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I am considering nitrogen fixation and in my lecture notes, there is the statement
The glutamine synthetase- glutamate synthase system requires use of an ATP molecule as well as reducing power. Although it is energy intensive compared with glutamine synthase, the Km of glutamine synthetase is much lower than that of glutamate dehydrogenase. When NH3 is scarce, the additional ATP investment is worthwhile.
Try as I might, I can't find anything about glutamine synthase. Searches online seem to only return results for glutamine synthetase. I was wondering if this is a mistake in my notes? If so, I can only think that this should be 'glutamate dehydrogenase', in which case the statement is about comparing the method used by animals and fungi (which use glutamate dehydrogenase to fix nitrogen via a Schiff base) compared with plants and other microorganisms (which use the glutamine synthetase glutamate synthase method).
This is my first exposure to learning about nitrogen fixation, so I would very much are coated any clarification.
Also, I was wondering what the general difference is between a 'synthase' enzyme and 'synthetase', if there is one.
Probably a mistake in your notes, or in the lecture notes for the class, but not a very important error in either case aside from the confusion it has caused you.
See the wikipedia article on Synthase and on wikipedia article on Ligase for a note on the terminology: historically synthetases use ATP, synthases do not, but this terminology is not adhered to strictly, and the current standard is that the terms are essentially synonymous; in my experience, however, a particular nomenclature dominates the literature even if the synonym is technically correct.
I'll deal with the second question first. There is a difference between synthetases and synthases, but because of the confusion the International Enzyme Commission now discourages the use of the term synthetase. The section on ligases states (my emphasis):
Class 6. Ligases. Ligases are enzymes catalysing the joining together of two molecules coupled with the hydrolysis of a diphosphate bond in ATP or a similar triphosphate. The systematic names are formed on the system X:Y ligase (ADP-forming). In earlier editions of the list the term synthetase has been used for the common names. Many authors have been confused by the use of the terms synthetase (used only for Group 6) and synthase (used throughout the list when it is desired to emphasis the synthetic nature of the reaction). Consequently NC-IUB decided in 1983 to abandon the use of synthetase for common names, and to replace them with names of the type X-Y ligase. In a few cases in Group 6, where the reaction is more complex or there is a common name for the product, a synthase name is used (e.g. EC 184.108.40.206 and EC 220.127.116.11). It is recommended that if the term synthetase is used by authors, it should continue to be restricted to the ligase group.
But the old nomenclature dies hard.
As regards your first question, this is an obvious mistake, as others have pointed out. For the record, I post diagrams from my own lectures of the two alternative methods of aminating ketoacids (oxo-acids).
This short review outlines the central role of glutamine synthetase (GS) in plant nitrogen metabolism and discusses some possibilities for crop improvement. GS functions as the major assimilatory enzyme for ammonia produced from N fixation, and nitrate or ammonia nutrition. It also reassimilates ammonia released as a result of photorespiration and the breakdown of proteins and nitrogen transport compounds. GS is distributed in different subcellular locations (chloroplast and cytoplasm) and in different tissues and organs. This distribution probably changes as a function of the development of the tissue, for example, GS1 appears to play a key role in leaf senescence. The enzyme is the product of multiple genes with complex promoters that ensure the expression of the genes in an organ‐ and tissue‐specific manner and in response to a number of environmental variables affecting the nutritional status of the cell. GS activity is also regulated post‐translationally in a manner that involves 14‐3‐3 proteins and phosphorylation. GS and plant nitrogen metabolism is best viewed as a complex matrix continually changing during the development cycle of plants. Along with GS, a number of other enzymes play key roles in maintaining the balance of carbon and nitrogen. It is proposed that one of these is glutamate dehydrogenase (GDH). There is considerable evidence for a GDH shunt to return the carbon in amino acids back into reactions of carbon metabolism and the tri‐carboxylic acid cycle. Results with transgenic plants containing transferred GS genes suggest that there may be ways in which it is possible to improve the efficiency with which crop plants use nitrogen. Marker‐assisted breeding may also bring about such improvements.
