd-dependent enzyme is normally present in high activities in the chloroplasts of photosynthetic tissues, where it is able to utilize light energy directly as a supply of reductant. The NADH-dependent enzyme, which is also present in plastids, is located predominantly in non-photosynthesizing cells, where reductant is supplied by the pentose phosphate pathway ( Bowsher et al., 2007). The Fd- and NADH-dependent forms would appear to be expressed differently in separate plant tissues. However, the situation is now more complex, as evidence is accumulating to suggest that in most plants there are two genes that encode each form of glutamate synthase. Two recent reviews have covered the early history of the discovery of the two enzymes, their structure, and gene regulation ( Lea and Miflin, 2003 Suzuki and Knaff, 2005). In this volume, Tabuchi et al. (2007) and Cánovas et al. (2007) discuss the role of glutamate synthase in rice and conifers. Clear evidence of perturbations in amino acid metabolism have been demonstrated in plants that have reduced amounts of either form of enzyme activity, caused by mutation or gene knockout ( Somerville and Ogren, 1980 Blackwell et al., 1988 Leegood et al., 1995 Ferrario-Méry et al., 2002a, b Lancien et al., 2002).

The nitrogen-containing substrate for the glutamate synthase reaction is provided in the form of glutamine. This itself is synthesized in the ATP-dependent combination of glutamate and ammonia catalysed by glutamine synthetase (GS EC 6.3.1.2). The ammonia may have been generated by direct primary nitrate assimilation, or from secondary metabolism such as photorespiration ( Leegood et al., 1995). GS activity is located in both the cytoplasm and chloroplasts/plastids in most higher plants, except conifers. The enzyme proteins can be readily separated by standard chromatographic localization and western blotting techniques into cytoplasmic (GS1) and plastidic (GS2) forms. However, this distinction is not as simple as was first thought, as although only one gene has been shown to encode the plastidic form, a small family of up to five genes is now known to encode the cytoplasmic form. Recent information on the regulation of the genes encoding GS in conifers ( Cánovas et al., 2007), maize ( Hirel et al., 2007), and rice ( Tabuchi et al., 2007) is included in this volume. Limited success in the improvement of growth rates has been demonstrated following the overexpression of GS1 genes in Lotus corniculatus, poplar trees, tobacco, and maize ( Martin et al., 2006 Tabuchi et al., 2007). Early mutants of barley lacking chloroplastic GS2 exhibited a severe phenotype and were only able to grow under non-photorespiratory conditions ( Blackwell et al., 1988 Leegood et al., 1995). However, more recently, GS1 knockout lines have been isolated and characterized in both rice and maize. The growth rate, spikelet number, and weight were considerably reduced in a homozygous knockout line of OsGS11 in rice ( Tabuchi et al., 2005). In contrast, maize mutants with knockouts of the gln1-3 and gln1-4 genes appeared to grow normally until grain filling ( Martin et al., 2006). However, there was a reduction of kernel size in gln1-4 and of kernel number in gln1-3 mutants. In the gln1-3/1-4 double mutant, a cumulative effect of the two mutations was observed ( Martin et al., 2006). The data from these two experiments clearly show that individual GS1 proteins have a non-redundant role in plant metabolism.

Glutamate dehydrogenase

The two enzymes involved in glutamate synthesis discussed previously catalyse irreversible reactions. A third enzyme, glutamate dehydrogenase (GDH EC 1.4.1.2), catalyses a reversible amination/deamination reaction, which could lead to either the synthesis or the catabolism of glutamate. During the last 33 years, the role of GDH in glutamate metabolism in plants has been the subject of continued controversy ( Oaks and Hirel, 1985 Dubois et al., 2003 Tercé-Laforgue et al., 2004b). However, following recent investigations into the regulation of the genes encoding the enzyme protein, the presence of overexpressing and antisense lines and the use of nuclear magnetic resonance (NMR) and gas chromatography–mass spectrometry (GC-MS) techniques, the role is becoming clearer. Very recently, Masclaux-Daubresse et al. (2006) have reconfirmed that in both young and old tobacco leaves, glutamate is synthesized via the combined action of GS and glutamate synthase, whilst GDH is responsible for the deamination of glutamate.

