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Chapter 25
Nitrogen Acquisition and
Amino Acid Metabolism
Biochemistry
by
Reginald Garrett and Charles Grisham
Essential Question
• What are the biochemical pathways that form
ammonium from inorganic nitrogen
compounds prevalent in the inanimate
environment?
• How is ammonium incorporated into organic
compounds?
• How are amino acids synthesized and
degraded?
Outline
1. Which Metabolic Pathways Allow Organisms
to Live on Inorganic Forms of Nitrogen?
2. What Is The Metabolic Fate of Ammonium?
3. What Regulatory Mechanisms Act on
Escherichia coli Glutamine Synthetase?
4. How Do Organisms Synthesize Amino
Acids?
5. How Does Amino Acid Catabolism Lead into
Pathways of Energy Production?
25.1 – Which Metabolic Pathways
Allow Organisms to Live on
Inorganic Forms of Nitrogen?
Nitrogen is cycled between organisms and
inanimate enviroment
• The principal inorganic forms of N are in an
oxidized state
– As N2 in the atmosphere
– As nitrate (NO3-) in the soils and ocean
• All biological compounds contain N in a
reduced form (NH4+)
• Thus, Nitrogen acquisition must involve
1. The Reduction of the oxidized forms (N2 and
NO3-) to NH4+
2. The incorporation of NH4+ into organic linkage
as amino or amido groups
• The reduction occurs in microorganisms and
green plants. But animals gain N through
diet.
(+3)
(+5)
(-3)
(+2)
(+1) (0)
The nitrogen cycle
Figure 25.1
The nitrogen cycle. Organic nitrogenous compounds are formed by the incorporation of NH4+
into carbon skeletons. Ammonium can be formed from oxidized inorganic percursors by
reductive reactions: nitrogen fixation reduces N2 to NH4+; nitrate assimilation reduces NO3- to
NH4+. Nitrifying bacteria can oxidize NH4+ back to NO3- and obtain energy for growth in the
process of nitrification. Denitrification is a form of bacterial respiration whereby nitrogen
oxides serve as electron acceptors in the place of O2 under anaerobic conditions.
The reduction of Nitrogen
Nitrogen assimilation and nitrogen fixation
1. Nitrate assimilation occurs in two steps:
–
–
•
2e- reduction of nitrate to nitrite
6e- reduction of nitrite to ammonium (fig 25.1)
Nitrate assimilation accounts for 99% of N
acquisition by the biosphere
2. Nitrogen fixation involves reduction of N2 in
prokaryotes by nitrogenase
Nitrate Assimilation
•
Two steps:
1. Nitrate reductase
NO3- + 2 H+ + 2 e- → NO2- + H2O
2. Nitrite reductase
NO2- + 8 H+ + 6 e- → NH4+ + 2 H2O
•
Electrons are transferred from NADH to
nitrate
Nitrate reductase
•
•
•
Pathway involves -SH of enzyme, FAD,
cytochrome b and Molybdenm cofactor - all
protein-bound
Nitrate reductases are cytosolic 220 kD
dimeric protein
MoCo required both for reductase activity
and for assembly of enzyme subunits to
active dimer
NO3-
NADH
[-SH →FAD→cytochrome b557 →MoCo]
NADH+
NO2-
Figure 25.2
The novel prosthetic groups of nitrate reductase and nitrite reductase. (a) The molybdenum
cofactor of nitrate reductase. The molybdenum-free version of this compound is a pterin
derivative called molybdopterin. (b) Siroheme, a uroporphyrin derivative, is a member of the
isobacteriochlorin class of hemes, a group of porphyrins in which adjacent pyrrole rings are
reduced. Siroheme is novel in having eight carboxylate-containing side chains. These
carboxylate groups may act as H+ donors during the reduction of NO2- to NH4+.