The improvement of nitrogen use efficiency, particularly in cereals, is a major goal of crop improvement. Such improved crops would make better use of the nitrogen fertilizer supplied they would also produce higher yields with better protein content. This might be achieved, at least in part, by a better understanding of nitrogen metabolism and its regulation, and by identifying likely target genes for manipulation by either direct gene transfer or marker‐assisted breeding.
Frontotemporal dementia (FTD) is a leading type of early-onset dementia and involves progressive brain atrophy largely affecting the frontal and temporal lobes of the brain [1, 2]. While FTD has conventionally been regarded as a syndrome characterized by behavioural and cognitive perturbations, a critical involvement of cerebral metabolic alterations in FTD pathology is now being increasingly recognized [3,4,5].
Normal brain function requires a large and continuous supply of oxygen and glucose for energy production used mainly to satisfy the high demand of electrical activity and synaptic functions in neurons . Moreover, strict regulation of glutamate and glutamine metabolism is vital both for energy homeostasis and for excitatory neurotransmission . Several neurodegenerative disorders are associated with critical metabolic impairments including diminished glucose uptake and utilization, as well as hampered mitochondrial activity, with a subsequent decrease in ATP production even preceding the presentation of major pathophysiological phenotypes and symptoms [3, 8,9,10,11]. Hence, hypometabolism of glucose detected by positron emission tomography has been utilized as a reliable biomarker of disease progression in both patients and animal models of neurodegeneration . Nonetheless, the changes in the metabolic landscape at a cellular level in such neurodegenerative disorders remain poorly characterized.
Most FTD cases are sporadic, yet approximately 20–30% is genetically linked. Mutations in specific genes associated with such familial FTD cases have been identified . Amongst these, the CHMP2B gene encoding charged multivesicular body protein 2B located on chromosome 3 and causative for FTD3 is of particular interest . CHMP2B is a component of the “Endosomal Sorting Complex Required for Transport III” (ESCRT-III) involved in endo-lysosomal trafficking . Thus, in FTD3, a dominant gain-of-function mutation of CHMP2B affects endo-lysosomal-mediated functions such as recycling or degradation of cell surface receptors. Even though mutation carriers can be easily identified, there is currently no therapy to cure, halt or even decelerate disease progression. This is in part reflective of a paucity of human disease models with which to dissect the mechanisms underlying FTD3 pathogenesis. In an attempt to meet such a need, we have recently developed a human disease model using human induced pluripotent stem cells (hiPSCs) from patients carrying mutations in CHMP2B and isogenic controls generated via the CRISPR/Cas9 system with subsequent differentiation into functional neurons expressing typical markers for the frontal and temporal lobe . Pathologies observed in patients and animal models such as dysregulation of the endosome-like structure functionality [15, 16] were validated in our model. Furthermore, our results revealed novel disease-relevant phenotypes including abnormal mitochondrial morphology and function . We therefore sought to gain a more comprehensive understanding of the molecular pathogenesis in FTD3 and to identify key cellular changes in neuronal and glial energy metabolism. Using an integrative approach comprising quantitative proteomics, metabolic mapping via stable isotope 13 C-labeled energy substrates and gas chromatography coupled to mass spectrometry, as well as live-cell bioenergetics analysis, we have identified a constellation of metabolic changes that lead us to conclusively identify two major enzymes linking neurotransmitter and energy metabolism as crucial players in FTD3 pathology.
Martin Kohlmeier , in Nutrient Metabolism , 2003
Digestion and absorption
Denaturation and hydrolysis of Gln-containing proteins begins with mastication of foods in the mouth and continues in the stomach under the influence of hydrochloric acid, pepsin A (EC18.104.22.168), and gastricin (22.214.171.124). Several pancreatic and brush border enzymes continue protein hydrolysis, though none specifically cleaves peptide bonds adjacent to Gln.
Gln is taken up from the small intestinal lumen mainly via the sodium-amino acid cotransport system Ba ° (ASCT2 Avissar et al., 2001 Bode, 2001 ). The sodium-independent transport system b °,+ , comprised of a light subunit BAT1 (SLC7A9) and a heavy subunit rBAT (SLC3A1), exchanges Gln for another neutral amino acid. Gln as a component of di- or tripeptides can also be taken up via the hydrogen ion/peptide cotransporters 1 (SLC15A1, PepT1) and 2 (SLC15A2, PepT2).
