Amino Acid Biosynthesis

Essential and Nonessential Amino Acids

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
Alanine    
Asparagine
Aspartate
Cysteine    (requires sulfhydryl group from methionine)
Glutamate
Glutamine
Glycine
Proline
Serine
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)
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Tryptophan
Valine

Arginine is synthesized by mammals in the urea cycle, but most of it hydrolyzed to urea and ornithine:

 (http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/amino.htm)

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 for 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:

histidine

tryptophan

carbamoyl phosphate (synthesized from carbamoyl phosphate synthetase II)

glucosamine-6-phosphate

AMP

CTP

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: 

 

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.

 

Exercise: Use the regulatory pathway to explain the effect of a rising level of glutamine on the activity of bacterial glutamine synthetase.

 

 

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

    (4) show species variation

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

    (4) Histidine

 

The Aspartate Family

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 9 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 amion 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 N5-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 anergy source.This reaction can also be looked at as the transfer of a methyl group from N5-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.

N5-methyl-THF ,as we have just seen, transfers  the methyl group (-CH3), in which the oxidation level of C is that of methanol (-4).  N5,N10-methylene-THF carries a methylene group (-CH2-) and the oxidation level is that of formaldehyde (0), while N5-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 N5 and N10 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 N5,N10-methylene-THF is the methyl donor.