Nitrite Reductase
Light drives reduction of ferredoxins and
electrons flow to 4Fe-4S and siroheme and
then to nitrite
• Nitrite is reduced to ammonium while still
bound to siroheme
• In higher plants, nitrite reductase is in
chloroplasts, but nitrate reductase is cytosolic
Light → 6 Fdred
NO2[(4Fe-4S → siroheme]
6 Fdox
NH4+
Figure 25.3
Domain organization
within the enzymes
of nitrate
assimilation. The
numbers denote
residue number
along the amino acid
sequence of the
proteins. The
numbering for
nitrate reductase is
that from the green
plant Arabidopsis
thaliana; the plant
nitrite reductase
sequence shown
here is spinach; the
fungal nitrite
reductase is
Neurospora crassa.
(Adapted in part from
Campbell & Kinghorn,
1990. Trends in
Biochemical Sciences
15:315-319.)
Nitrogen fixation
•
•
•
•
N2 + 10 H+ + 8 e- → 2 NH4+ + H2
Only occurs in certain prokaryotes
Rhizobia fix nitrogen in symbiotic association
with leguminous plants
Rhizobia fix N for the plant and plant provides
Rhizobia with carbon substrates
Fundamental requirements:
1.
2.
3.
4.
Nitrogenase
A strong reductant (reduced ferredoxin)
ATP
O-free conditions
Nitrogenase Complex
Two metalloprotein components:
nitrogenase reductase and nitrogenase
• Nitrogenase reductase
– Fe-protein
– is a 60 kD homodimer with a single 4Fe-4S cluster
• Very oxygen-sensitive
• Binds MgATP and hydrolyzes 2 ATPs per
electron transferred
• Reduction of N2 to 2NH3 + H2 requires 4 pairs
of electrons, so 16 ATP are consumed per N2
Figure 25.4
The triple bond in
N2 must be
broken during
nitrogen fixation.
A substantial
energy input is
needed to
overcome this
thermodynamic
barrier, even
though the overall
free energy
change (DG°‘)for
biological N2
reduction is
negative.
• N2 reduction to ammonia is thermodynamically favorable
• However, the activation barrier for breaking the N-N triple
bond is enormous
• 16 ATP provide the needed activation energy
Nitrogenase
• MoFe-protein
• A 220 kD a2b2 heterotetramer
• Two types of metal centers (fig 25.5)
– P-cluster: 8Fe-7S
– FeMo-cofactor; 7Fe-1Mo-9S
• Nitrogenase is slow enzyme
– 12 e- pairs per second, i.e., only three molecules
of N2 per second
– As much as 5% of cellular protein may be
nitrogenase
Figure 25.5
Structures of the two types of
metal clusters found in
nitrogenase.
(a) The P-cluster. Two Fe4S3
clusters share a fourth S and
are bridged by two thiol
ligands from the protein
(Cysa88 and Cysb95). (b) The
FeMo-cofactor. This novel
molybdenum-containing Fe-S
complex contains 1 Mo, 7 Fe,
and 9 S atoms; it is liganded
to the protein via a Cysa275-S
linkage to an Fe atom and a
Hisa442-N linkage to the Mo
atom. Homocitrate provides
two oxo ligands to the Mo
atom. (Adapted from Leigh, G. J.,
1995. The mechanism of
dinitrogen reduction by
molybdenum nitrogenases.
European Journal of Biochemistry
229:14-20.)
Figure 25.6
The nitrogenase reaction. Depending on the bacterium, electrons for N2 reduction may
come from light, NADH, hydrogen gas, or pyruvate. The primary e- donor for the
nitrogenase system is reduced ferredoxin. Reduced ferredoxin passes electrons directly to
nitrogenase reductase. A total of six electrons is required to reduce N2 to 2 NH4+, and
another two electrons are consumed in the obligatory reduction of 2 H+ to H2. Nitrogenase
reductase transfers e- to nitrogenase one electron at a time. N2 is bound at the critical
FeMoCo prosthetic group of nitrogenase until all electrons and protons are added; no free
intermediates such as HN=NH or H2N-NH2 are detectable.