Figure 8.11 . Intestinal absorption of L- glutamine
Export across the basolateral membrane uses the sodium-amino acid cotransport systems ATA2 ( Sugawara et al., 2000 ) and the sodium-independent transporter heterodimers LAT2 + 4F2 (SLC7A8 + SLC3A2), y + LAT1 + 4F2 (SLC7A7 + SLC3A2), y + LAT2 + 4F2 (SLC7A6 + SLC3A2).
Starvation increases expression of the transport systems A and L, whereas ASC and non-mediated uptake are not affected ( Muniz et al., 1993 ).
Cooperativity in Enzymes (With Diagram)
When enzymes contain more than one active site, the binding of a substrate molecule to the first site may influence substrate binding to a second site.
Binding of the second substrate may influence binding of a third, and so on. This phenomenon is called cooperativity.
The influence may be positive in that binding of the first substrate molecule facilitates binding of subsequent substrate molecules (called “positive cooperativity”) or the influence may be negative in that binding of a second or subsequent substrate molecule occurs less readily than binding of the first (called “negative cooperativity”).
In a sense, the substrate itself is acting as either a positive or negative effector for the enzyme. These relationships are depicted in Figure 8-24.
Cooperative effects are not restricted to enzymes but are observed with other proteins. We considered the cooperativity that exists among the globin chains of hemoglobin, a cooperativity that facilitates successive binding of oxygen molecules to the alpha and beta globin chains (i.e., positive cooperativity).
Both cooperative effects involving active sites on neighboring subunits of an enzyme and true allosteric effects involving regulatory sites may occur in a single enzyme molecule. A case in point is that of cytidine triphosphate synthetase, an enzyme involved in nucleic acid metabolism and consisting of four sub- units.
Two of these subunits contain active sites that bind the substrate glutamine and the other two have regulatory sites that bind GTR When glutamine is bound to the active site of one subunit, a conformational change transmitted through the enzyme to another subunit renders the latter’s active site unable to bind to glutamine (i.e., negative cooperativity).
On the other hand, when GTP is bound to the regulatory site of one subunit, this has a positive effect on glutarnine binding but it negatively affects GTP binding at the other regulatory site. In other words, the effector GTP serves to activate catalysis but to inhibit further GTP binding.
Through comparative analyses of gene expression data of 11 types of cancer, we computationally predicted how glutamine and glutamate contribute to cancer biology. Specifically, we observed that (i) the increased influx of glutamine in cancer is mainly due to up-regulated importers, whereas increased influx of glutamate is due to both increased conversion from glutamine and increased uptake, depending on specific types of cancer (ii) glutamine and glutamate metabolisms are mostly increased in cancer, and the level of change in glutamine strongly correlates with the 5-year survival rate (iii) our analyses in terms of the levels of statistical contributions by glutamine and/or glutamate to seven pathways reveal the following novel information: (1) glutamine generally does not contribute to purine synthesis in cancer except for BRCA, similarly not to pyridine synthesis except for KIRC (2) glutamine generally does not contribute to ATP production in cancer (3) the contribution to nucleotide synthesis by glutamine is minimal if any in cancer (4) glutamine does not contribute to asparagine synthesis in cancer except for BLCA and LUAD and (5) glutamate generally does not contribute to serine synthesis except for BLCA and (iv) strong correlations between increased glutamine and glutamate metabolisms and increased ROS level suggest an anti-oxidation function of glutamine and glutamate.
Different from cell line-based studies, our analysis was conducted on gene expression data of cancer and control tissues. Hence, the analysis results offered a more accurate reflection of the functional roles played by glutamine and glutamate in cancer. In the meantime, tissue-based gene expression data are considerably more complex than cell line data, as the observed gene expression data have contributions from non-cancer cells, such as immune cells, stromal cells, and fat cells, which clearly raises an issue of how reliable the estimated results are, particularly when the percentage of cancer cells in different tissues may vary, in some cases substantially. Knowing this information, we have to note that the present study is limited by the complications of multiple types of cells in cancer tissues, because subtle changes in terms of differential expression may not be detectable using our present analyses of the tissue-based data. Hence up- or down-regulated genes should be considered to be substantially up- or down-regulated. To overcome this limitation, further studies to tease out the true expression of cancer cells in the tissue data are necessary, before we could detect more subtle changes within cancer cells.
