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Molecular Biochemistry II

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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 Glutamate Glutamine Glycine Proline Serine Tyrosine
(synthesized from phenylalanine) (requires sulfhydryl group from methionine)

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 ketoacids. Therefore, it could be possible to synthesize any one of the nonessential amino acids directly by transaminating its corresponding -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 -carbon of a ketoacid, is catalyzed by an aminotransferase. Three very common ketoacids can be transaminated in one step to their corresponding amino acid: Pyruvate (glycolytic end product) --> alanine Oxaloacetate (citric acid cycle intermediate) --> aspartate 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 -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 -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 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 ketoglutarate are produced. The -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 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 (see next lecture) CTP (see next lecture) 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 P II, 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 -ketoglutarate and ATP, while it is inhibited by glutamine and Pi. The following diagram summarizes the regulation of bacterial glutamine synthetase: see text pg 643

We can "walk through" this regulatory cascade by looking at a specific example, namely increased levels of -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) -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.

Proline, Ornithine and Arginine are derived from Glutamate The first step involves phosphorylation of glutamate by ATP with the enzyme -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, -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 + N5,N10 -methylene-THF hydroxymethyltransferase) Glycine is also formed in a condensation reaction as follows: N5,N10 -methylene-THF + CO2 + NH4+ --> glycine requires NADH) (enzyme: glycine synthase; (enzyme: serine

Cysteine is synthesized from serine and homocysteine (methionine breakdown product): ser + homocysteine -> cystathionine + H2O cystathionine + H2O --> -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 (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--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: -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 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 energy source.This reaction can also be looked at as the transfer of a methyl group from N5methyl-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 SAdenosylmethionine (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.

The Pyruvate Family 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 -ketoacid dehydrogenase, resulting in a buildup of branched-chain -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 -acetolactate, in which case the pathway heads toward valine and isoleucine, or it can react with -ketobutyrate, in which case the pathway leads to isoleucine. There is a branch point at -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 -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. The Aromatic Amino Acids: Phosphoenolpyruvate (PEP), a glycolytic intermediate, condenses with erythrose-4-phosphate, a pentose-phosphate pathway intermediate, to form 2-keto-3-deoxyarabinoheptulosonate-7phosphate 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. Clinical Correlation: Defective or absent phenylalanine hydroxylase results in buildup of phenylalanine, which is subsequently transaminated to phenylpyruvate and excreted in the urine. The disease "phenylketonuria" rapidly leads to severe mental retardation if not treated soon after birth with a low phenylalanine diet. Universal screening of newborns in the U.S. for this condition by blood analysis has greatly decreased the morbidity of the untreated condition.

Some very important physiologically active amines are derived from tyrosine, and these are LDOPA, 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:

Histidine Biosynthesis: We will look at this pathway in a bit more detail, because it involves the molecule 5phosphoribosyl--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, N1-5'-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 5aminoamidazole-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 the synthesis of nucleotides. Since we have just identified histidine as a likely evolutionary transition point from purines to amino acids and proteins, this is a convenient point at which to transition from one lecture to another.