The regulation of nitrogen Fixation
•
1.
2.
•
Two regulatory controls
ADP inhibits the activity of nitrogease
NH4+ represses the expression of nif genes
Some organism, ADP-ribosylation of
nitrogenase reductase
Figure 25.8
Regulation of
nitrogen fixation. (a)
ADP inhibits
nitrogenase activity.
(b) NH4+ represses
nif gene expression.
(c) In some
organisms, the
nitrogenase complex
is regulated by
covalent
modification. ADPribosylation of
nitrogenase
reductase leads to its
inactivation.
Nitrogenase
reductase is a distant
relative of the signaltransducing Gprotein superfamily.
25.2 – What Is The Metabolic Fate of
Ammonium?
Ammonium enters organic linkage via
three major reactions in all cells
1.
2.
3.
•
Carbamoyl-phosphate synthetase (CPS)
Glutamate dehydrogenase (GDH)
Glutamine synthetase (GS)
Asparagine synthetase (some
microorganisms)
Carbamoyl-phosphate synthetase (CPS)
NH4+ + HCO3- + 2 ATP →
carbamoyl phosphate + 2 ADP + Pi + 2 H+
•
Two ATP required
– one to activate bicarbonate
– one to phosphorylate carbamate
• N-acetylglutamate is an essential allosteric
activator
Glutamate dehydrogenase
NH4+ + a-ketoglutarate + NADPH + 2 H+ →
glutamate + NADP+ + H2O
•
Reductive amination of a-ketoglutarate to
form glutamate
Glutamine synthetase
NH4+ + glutamate + ATP →
glutamine + ADP + Pi
• ATP-dependent amidation of g-carboxyl of
glutamate to glutamine
• Glutamine is a major N donor in the
biosynthesis of many organic N compounds,
therefore GS activity is tightly regulated
Figure 25.10
(a) The enzymatic reaction catalyzed by glutamine synthetase. (b) The reaction proceeds by
(a) activation of the g-carboxyl group of Glu by ATP, followed by (b) amidation by NH4+.
The major pathways of Ammonium
Assimilation
Two principal pathways
1. Principal route: GDH/GS in organisms rich
in N
• See Figure 25.11 - both steps assimilate N
The major pathways of Ammonium
Assimilation
Two principal pathways
1. Principal route: GDH/GS in organisms
rich in N
2. Secondary route: GS/GOGAT in
organisms confronting N limitation
• GOGAT is glutamate synthase or
glutamate:oxo-glutarate amino transferase
• See Figures 25.12 and 25.13
Figure 25.12
The glutamate synthase reaction, showing the reductants exploited by different
organisms in this reductive amination reaction.
Figure 25.11 The GDH/GS pathway of ammonium assimilation.
Figure 25.13 The GS/GOGAT pathway of ammonium assimilation. The sum
of these reactions results in the conversion of 1 a-ketoglutarate to 1 glutamine at the
expense of 2 ATP and 1 NADPH.
25.3 – What Regulatory Mechanisms
Act on Glutamine Synthetase
•
GS in E. coli is regulated in three ways:
1. Feedback inhibition
2. Covalent modification (interconverts between
inactive and active forms)
3. Regulation of gene expression and protein
synthesis control the amount of GS in cells
•
But no such regulation occurs in eukaryotic
versions of GS
Figure 25.14
The subunit organization
of bacterial glutamine
synthetase.
(a) Diagram showing its
dodecameric structure as
a stack of two hexagons.
(b) Molecular structure of
glutamine synthetase
from Salmonella
typhimurium (a close
relative of E.coli), as
revealed by X-ray
crystallographic analysis.
(From Almassy, R. J.,
Janson, C. A., Hamlin, R.,
Xuong, N.-H., and Eisenberg,
D., 1986. Novel subunitsubunit interactions in the
structure of glutamine
synthetase.Nature 323:304.