Regulation of hepatic glutamine and glutamate metabolism
Hepatic glutamine utilization is regulated by control at three key sites. Low et al. (1993) used control strength analysis to determine that glutamine (control strength 0.96) and system N transport (control strength 0.5) were important in changing the rate of glutamine utilization. However, these were countered by glutamine efflux from the cell (control strength −0.4), which could effectively decrease the rate of glutamine degradation. Thus, changing any one of these variables will bring about large changes in the rate of glutamine degradation.
Short-term stimulation of glutaminase [see Brosnan et al. (1995)] is seen in response to hormones such as glucagon or epinephrine, acting via cAMP, and vasopressin, acting via calcium. Interestingly, these increases are maintained in mitochondria isolated from liver after treatment in vivo or in vitro, but are lost on disruption of the mitochondria. Similarly, from work in isolated, perfused livers or hepatocytes, agents such as ammonia or bicarbonate (as well as increasing pH) stimulate glutamine breakdown and urea synthesis. Purified hepatic glutaminase exhibits a flat pH curve and is not affected by many of the agents that appear to regulate it in intact cells or mitochondria ( Smith and Watford 1988). Indeed, McGivan (1988) proposed that the tight association of the enzyme with the mitochondrial inner membrane is responsible for many of its kinetic characteristics in vivo. There is also extensive evidence of acute regulation of glutamine synthetase activity in intact cell or tissue preparations, with increased flux during acidosis and decreased flux at higher pH levels ( Haussinger, 1990, Lueck and Miller 1970). However, the mechanisms involved have not yet been defined, and such changes have not been demonstrated in vivo (see below).
The long-term regulation of rat hepatic glutaminase is well documented ( Curthoys and Watford 1995). Increased activity occurs in diabetes, starvation and with the feeding of high protein diets, whereas decreased activity occurs with the feeding of low protein diets. To date, all such changes suggest transcriptional regulation of the glutaminase gene to be the major control point. Studies with the promoter region 5′ to the hepatic glutaminase gene confirmed that dexamethasone was a strong inducer of expression in HepG2 cells ( Chung-Bok et al. 1997), with the response requiring a region −252 to −103 upstream of the start site of transcription. Surprisingly, in contrast to the evidence from experiments in vivo, ammonium chloride repressed gene expression ( Chung-Bok and Watford 1997), whereas glucagon and cAMP analogs did not affect the activity of the promoter. However, this negative finding may be due to the short (1000 bp) promoter sequence analyzed.
Hepatic glutamine synthetase activity appears unresponsive to physiologic and pathophysiologic changes in vivo (it is unchanged in diabetes, acidosis or after changes in dietary protein intake), although a slight increase has been reported in starvation (Arola et al. 1991). The activity is lower in hypophysectomized, adrenalectomized and thyroidectomized rats, and can be restored by growth hormone, glucocorticoids or thyroid hormones (respectively), but these hormones have no effect in intact animals [see Lie-Venema (1997)]. Hepatic glutamine synthetase activity is decreased in conditions of liver atrophy brought about by energy or protein restriction, but the effect follows from an interesting mechanism, i.e., a decrease in the number of glutamine synthetase–positive cells [rather than a reduction in enzyme activity within the cells themselves see Lie-Venema (1997)].
Glutamine transport on system N has been proposed to be a major control site for hepatic glutamine utilization. Consistent with such a role, system N is up-regulated in conditions of increased urea synthesis, such as diabetes and burn injury ( Lohmann et al. 1998, Meijer et al. 1990), and some evidence indicates that the sodium-independent system “n” is up-regulated in tumor-bearing rats ( Inoue et al. 1995). Perivenous sodium-linked glutamate transport has also been reported to decline in response to acidosis, an action at odds with the increased requirement for glutamate in such cells [ see Meijer et al. (1990)].
The enzyme glutamine synthetase is a key enzyme controlling the use of nitrogen inside cells. Glutamine, as well as being used to build proteins, delivers nitrogen atoms to enzymes that build nitrogen-rich molecules, such as DNA bases and amino acids. So, glutamine synthetase, the enzyme that builds glutamine, must be carefully controlled. When nitrogen is needed, it must be turned on so that the cell does not starve. But when the cell has enough nitrogen, it needs to be turned off to avoid a glut.
