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Biosynthesis of Amino
Acids, Nucleotides, and
Related Molecules
chapter
22
S-258
1. ATP Consumption by Root Nodules in Legumes Bacteria residing in the root nodules of the pea
plant consume more than 20% of the ATP produced by the plant. Suggest why these bacteria consume
so much ATP.
2. Glutamate Dehydrogenase and Protein Synthesis The bacterium Methylophilus methylotro-
phus can synthesize protein from methanol and ammonia. Recombinant DNA techniques have im-
proved the yield of protein by introducing into M. methylotrophus the glutamate dehydrogenase
gene from E. coli. Why does this genetic manipulation increase the protein yield?
3. PLP Reaction Mechanisms Pyridoxal phosphate can help catalyze transformations one or two car-
bons removed from the ␣carbon of an amino acid. The enzyme threonine synthase (see Fig. 22–17)
promotes the PLP-dependent conversion of phosphohomoserine to threonine. Suggest a mechanism
for this reaction.
4. Transformation of Aspartate to Asparagine There are two routes for transforming aspartate to
asparagine at the expense of ATP. Many bacteria have an asparagine synthetase that uses ammonium
ion as the nitrogen donor. Mammals have an asparagine synthetase that uses glutamine as the nitrogen
donor. Given that the latter requires an extra ATP (for the synthesis of glutamine), why do mammals
use this route?
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5. Equation for the Synthesis of Aspartate from Glucose Write the net equation for the synthesis
of aspartate (a nonessential amino acid) from glucose, carbon dioxide, and ammonia.
Answer We can approach this problem by working “backward” from aspartate to glucose as
follows. Aspartate is synthesized from oxaloacetate by transamination from glutamate;
glutamate is synthesized from a-ketoglutarate by glutamate dehydrogenase:
6. Asparagine Synthetase Inhibitors in Leukemia Therapy Mammalian asparagine synthetase
is a glutamine-dependent amidotransferase. Efforts to identify an effective inhibitor of human
asparagine synthetase for use in chemotherapy for patients with leukemia has focused not on the
amino-terminal glutaminase domain but on the carboxyl-terminal synthetase active site. Explain why
the glutaminase domain is not a promising target for a useful drug.
7. Phenylalanine Hydroxylase Deficiency and Diet Tyrosine is normally a nonessential amino acid,
but individuals with a genetic defect in phenylalanine hydroxylase require tyrosine in their diet for
normal growth. Explain.
8. Cofactors for One-Carbon Transfer Reactions Most one-carbon transfers are promoted by one of
three cofactors: biotin, tetrahydrofolate, or S-adenosylmethionine (Chapter 18). S-Adenosylmethionine
is generally used as a methyl group donor; the transfer potential of the methyl group in N
5
-methylte-
trahydrofolate is insufficient for most biosynthetic reactions. However, one example of the use of N
5
-
methyltetrahydrofolate in methyl group transfer is in methionine formation by the methionine synthase
reaction (step 9 of Fig. 22–17); methionine is the immediate precursor of S-adenosylmethionine (see
Fig. 18–18). Explain how the methyl group of S-adenosylmethionine can be derived from N
5
-methylte-
trahydrofolate, even though the transfer potential of the methyl group in N
5
-methyltetrahydrofolate is
one one-thousandth of that in S-adenosylmethionine.
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S-260 Chapter 22 Biosynthesis of Amino Acids, Nucleotides, and Related Molecules
9. Concerted Regulation in Amino Acid Biosynthesis The glutamine synthetase of E. coli is indepen-
dently modulated by various products of glutamine metabolism (see Fig. 22–8). In this concerted inhibition,
the extent of enzyme inhibition is greater than the sum of the separate inhibitions caused by each prod-
uct. For E. coli grown in a medium rich in histidine, what would be the advantage of concerted inhibition?
10. Relationship between Folic Acid Deficiency and Anemia Folic acid deficiency, believed to be
the most common vitamin deficiency, causes a type of anemia in which hemoglobin synthesis is
impaired and erythrocytes do not mature properly. What is the metabolic relationship between hemo-
globin synthesis and folic acid deficiency?
