View
227
Download
0
Category
Preview:
Citation preview
8/2/2019 Molecular Biochemistry II
1/19
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)
http://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/index.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/index.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/index.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/rna,viral_str%27.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/rna,viral_str%27.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/rna,viral_str%27.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/new_page_2.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/new_page_2.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/new_page_2.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/nucleotides.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/nucleotides.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/nucleotides.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/new_page_1.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/new_page_1.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/new_page_1.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/transcription.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/transcription.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/transcription.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/dna_replicn%27.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/dna_replicn%27.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/dna_replicn%27.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/photosynthesis.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/photosynthesis.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/photosynthesis.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/bioch__comm_.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/bioch__comm_.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/bioch__comm_.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/metabolism.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/metabolism.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/metabolism.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/metabolism.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/bioch__comm_.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/photosynthesis.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/dna_replicn%27.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/transcription.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/new_page_1.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/nucleotides.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/new_page_2.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/rna,viral_str%27.htmhttp://rpi.edu/dept/bcbp/molbiochem/BiochSci/sbello/index.htm8/2/2019 Molecular Biochemistry II
2/19
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.htm8/2/2019 Molecular Biochemistry II
3/19
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:
8/2/2019 Molecular Biochemistry II
4/19
8/2/2019 Molecular Biochemistry II
5/19
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:
8/2/2019 Molecular Biochemistry II
6/19
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
8/2/2019 Molecular Biochemistry II
7/19
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
8/2/2019 Molecular Biochemistry II
8/19
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:
8/2/2019 Molecular Biochemistry II
9/19
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)
8/2/2019 Molecular Biochemistry II
10/19
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
8/2/2019 Molecular Biochemistry II
11/19
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 :
8/2/2019 Molecular Biochemistry II
12/19
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:
8/2/2019 Molecular Biochemistry II
13/19
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
8/2/2019 Molecular Biochemistry II
14/19
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
8/2/2019 Molecular Biochemistry II
15/19
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:
8/2/2019 Molecular Biochemistry II
16/19
The formation of epinephrine from norepinephrine involves the transfer of the highly reactive
methyl group of S-adenosylmethionine to norepinephrine:
8/2/2019 Molecular Biochemistry II
17/19
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.
8/2/2019 Molecular Biochemistry II
18/19
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.
8/2/2019 Molecular Biochemistry II
19/19
Recommended