Glutamine synthetase acts like a tiny molecular computer, monitoring the amounts of nitrogen-rich molecules. It watches levels of amino acids like glycine, alanine, histidine and tryptophan, and levels of nucleotides like AMP and CTP. If too much of one of these molecules is made, glutamine synthetase senses this and slows production slightly. But as levels of all of these nucleotides and amino acids rise, together they slow glutamine synthetase more and more. Eventually, the enzyme grinds to a halt when the supply meets the demand.
Nomenclature of enzymes involved in synthesis of glutamine - Biology
Nonessential amino acids are those that are synthesized by mammals, while the essential amino acids must be obtained from dietary sources. Why would an organism evolve in such a way that it could not exist in the absence of certain amino acids? Most likely, the ready availability of these amino acids in lower organisms (plants and microorganisms) obviated the need for the higher organism to continue to produce them. The pathways for their synthesis were selected out. Not having to synthesize an additional ten amino acids (and regulate their synthesis) represents a major economy, then. Nevertheless, it remains for us to become familiar with the synthetic pathways for these essential amino acids in plants and microorganisms, and it turns out that they are generally more complicated that the pathways for nonessential amino acid synthesis and they are also species-specific.
The twenty amino acids can be divided into two groups of 10 amino acids. Ten are essential and 10 are nonessential. However, this is really not an accurate dichotomy, as there is overlap between the two groups, as is indicated in the text accompanying the following two charts:
|The Ten "Nonessential" Amino Acids|
|Cysteine (requires sulfhydryl group from methionine)|
|Tyrosine (synthesized from phenylalanine)|
Note that tyrosine is really an essential amino acid, as it is synthesized by the hydroxylation of phenylalanine, an essential amino acid. Also, in animals, the sulfhydryl group of cysteine is derived from methionine, which is an essential amino acid, so cysteine can also be considered essential.
The ten "essential" amino acids are:
|The Ten "Essential" Amino Acids|
|Arginine (see below)|
Arginine is synthesized by mammals in the urea cycle, but most of it hydrolyzed to urea and ornithine:
(Link to Dr. Diwan's webpage on Amino Acid Catabolism for more information about the hydrolysis of urea, as well as for review of amino acid catabolism)
Because mammals cannot synthesize enough arginine to meet the metabolic needs of infants and children, it is classified as an essential amino acid.
Synthesis of Nonessential Amino Acids
Ignoring tyrosine (as it's immediate precursor is phenylalanine, an essential amino acid), all of the nonessential amino acids (and we will include arginine here) are synthesized from intermediates of major metabolic pathways. Furthermore, the carbon skeletons of these amino acids are traceable to their corresponding a- ketoacids. Therefore, it could be possible to synthesize any one of the nonessential amino acids directly by transaminating its corresponding a -ketoacid, if that ketoacid exists as a common intermediate. A "transamination reaction", in which an amino group is transferred from an amino acid to the a -carbon of a ketoacid, is catalyzed by an aminotransferase .
Three very common a- ketoacids can be transaminated in one step to their corresponding amino acid:
Pyruvate (glycolytic end product) --> alanine
Oxaloacetate (citric acid cycle intermediate) --> aspartate
a- ketoglutarate (citric acid cycle intermediate) --> glutamate
The individual reactions are:
Asparagine and glutamine are the products of amidations of aspartate and glutamate, respectively. Thus, asparagine and glutamine, and the remaining nonessential amino acids are not directly the result of transamination of a -ketoacids because these are not common intermediates of the other pathways. Still, we will be able to trace the carbon skeletons of all of these back to an a -ketoacid. I make this point not because of any profound implications inherent in it, but rather as a way to simplify the learning of synthetic pathways of the nonessential amino acids.