GDH is located in the mitochondria, and on occasion the cytoplasm, within the phloem companion cells of shoots ( Tercé-Laforgue et al., 2004a Fontaine et al., 2006). GDH extracted from most plant species can be readily separated into seven isoenzymic forms following native gel electrophoresis ( Thurman et al., 1965 Loulakakis and Roubelakis-Angelakis, 1996). The reason for this is that GDH comprises two distinct subunits (α and β) that are able to assemble, apparently at random, into enzymatically active hexamers. The relative proportion of the α- and β-subunits and hence the isoenzyme pattern observed varies with plant organ and nitrogen source ( Loulakakis and Roubelakis-Angelakis, 1991 Turano et al., 1997). The α-subunit is encoded by GDHA in Nicotiana plumbaginifolia and GDH2 in Arabidopsis, and the β-subunit by GDHB in N. plumbaginifolia and GDH1 in Arabidopsis ( Purnell et al., 2005).

Antisense and mutant lines of both genes in tobacco and Arabidopsis have been constructed, but a full metabolic analysis of the plants has not yet been carried out ( Fontaine et al., 2006). Tobacco lines overexpressing the GDH β-subunit exhibited a greater capacity to catabolize glutamate but were not able to assimilate ammonia, when GS was inhibited ( Purnell and Botella, 2007). GDH activity has long been known to increase during senescence and following the application of a range of stresses. It has recently been shown that high NaCl induces the formation of reactive oxygen species, which in turn induces the synthesis of the α-subunit of GDH in tobacco and grapevine. When GS was inhibited, there was evidence of incorporation of ammonia via GDH into [ 15 N]glutamate and [ 15 N]proline in the presence of high salt ( Skopelitis et al., 2006).

Glutamate as a substrate for aminotransferase reactions

Following the formation of glutamate, the α-amino group can be transferred to a wide variety of 2-oxo acid acceptors to form amino acids, and similarly the α-amino group can be transferred back to form glutamate when 2-oxoglutarate and other amino acids are available. The reactions are carried out by reversible pyridoxal-5′-phosphate-containing enzymes termed aminotransferases (EC 2.6.1.x), also known as transaminases. It is probably the reversibility of these enzyme reactions that accounts for the relative stability of the glutamate concentration found in plants, which will be discussed in a later section. Studies on the substrate specificity of aminotransferases in the past have been confused by the use of impure plant extracts however, there is now substantial evidence that aminotransferases can exhibit wide preferences for both the amino acid and oxo acid substrate. Following the publication of the genome sequence of Arabidopsis thaliana, it was calculated that there were 44 genes that encoded aminotransferases ( Liepman and Olsen, 2004). Such a large number of enzymes is clearly beyond the scope of this article, and for this reason only a few reviews and key papers will be discussed.

The α-amino group of glutamate may be transferred to oxaloacetate to form aspartate by aspartate aminotransferase (EC 2.6.1.1). Aspartate is a precursor of asparagine ( Lea et al., 2007) and the aspartate family of amino acids, lysine, threonine, methionine, and isoleucine ( Azevedo et al., 2006). Aspartate aminotransferase also plays a key role in C4 photosynthesis ( Edwards et al., 2004). In Arabidopsis there are five distinct forms of aspartate aminotransferase that are located in the cytosol, mitochondria, plastids, and peroxisomes ( Wadsworth, 1997 Liepman and Olsen, 2004). Very recently, a plastid-localized aspartate aminotransferase has been identified that is unrelated to other plant forms of the enzyme, but is similar to a prokaryotic enzyme ( De la Torre et al., 2006).