11. Nucleotide Biosynthesis in Amino Acid Auxotrophic Bacteria Wild-type E. coli cells can synthe-
size all 20 common amino acids, but some mutants, called amino acid auxotrophs, are unable to synthe-
size a specific amino acid and require its addition to the culture medium for optimal growth. Besides
their role in protein synthesis, some amino acids are also precursors for other nitrogenous cell products.
Consider the three amino acid auxotrophs that are unable to synthesize glycine, glutamine, and aspartate,
respectively. For each mutant, what nitrogenous products other than proteins would the cell fail to
synthesize?
Answer Glycine, glutamine, and aspartate are required for the de novo synthesis of purine
12. Inhibitors of Nucleotide Biosynthesis Suggest mechanisms for the inhibition of (a) alanine race-
mase by
L
-fluoroalanine and (b) glutamine amidotransferases by azaserine.
13. Mode of Action of Sulfa Drugs Some bacteria require p-aminobenzoate in the culture medium for
normal growth, and their growth is severely inhibited by the addition of sulfanilamide, one of the earli-
est sulfa drugs. Moreover, in the presence of this drug, 5-aminoimidazole-4-carboxamide ribonucleotide
(AICAR; see Fig. 22–35) accumulates in the culture medium. These effects are reversed by addition of
excess p-aminobenzoate.
Answer
(a) What is the role of p-aminobenzoate in these bacteria? (Hint: see Fig. 18–16.)
(b) Why does AICAR accumulate in the presence of sulfanilamide?
(c) Why are the inhibition and accumulation reversed by addition of excess p-aminobenzoate?
Answer
Chapter 22 Biosynthesis of Amino Acids, Nucleotides, and Related Molecules S-261
OO
SCH
2
NH
2
OO
NNH
2
p-Aminobenzoate Sulfanilamide
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S-262 Chapter 22 Biosynthesis of Amino Acids, Nucleotides, and Related Molecules
14. Pathway of Carbon in Pyrimidine Biosynthesis Predict the locations of
14
C in orotate isolated
from cells grown on a small amount of uniformly labeled [
14
C]succinate. Justify your prediction.
Answer
15. Nucleotides as Poor Sources of Energy Under starvation conditions, organisms can use proteins
and amino acids as sources of energy. Deamination of amino acids produces carbon skeletons that can
enter the glycolytic pathway and the citric acid cycle to produce energy in the form of ATP. Nu-
cleotides, on the other hand, are not similarly degraded for use as energy-yielding fuels. What observations
about cellular physiology support this statement? What aspect of the structure of nucleotides makes
them a relatively poor source of energy?
16. Treatment of Gout Allopurinol (see Fig. 22–50), an inhibitor of xanthine oxidase, is used to treat
chronic gout. Explain the biochemical basis for this treatment. Patients treated with allopurinol some-
times develop xanthine stones in the kidneys, although the incidence of kidney damage is much lower
than in untreated gout. Explain this observation in the light of the following solubilities in urine: uric
acid, 0.15 g/L; xanthine, 0.05 g/L; and hypoxanthine, 1.4 g/L.
17. Inhibition of Nucleotide Synthesis by Azaserine The diazo compound O-(2-diazoacetyl)-
L
-ser-
ine, known also as azaserine (see Fig. 22–51), is a powerful inhibitor of glutamine amidotransferases. If
growing cells are treated with azaserine, what intermediates of nucleotide biosynthesis will accumu-
late? Explain.
Data Analysis Problem
18. Use of Modern Molecular Techniques to Determine the Synthetic Pathway of a Novel Amino
Acid Most of the biosynthetic pathways described in this chapter were determined before the develop-
ment of recombinant DNA technology and genomics, so the techniques were quite different from those
that researchers would use today. Here we explore an example of the use of modern molecular tech-
niques to investigate the pathway of synthesis of a novel amino acid, (2S)-4-amino-2-hydroxybutyrate
(AHBA). The techniques mentioned here are described in various places in the book; this problem is de-
signed to show how they can be integrated in a comprehensive study.