Aspartate is transaminated to asparagine in an ATP-dependent reaction catalyzed by asparagine synthetase, and glutamine is the amino group donor:
The synthesis of glutamine is a two-step one in which glutamate is first "activated" to a g- glutamylphosphate intermediate, followed by a reaction in which NH3 displaces the phosphate group:
So, the synthesis of asparagine is intrinsically tied to that of glutamine, and it turns out that glutamine is the amino group donor in the formation of numerous biosynthetic products, as well as being a storage form of NH3 . Therefore, one would expect that glutamine synthetase, the enzyme responsible for the amidation of glutamate, plays a central role in the regulation of nitrogen metabolism. We will now look into this control in more detail, before proceeding to the biosynthesis of the remaining nonessential amino acids.
You have previously studied the oxidative deamination of glutamate by glutamate dehydrogenase, in which NH3 and a- ketoglutarate are produced. The a -ketoglutarate produced is then available for accepting amino groups in other transamination reactions, but the accumulation of ammonia as the other product of this reaction is a problem because, in high concentrations, it is toxic. To keep the level of NH3 in a controlled range, a rising level of a -ketoglutarate activates glutamine synthetase, increasing the production of glutamine, which donates its amino group in various other reactions.
The regulation of glutamine synthetase has been studied in E.Coli and, although complicated, it is worthwhile to look at some of its features because this will give us more insight into regulation of intersecting metabolic pathways. Xray diffraction of crystals of the enzyme reveals a hexagonal prism structure (D6 symmetry) composed of 12 identical subunits. The activity of the enzyme is controlled by 9 allosteric feedback inhibitors, 6 of which are end products of pathways involving glutamine:
carbamoyl phosphate (synthesized from carbamoyl phosphate synthetase II)
The other three effectors are alanine, serine and glycine , which carry information regarding the cellular nitrogen level.
The enzyme is also regulated by covalent modification (adenylylation of a Tyr residue), which results in an increase sensitivity to the cumulative feedback inhibition by the above nine effectors. Adenylyltransferase is the enzyme which catalyzes both the adenylylation and deadenylylation of E. coli glutamine synthetase, and this enzyme is complexed with a tetrameric regulatory protein, PII. Regulation of the adenylylation and its reverse occurs at the level of PII, depending upon the uridylylation of another Tyr residue, located on PII. When PII is uridylylated, glutamine synthetase is deadenylylated the reverse occurs when UMP is covalently attached to the Tyr residue of PII. The level of uridylylation is, in turn, regulated by the activities of the two enzymes, uridylyltransferase and uridylyl-removing enzyme, both located on the same protein. Uridylyltransferase is activated by a -ketoglutarate and ATP, while it is inhibited by glutamine and Pi.
The following diagram summarizes the regulation of bacterial glutamine synthetase (see text page 1035) :
We can "walk through" this regulatory cascade by looking at a specific example, namely increased levels of a -ketoglutarate ( reflecting a corresponding increase in NH3) levels:
(1) Uridylyltransferase activity is increased
(2) PII (in complex with adenylyltransferase) is uridylylated
(3) Glutamine synthetase is deadenylylated
(4) a -ketoglutarate and NH3 form glutamine and Pi
That the control of bacterial glutamine synthetase is exquisitely sensitive to the level of the cell's nitrogen metabolites is illustrated by the fact that the glutamine just produced in the above cascade is now an inhibitor of further glutamine production.
In Class Exercise: Use the regulatory pathway to explain the effect of a rising level of glutamine on the activity of bacterial glutamine synthetase.
Proline, Ornithine and Arginine are derived from Glutamate
The first step involves phosphorylation of glutamate by ATP with the enzyme g -glutamyl kinase, followed by reduction to glutamate-5-semialdehyde which spontaneously cyclizes (no enzyme required) to an internal Schiff base. The formation of the semialdehyde also requires the presence of either NADP or NADPH.
The semialdehyde is a branch point, however. One branch leads to proline while the other branch leads to ornithine and arginine. Glutamate-5-semialdehyde is transaminated to ornithine and glutamate is the amino group donor. Ornithine, a urea cycle intermediate, is converted to arginine through the urea cycle.
To further highlight the importance of glutamate, it is converted to the physiologically active amine, g -aminobutyric acid (GABA), the major inhibitory neurotransmitter in the brain:
The glycolytic intermediate, 3-phosphoglycerate, is converted to serine, cysteine and glycine.
