Molecular Biochemistry II

8/2/2019 Molecular Biochemistry II

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Molecular Biochemistry II[Home][Introduction][Amino Acid Synthesis][Nucleotides][DNA Structure and Topology]

[Transcription][DNA Replication][Photosynthesis][Biochemical Communication]

[Metabolism]

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 thehigher 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 outthat 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 overlapbetween 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)

GlutamateGlutamine

Glycine

Proline

Serine

Tyrosine (synthesized from phenylalanine)
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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 andchildren, 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 tosynthesize 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
http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/amino.htmhttp://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/amino.htmhttp://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/amino.htmhttp://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/amino.htm


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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:


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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 commonintermediates 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 asparaginesynthetase, and glutamine is the amino group donor:


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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 phosphategroup:

So, the synthesis of asparagine is intrinsically tied to that of glutamine, and it turns out thatglutamine 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 thebiosynthesis 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, itis 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. Theactivity of the enzyme is controlled by 9 allosteric feedback inhibitors, 6 of which are end


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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 thecellular nitrogen level.

The enzyme is also regulated by covalent modification (adenylylation of a Tyr residue), whichresults 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 tetramericregulatory 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 P II. When PII is uridylylated,

glutamine synthetase is deadenylylated; the reverse occurs when UMP is covalently attached tothe 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


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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 theactivity 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 ornithineand 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:


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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,N

10-methylene-THF (enzyme: serine

hydroxymethyltransferase)

Glycine is also formed in a condensation reaction as follows:

N5,N

10-methylene-THF + CO2 + NH4

+--> glycine (enzyme: glycine synthase;

requires NADH)


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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


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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 byaspartokinase:

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 methioninesynthase :


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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 anenergy 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 homocysteinemethyltransferase.

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 topyruvate 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 SAMmethylations, 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 havethe 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:


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We will see THF again when we study the synthesis of thymidylate from dUMP, catalyzed bythe enzymethymidylate 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 whichcase the pathway leads to isoleucine.

There is a branch point at -ketoisovalerate which, in one direction leads to valine and, in the


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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 aminoacids.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-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


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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

henylalanine, which is subsequently transaminated to phenylpyruvate and excreted in the

urine. The disease "phenylketonuria" rapidly leads to severe mental retardation if not treatedsoon after birth with a low phenylalanine diet. Universal screening of newborns in the U.S.

or 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 L-DOPA, dopamine, norepinephrine and epinephrine. The pathway from tyrosine to

norepinephrine is shown below:


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The formation of epinephrine from norepinephrine involves the transfer of the highly reactive

methyl group of S-adenosylmethionine to norepinephrine:


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Histidine Biosynthesis:

We will look at this pathway in a bit more detail, because it involves the molecule 5-

phosphoribosyl--pyrophosphate (which we will refer to as "PRPP" from now on). PRPP is alsoinvolved 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 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 nucleotideformation. 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 theorigin of histidine biosynthesis.


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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 identifiedhistidine 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.


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Molecular Biochemistry II

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