AHBA is a ␥-amino acid that is a component of some aminoglycoside antibiotics, including the
antibiotic butirosin. Antibiotics modified by the addition of an AHBA residue are often more resis-
tant to inactivation by bacterial antibiotic-resistance enzymes. As a result, understanding how
AHBA is synthesized and added to antibiotics is useful in the design of pharmaceuticals.
In an article published in 2005, Li and coworkers describe how they determined the synthetic
pathway of AHBA from glutamate.
(a) Briefly describe the chemical transformations needed to convert glutamate to AHBA. At this
point, don’t be concerned about the order of the reactions.
Li and colleagues began by cloning the butirosin biosynthetic gene cluster from the bacterium Bacillus
circulans, which makes large quantities of butirosin. They identified five genes that are essential for the
pathway: btrI,btrJ,btrK,btrO, and btrV. They cloned these genes into E. coli plasmids that allow overex-
pression of the genes, producing proteins with “histidine tags” fused to their amino termini to facilitate
purification (see Section 9.1).
The predicted amino acid sequence of the BtrI protein showed strong homology to known acyl
carrier proteins (see Fig. 21–5). Using mass spectrometry (see Box 3–2), Li and colleagues found a
molecular mass of 11,812 for the purified BtrI protein (including the His tag). When the purified BtrI
was incubated with coenzyme A and an enzyme known to attach CoA to other acyl carrier proteins,
the majority molecular species had an M
r
of 12,153.
Chapter 22 Biosynthesis of Amino Acids, Nucleotides, and Related Molecules S-263
O
C C O
NH3
Glutamate AHBA
CO
NH3
OH
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S-264 Chapter 22 Biosynthesis of Amino Acids, Nucleotides, and Related Molecules
(b) How would you use these data to argue that BtrI can function as an acyl carrier protein with a
CoA prosthetic group?
Using standard terminology, Li and coauthors called the form of the protein lacking CoA apo-BtrI
and the form with CoA (linked as in Fig. 21–5) holo-BtrI. When holo-BtrI was incubated with glutamine,
ATP, and purified BtrJ protein, the holo-BtrI species of M
r
12,153 was replaced with a species of M
r
12,281, corresponding to the thioester of glutamate and holo-BtrI. Based on these data, the authors
proposed the following structure for the M
r
12,281 species (␥-glutamyl-S-BtrI):
BtrI
OS
C C O
NH3
(c) What other structure(s) is (are) consistent with the data above?
(d) Li and coauthors argued that the structure shown here (␥-glutamyl-S-BtrI) is likely to be correct
because the ␣-carboxyl group must be removed at some point in the synthetic process. Explain
the chemical basis of this argument. (Hint: See Fig. 18–6, reaction C.)
The BtrK protein showed significant homology to PLP-dependent amino acid decarboxylases, and
BtrK isolated from E. coli was found to contain tightly bound PLP. When ␥-glutamyl-S-BtrI was incu-
bated with purified BtrK, a molecular species of M
r
12,240 was produced.
(e) What is the most likely structure of this species?
(f) Interestingly, when the investigators incubated glutamate and ATP with purified BtrI, BtrJ, and
BtrK, they found a molecular species of M
r
12,370. What is the most likely structure of this
species? Hint: Remember that BtrJ can use ATP to ␥-glutamylate nucleophilic groups.
Li and colleagues found that BtrO is homologous to monooxygenase enzymes (see Box 21–1) that
hydroxylate alkanes, using FMN as a cofactor, and BtrV is homologous to an NAD(P)H oxidoreductase.
Two other genes in the cluster, btrG and btrH, probably encode enzymes that remove the ␥-glutamyl
group and attach AHBA to the target antibiotic molecule.
(g) Based on these data, propose a plausible pathway for the synthesis of AHBA and its addition to
the target antibiotic. Include the enzymes that catalyze each step and any other substrates or co-
factors needed (ATP, NAD, etc.).
Answer
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