The α-amino group of glutamate may also be transferred to pyruvate to form alanine by the action of alanine aminotransferase (EC 2.6.1.2). Alanine synthesis has been shown to play a key role in the response to hypoxia/anoxia ( Ricoult et al., 2006) and in C4 photosynthesis ( Edwards et al., 2004). Four genes encoding alanine aminotransferases have been identified in Arabidopsis ( Liepman and Olsen, 2004) and Medicago truncatula ( Ricoult et al., 2006), with the enzymes being located in the cytosol, mitochondria, and peroxisomes. The transfer of amino groups to glyoxylate to form glycine in the peroxisomes in the photorespiratory nitrogen cycle ( Keys, 2006 Reumann and Weber, 2006) is an unusual case, as the reaction is considered to be unidirectional. As well as a serine:glyoxylate aminotransferase, two forms of glutamate:glyoxylate aminotransferase (GGAT) are present in the peroxisome. Recombinant GGT1 was shown to have glutamate:glyoxylate, alanine:glyoxylate, glutamate:pyruvate, and alanine:2-oxoglutarate activities ( Liepman and Olsen, 2004 Reumann and Weber, 2006). Aminotransferases involved in the metabolism of GABA, proline, and arginine will be discussed in later sections. At least six aminotransferases are involved in the synthesis and metabolism of branched chain amino acids ( Binder et al., 2007) and two in the formation of histidinol phosphate in the synthesis of histidine ( Mo et al., 2006). Very recently, a totally new aminotransferase capable of converting tetrahydrodipicolinate to LL -diaminopimelate, bridging three enzyme reactions, has been identified in the lysine synthetic pathway of Arabidopsis ( Hudson et al., 2006).

GABA, arginine, and proline

Glutamate may be converted to GABA by the irreversible action of glutamate decarboxylase (GAD EC 4.1.1.15) in the cytoplasm. GAD was initially shown to have a low pH optimum of <6.0. However, it is now known that the enzyme protein has a Ca 2+ /calmodulin-binding site at the C-terminus ( Zik et al., 2006), which allows the enzyme to be stimulated in the presence of Ca 2+ /calmodulin at pH values >7.0. GABA accumulates in higher plants following the onset of a variety of stresses such as acidification, oxygen deficiency, low temperature, heat shock, mechanical stimulation, pathogen attack, and drought ( Shelp et al., 1999 Bouché and Fromm, 2004 Bown et al., 2006). Shelp et al. (2006) have presented evidence that GABA is involved in communication between plants and animals, fungi, bacteria, and other plants. Very recently, Lancien and Roberts (2006) have also shown that GABA can down-regulate the expression of genes encoding 14-3-3 proteins in a calcium-, ethylene-, and abscisic acid-dependent manner.

Glutamate is the precursor of arginine and is metabolized via acetylated derivatives to ornithine, citrulline, and arginosuccinate in a nine-step process ( Slocum, 2005). Arginine has a high N:C ratio (4:6), and along with asparagine (2:4) acts as a major nitrogen storage compound in higher plants, where it occurs in both the protein and soluble form. Arginine plays a key metabolic role in seed maturation and germination, phloem and xylem transport, particularly in conifer trees, and accumulates under stress and deficiency conditions ( Lea et al., 2007). Arginine and ornithine may also act as precursors of polyamines, which can play an important role in the response of plants to stress ( Alcázar et al., 2006). Early studies indicated that the major control point of arginine synthesis was at N-acetylglutamate kinase (NAGK EC 2.7.2.8), the activity of which was inhibited by arginine and activated by N-acetylglutamate. There is now strong evidence that this control is mediated through the binding of PII, a 2-oxoglutarate- and amino acid-sensing protein ( Lam et al., 2006), which is able to relieve the inhibition of NAGK by the end-product arginine ( Sugiyama et al., 2004 Chen et al., 2006 Ferrario-Méry et al., 2006).

Glutamate can act as the precursor of proline in a pathway that requires three enzyme-catalysed reactions and a spontaneous chemical reaction. Proline is a cyclic amino acid that has been shown to accumulate following a wide range of abiotic stresses such as that induced by drought, salinity, low and high temperatures, and heavy metals ( Munns, 2005 Sharma and Dietz, 2006). There is also evidence that proline is a major component of the nitrogen transport stream in both the xylem and phloem ( Brugière et al., 1999). The bifunctional enzyme protein Δ 1 -pyrroline-5-carboxylate synthetase (P5CS) catalyses two reactions (EC 2.7.2.11 and EC 1.2.1.41), the first of which, the phosphorylation of glutamate, is subject to feedback inhibition by proline ( Delauney and Verma, 1993 Strizhov et al., 1997 Hong et al., 2000 Armengaud et al., 2004).


<p>This section provides any useful information about the protein, mostly biological knowledge.<p><a href='/help/function_section' target='_top'>More. </a></p> Function i

Devoted to catabolic function of glutamate (and other amino acids of the glutamate family) utilization as sole nitrogen source. It is not involved in anabolic function of glutamate biosynthesis since B.subtilis possesses only one route of glutamate biosynthesis from ammonia, catalyzed by glutamate synthase. RocG is unable to utilize glutamate or glutamine as sole carbon source and to synthesize glutamate, but it is involved in the utilization of arginine, and proline as carbon or nitrogen source. The catabolic RocG is essential for controlling gltAB expression via an inhibitory interactions with the transcriptional regulator GltC in response to the availability of sugars.