Note the participation of glutamate as the amino group donor. Serine is converted to glycine in the following reaction:
serine + THF --> glycine + N 5 ,N 10 -methylene-THF (enzyme: serine hydroxymethyltransferase)
Glycine is also formed in a condensation reaction as follows:
N 5 ,N 10 -methylene-THF + CO2 + NH4 + --> glycine (enzyme: glycine synthase requires NADH)
Cysteine is synthesized from serine and homocysteine (methionine breakdown product):
ser + homocysteine -> cystathionine + H2O
cystathionine + H2O --> a -ketobutyrate + cysteine + NH3
Synthesis of Essential Amino Acids
The synthetic pathways for the essential amino acids are:
(1) present only in microorgansims
(2) considerably more complex than for nonessential amino acids
(3) use familiar metabolic precursors
For purposes of classification, consider the following 4 "families" which are based upon common precursors:
(1) Aspartate Family : lysine, methionine,threonine
(2) Pyruvate Family : leucine, isoleucine, valine
(3) Aromatic Family : phenylalanine, Tyrosine, Tryptophan
The first committed step for the synthesis of Lys, Met and Thr is the first step, in which aspartate is phosphorylated to aspartyl- b -phosphate, catalyzed by aspartokinase :
E.coli has 3 isozymes of aspartokinase that respond differently to each of the 3 amino acids, with regard to enzyme inhibition and feedback inhibition. The biosynthesis of lysine, methionine and threonine are not, then, controlled as a group.
The pathway from aspartate to lysine has 10 steps.
The pathway from aspartate to threonine has 5 steps
The pathway from aspartate to methionine has 7 steps
Regulation of the three pathways also occurs at the two branch points:
b -Aspartate-semialdehyde (homoserine and lysine)
Homoserine (threonine and methionine)
The regulation results from feedback inhibition by the amino acid products of the branches, indicated in the brackets above.
We will consider one important step in the synthesis of this group of 3 amino acids, namely the step in which homocysteine is converted to methionine, catalyzed by the enzyme methionine synthase :
In this reaction, homocysteine is methylated to methionine, and the C1 donor is N 5 -methyl-THF. Note that the enzyme is called a "synthase" rather than a synthetase, because the reaction is a condensation reaction in which ATP (or another nucleoside triphosphate) is not used as an energy source. This is to be compared to a "synthetase" in which an NTP is required as an energy source.This reaction can also be looked at as the transfer of a methyl group from N 5 -methyl-THF to homocysteine, so another name for the enzyme catalyzing it is homocysteine methyltransferase.
It is reasonable to review reactions in which a C1 unit is added to a metabolic precursor , as these reactions are seen very commonly in our study of biochemical pathways. You have already seen the transfer of a carboxyl group from the biotin cofactor of pyruvate carboxylase to pyruvate to form oxaloacetate (why isn't this called a "transferase" or a "synthase"?). Most carboxylation reactions use biotin as a cofactor. You have also studied methionine breakdown, in which the first step involves the transfer of adenosine to methionine to form S-Adenosylmethionine (SAM). The methyl group on the sulfonium ion of SAM is highly reactive, so it is not surprising to find that SAM is a methylating agent in some reactions. Tetrahydrofolates are also C1 donating agents and, unlike the carboxylations and the SAM methylations, the THFs can transfer C1 units in more than one oxidation state.
N 5 -methyl-THF ,as we have just seen, transfers the methyl group (-CH3), in which the oxidation level of C is that of methanol (-4). N 5 ,N 10 -methylene-THF carries a methylene group (-CH2-) and the oxidation level is that of formaldehyde (0), while N 5 -formimino-THF transfers the formimino group (-CH=NH), in which the oxidation level of the C atom is that of formate. Formyl (-CH=O) and methenyl (-CH=) groups are also transfered by THF and these both have the C in the oxidation level of formate (+2). The structure of THF is suited for these transfers by virtue of its N 5 and N 10 groups as shown in the following chemical structure:
We will see THF again when we study the synthesis of thymidylate from dUMP, catalyzed by the enzyme thymidylate synthase in which N 5 ,N 10 -methylene-THF is the methyl donor.
These are the "branched chain" amino acids, and it's helpful to remember them as a group, not only because they all originate from the pyruvate carbon skeleton, but also because the disease "maple syrup urine disease" (MSUD) is a result of deficiency of branched-chain a -ketoacid dehydrogenase, resulting in a buildup of branched-chain a -keto acids.