Photos courtesy of S.-H.
Liaw and D. Eisenberg.)
Allosteric Regulation
of Glutamine Synthetase
• Nine different feedback inhibitors: Gly, Ala,
Ser, His, Trp, CTP, AMP, carbamoyl-P and
glucosamine-6-P
• Gly, Ala, Ser are indicators of amino acid
metabolism in cells
• Other six are end products of a biochemical
pathway
• This effectively controls glutamine’s
contributions to metabolism
Figure 25.15
The allosteric
regulation of
glutamine synthetase
activity by feedback
inhibition.
Covalent Modification
of Glutamine Synthetase
• Each subunit is adenylylated at Tyr-397
• Adenylylation inactivates GS by adenylyl
transferase
• Adenylyl transferase catalyzes both the
adenylylation and deadenylylation
– PII (regulatory protein) controls these
• AT:PIIA catalyzes adenylylation
• AT:PIID catalyzes deadenylylation
• a-Ketoglutarate and Gln also affect
Figure 25.16
Covalent modification of GS: Adenylylation of Tyr397 in the glutamine synthetase polypeptide
via an ATP-dependent reaction catalyzed by the converter enzyme adenylyl transferase (AT).
From 1 through 12 GS monomers in the GS holoenzyme can be modified, with progressive
inactivation as the ratio of [modified]/[unmodified] GS subunits increases.
Figure 25.17
The cyclic cascade system regulating the covalent modification of GS.
Gene Expression regulates GS
• Gene GlnA is actively transcribed only if
transcriptional enhancer NRI is in its
phosphorylated form, NRI-P
• NRI is phosphorylated by NRII, a protein
kinase
• If NRII is complexed with PIIA it acts as a
phosphatase, not a kinase
Figure 25.18
Transcriptional regulation of GlnA expression through the reversible phosphorylation of NRI, as
controlled by NRII and its association with PIIA.
(kinase)
(phosphatase)
25.4 – Amino Acid Biosynthesis
• Plants and microorganisms can make all 20
amino acids and all other needed N metabolites
• In these organisms, glutamate is the source of N,
via transamination (aminotransferase) reactions
• Mammals can make only 10 of the 20 aas
• The others are classed as "essential" amino
acids and must be obtained in the diet
Amino acids are formed from aketo acids by transamination
Amino acid1 + a-keto acid2 → a-keto acid1 + Amino acid2
• Transamination (aminotransferase) reactions
• Named according their amino acid substrate
– Glutamate-asparate aminotransferase
Figure 25.19
Glutamatedependent
transamination of
a-keto acid carbon
skeletons is a
primary mechanism
for amino acid
synthesis. The
generic
transamination aminotransferase
reaction involves
the transfer of the
a-amino group of
glutamate to an
a-keto acid
acceptor (see
Figure 13.23). The
transamination of
oxaloacetate by
glutamate to yield
aspartate and
a-ketoglutarate is a
prime example.
The mechanism of PLP-catalyzed transamination reactions.
Amino Acid Biosynthesis can be
organized into families
• According to the intermediates that they are made
from
The a-Ketoglutarate Family
•
•
•
•
•
Glu, Gln, Pro, Arg, and sometimes Lys
Transamination of a-Ketoglutarate gives
glutamate
Amidation of glutamate gives glutamine
Proline is derived from glutamate (Figure
25.20)
Arginine are part of the urea cycle
Ornithine is also derived from glutamate
– the similarity to the proline pathway
(1) g-glutamyl kinase,
(2) glutamate-5-semialdehyde
dehydrogenase
(4) D1-pyrroline-5-carboxylate
reductase
Figure 25.20
The pathway of proline biosynthesis from glutamate. The enzymes are (1) g-glutamyl kinase,
(2) glutamate-5-semialdehyde dehydrogenase, and (4) D1-pyrroline-5-carboxylate
reductase; reaction (3) occurs nonenzymatically.