<p>Manually curated information for which there is published experimental evidence.</p> <p><a href="/manual/evidences#ECO:0000269">More. </a></p> Manual assertion based on experiment in i


DISCUSSION

This randomized cross-over trial in dietary-controlled, healthy subjects revealed that MSG added at nutritional doses to a standard diet elicited an antral distension and an elevation of plasma concentrations of several amino acids, including branched-chain amino acids, in the early postprandial phase. These physiological and metabolic effects were not associated with significant change in the metabolic fate of dietary N, the postprandial glucose and hormonal responses, and the gastrointestinal sensations of hunger and fullness.

The gastric distension following ingestion of a liquid, balanced meal providing approximately one-third of the daily energy intake was more pronounced in subjects receiving MSG at doses close to that commonly consumed than an equivalent dose of NaCl as control. The monitoring of antral area by two-dimension real-time ultrasound before and at regular intervals following the ingestion of the test meal has been proposed to represent a method to assess gastric emptying (5, 22), and the specific ultrasonographic monitoring of the antrum cross-sectional area is known to accurately detect variations in gastric volume (44, 48). MSG ingestion elicited an 18% higher AUC of antrum for the first 3 h after the meal ingestion. However, it is not obvious that the antral distension observed in our study after the MSG-supplemented meal really reflected a change in meal gastric emptying. Indeed, our results may rather be explained as a MSG-induced enhanced gastric secretion without increased gastric emptying resulting in increased antral distension. Our results are in contrast with that of the only available study that has evaluated the effect of MSG supplementation on gastric emptying using an oral dose of [ 13 C]sodium acetate (58). Using this latter method, the authors reported an acceleration of gastric emptying after MSG supplementation added to a protein-rich liquid diet, but no effect was observed when MSG was added to a carbohydrate-rich diet or to the same volume of water. Different hypotheses can be advanced to explain the discrepancy between Zai et al.'s (58) findings and the present results. We used a meal that contained more energy (700 vs. 400 kcal) and represented a larger volume (600 vs. 400 ml) than the meal used in their study, and both parameters are likely to influence gastric emptying (28). The concentration of MSG was lower in our study (16 vs. 30 mmol/l). Moreover, the protein source used in the study by Zai et al. was casein, whereas we used total milk proteins (35). Last but not least, as reported above, Zai et al. used [ 13 C]sodium acetate administration to measure gastric emptying while we used ultrasonographic monitoring of the antrum area.

Animal studies have been performed to assess the direct effect of MSG supplementation on gastric emptying or intestinal peristaltism. Toyomasu et al. (54) have shown stimulation of the upper gut motility through the vagus nerve after intragastric MSG stimulation, an effect that was associated with an acceleration of the gastric emptying rate. Furthermore, injection of MSG in stomach, duodenum, and portal vein increases gastric vagal afferent activity in rats (42, 56). However, it is worth noting that experimental conditions in animal and human studies are often very different, thus complicating comparison on the effects of MSG in these situations.

An interesting finding of the present study was the transient rise in plasma concentrations of many amino acids when subjects ingested the MSG-supplemented meal. This effect concerned in particular branched-chain amino acids, with significant 20–25% increases of circulating concentrations of leucine and isoleucine at 1 and 2 h after the meal. Branched-chain amino acid systemic availability has consistently been found to be increased in animals supplemented with MSG (24, 26, 30). This effect suggests a sparing effect of MSG on branched-chain amino acid first-pass uptake, through the implication of glutamate in reducing branched-chain amino acid transamination. Recently, enterocytes isolated from pig jejunum have been shown to extensively transaminate branched-chain amino acids, a phenomenon that is stimulated by α-ketoglutarate (8). In humans, leucine first-pass uptake may represent 13–37% of the dietary leucine intake (2, 10, 21, 23, 38), and it is conceivable that decreased transamination might translate into elevation of plasma concentrations. Increased postprandial glutamatemia, although of mild amplitude (11% increase of the postprandial AUC), is in line with previous results obtained in humans (51, 52).