We'll just look at the beginning and the end of the pathways:
The first step is common to all 3 amino acids:
Pyruvate + TPP --> Hydroxyethyl-TPP (catalyzed by acetolactate synthase)
Note that the central carbon atom in hydroxyethyl-TPP is a carbanion and it is stabilized by resonance forms.
Hydroxyethyl-TPP can react with another pyruvate to form a -acetolactate, in which case the pathway heads toward valine and isoleucine, or it can react with a -ketobutyrate, in which case the pathway leads to isoleucine.
There is a branch point at a -ketoisovalerate which, in one direction leads to valine and, in the other, to leucine.
The final step in the formation of each of these amino acids involves the transfer of an amino group from glutamate to the corresponding a -ketoacid of each of the 3 branched-chain amino acids.Here we see another example of the importance of one particular amino acid, namely glutamate, to the anabolic pathways for amino acids.
Phosphoenolpyruvate (PEP), a glycolytic intermediate, condenses with erythrose-4-phosphate, a pentose-phosphate pathway intermediate, to form 2-keto-3-deoxyarabinoheptulosonate-7-phosphate and inorganic phosphate. The enzyme involved is a synthase. This condensation product eventually cyclizes to chorismate.
From here, the pathway branches, ending up in the production of tryptophan at one branch end, and tyrosine and phenylalanine at the other end.
A few high points deserve mention. First, glutamine plays a role as the donor of an amino group to chorismate to form anthranilate at the tryptophan branch.The immediate precursor of tryptophan is indole:
The "indole ring" is the characterizing feature of the tryptophan structure. Note that serine is the donor of the amino group to indole to form tryptophan.
The branch that leads towards tyrosine and phenylalanine has another branch point at prephenate. The only difference between the 2 resulting amino acids is that the para carbon of the benzene ring of tyrosine is hydroxylated. Indeed, in mammals, phenylalanine is directly hydroxylated to tyrosine, catalyzed by the enzyme phenylalanine hydroxylase.
Some very important physiologically active amines are derived from tyrosine, and these are L-DOPA, dopamine, norepinephrine and epinephrine. The pathway from tyrosine to norepinephrine is shown below:
The formation of epinephrine from norepinephrine involves the transfer of the highly reactive methyl group of S-adenosylmethionine to norepinephrine:
Structure of S-Adenosyl Methionine Showing Its Reactive Methyl Group:
We will look at this pathway in a bit more detail, because it involves the molecule 5-phosphoribosyl- a -pyrophosphate (which we will refer to as "PRPP" from now on). PRPP is also involved in the synthesis of purines and pyrimidines, as we will soon see. In the first step of histidine synthesis, PRPP condenses with ATP to form a purine, N 1 -5 ' -phosphoribosyl ATP, in a reaction that is driven by the subsequent hydrolysis of the pyrophosphate that condenses out. Glutamine again plays a role as an amino group donor, this time resulting in the formation of 5-aminoamidazole-4-carboximide ribonucleotide (ACAIR), which is an intermediate in purine biosynthesis.
Histidine is special in that its biosynthesis is inherently linked to the pathways of nucleotide formation. Histidine residues are often found in enzyme active sites, where the chemistry of the imidazole ring of histidine makes it a nucleophile and a good acid/base catalyzer. We now know that RNA can have catalytic properties, and there has been speculation that life was originally RNA-based. Perhaps the transition to protein catalysis from RNA catalysis occurred at the origin of histidine biosynthesis.
The physiologically active amine, histamine, is formed from histidine:
In the next lecture, we will look at fuel regulation and organ specialization. This will give us a chance to tie together the metabolic pathways that you have studied thus far.
Glutamine and glutathione
Because it plays a role in glutathione production, it has been suggested that glutamine supplements may increase the amount of glutathione in the body. A study by Valencia et al. of Oxford Brooks University in the United Kingdom reported that orally administered glutamine increased the amount of glutamine in the blood but not the amount of glutathione, indicating that glutamine availability may not be the rate limiting factor in glutathione synthesis. Whether you are choosing glutathione or its precursor glutamine as a supplement, consult your doctor before you begin the regimen.