We also found an increase in other amino acids that are derived from glutamate, including ornithine, citrulline, and alanine, in accord with other studies (24, 26, 30, 53) and with the known capacity of isolated enterocytes to convert glutamate into ornithine and citrulline and to allow pyruvate transamination into alanine (3). In contrast, this study did not demonstrate a significant increase in glutamine, aspartate, proline, or arginine as previously reported (24, 51). Last, since the catabolism of cysteine and tyrosine involves transamination with α-ketoglutarate being converted to glutamate, it is conceivable that glutamate through a mere mass-action phenomenon would participate in the sparing of these amino acids and thus in their increased plasma concentration. Data obtained in pigs from arteriovenous differences in tyrosine plasma concentrations suggest that this amino acid is little degraded by the intestine in this experimental model (57). Regarding cysteine catabolism, from the low appearance of this amino acid in the portal blood (<20% of dietary intake), it has been suggested that piglet intestine extensively utilizes cyst(e)ine (49), a suggestion that is in line with the capacity of isolated enterocytes to catabolize this amino acid (9).

By contrast with the effects on aminoacidemia, we could not evidence any detectable difference in the postprandial metabolic fate of dietary proteins, whether the subjects ingested the meal with or without MSG. The 15 N-labeled protein was used in the experimental meals because we have previously shown that the postprandial appearance of dietary N in pools such as plasma amino acids or urea is very sensitive to the kinetics of intestinal delivery of dietary proteins (6, 13, 16, 29). Because the measure of postprandial dietary N retention gives a global picture of protein metabolism, we cannot infer from our results that protein synthesis and/or degradation rates were unaffected by the dietary treatment but only that the net utilization of dietary N was similar. The discrepancy between the MSG-induced increase in plasma amino acids and the absence of effect of MSG on dietary N appearance in plasma amino acids and urea indicates that the improved systemic availability of amino acids may rather be the result of a modified first-pass uptake than the result of changes in protein fluxes. However, it would be necessary to perform studies with tracers to clarify this point.

GLP-1 and ghrelin are two gastrointestinal hormones that are potent regulators of gastric emptying (18). However, in this study, postprandial GLP-1 and ghrelin concentrations were not significantly influenced by dietary MSG, and there was no clear association between these hormones and the functional response observed at the gastric level. The glucose and insulin postprandial responses were also unaffected by MSG, which is in accord with the GLP-1 results. Collectively, our results are in agreement with the only study that has assessed the effect of MSG (0.6%) administration to a high-protein meal on satiety, energy intake, and hormone circulating concentrations in humans (33). Accordingly, the MSG ingestion was not associated with any changes to the postprandial gastrointestinal sensations (fullness, hunger). Despite crossover design, our study may have lacked power to detect subtle changes in such sensations because visual analog scales represent a technique with high intersubject variability. Moreover, if modifications of the gastric volume are one component that intervenes in the sensations of fullness and hunger after a meal, they are not always sufficient to induce changes to satiety and hunger feelings (45).

In conclusion, we show that MSG supplementation at nutritional doses elicits in healthy humans a postprandial gastric distension and an elevation of several amino acid plasma concentrations that are possibly linked. These physiological and metabolic effects had, however, no measurable consequences in terms of satiety, hormone profiles, or dietary N metabolism. Thus, it can be hypothesized that the gastric distension did not translate into a delay of gastric emptying. The mechanisms involved in such distension are not known but may involve an increased water or acid secretion in the stomach in response to MSG supplementation. A recent study showing the stimulation of gastric acid secretion by MSG in dogs is in accordance with this hypothesis (59). Moreover, intraduodenal, but not intrajejunal, infusion of amino acids stimulates gastric secretion (32). Intravenous infusion of amino acids is also able to have this latter effect and acts on both acid and pepsin secretion (27). Our findings call for further studies to elucidate the mechanisms by which dietary glutamate impact systemic amino acid concentrations and the consequences of such modulations. Last, it is worth noting that, in the present study, amino acid concentrations were measured in blood plasma but not in erythrocytes, which can contribute to the interorgan transfer of amino acids (14). However, although some amino acids are highly concentrated in erythrocytes, it is generally considered that the exchanges with plasma proceed very slowly, and thus this intracellular pool is considered to contribute poorly to interorgan fluxes.