Figure 25.21
The bacterial
pathway of ornithine
biosynthesis from
glutamate. The
enzymes are (1) Nacetylglutamate
synthase, (2) Nacetylglutamate
kinase, (3) Nacetylglutamate-5semialdehyde
dehydrogenase, (4)
N-acetylornithine daminotransferase,
and (5) Nacetylornithine
deacetylase. In
mammals, ornithine
is synthesized
directly from
glutamate-5semialdehyde by a
pathway that does
not involve an Nacetyl block.
(1) N-acetylglutamate synthase
(2) N-acetylglutamate kinase
(3) N-acetylglutamate-5semialdehyde dehydrogenase
(4) N-acetylornithine d-aminotransferase
(5) N-acetylornithine deacetylase
The a-Ketoglutarate Family
•
Ornithine has three metabolic roles
1. To serve as precursor to arginine
2. To function as an intermediate in the urea cycle
3. To act as an intermediate in arginine degradation
Carbamoyl-phosphate synthetase I
•
Carbamoyl-phosphate synthetase I (CPS-I)
–
NH3-dependent mitochondrial CPS isozyme
1. HCO3- is activated via an ATP-dependent
phosphorylation
2. Ammonia attacks the carbonyl carbon of
carbonyl-P, displacing Pi to form carbamate
3. Carbamate is phosphorylated via a second ATP
to give carbamoyl-P
Figure 25.22
The mechanism of
action of CPS-I, the
NH3-dependent
mitochondrial CPS
isozyme. (1) HCO3- is
activated via an ATPdependent
phosphorylation. (2)
Ammonia attacks the
carbonyl carbon of
carbonyl-P,
displacing Pi to form
carbamate. (3)
Carbamate is
phosphorylated via a
second ATP to give
carbamoyl-P.
Carbamoyl-phosphate synthetase I
•
CPS-I represents the committed step in urea
cycle
•
Activated by N-acetylglutamate
–
Because N-acetylglutamate is a precursor to
orinithine synthesis and essential to the
operation of the urea cycle

Increase amino acid catabolism

Elevate glutamate level (N-acetylglutamate)

Stimulate CPS-I

Raise overall Urea cycle activity
The Urea Cycle
• The carbon skeleton of arginine is derived
from a-ketoglutarate
• N and C in the guanidino group of Arg
come from NH4+, HCO3- (carbamoyl-P), and
the a-NH2 of Glu and Asp
• Breakdown of Arg in the urea cycle releases
two N and one C as urea & ornithine
• Important N excretion mechanism in livers
of terrestrial vertebrates
• Urea cycle is linked to TCA by fumarate
The Urea Cycle
1.
2.
3.
4.
Ornithine transcarbamoylase (OTCase)
Argininosuccinate synthetase
Argininosuccinase
Arginase
Figure 25.23
The urea cycle series of reactions: Transfer
of the carbamoyl group of carbamoyl-P to
ornithine by ornithine transcarbamoylase
(OTCase, reaction 1) yields citrulline. The
citrulline ureido group is then activated by
reaction with ATP to give a citrullyl-AMP
intermediate (reaction 2a); AMP is then
displaced by aspartate, which is linked to
the carbon framework of citrulline via its aamino group (reaction 2b). The course of
reaction 2 was verified using 18O-labeled
citrulline. The 18O label (indicated by the
asterisk, *) was recovered in AMP. Citrulline
and AMP are joined via the ureido *O atom.
The product of this reaction is
argininosuccinate; the enzyme catalyzing
the two steps of reaction 2 is
argininosuccinate synthetase. The next
step (reaction 3) is carried out by
argininosuccinase, which catalyzes the
nonhydrolytic removal of fumarate from
argininosuccinate to give arginine.
Hydrolysis of Arg by arginase (reaction 4)
yields urea and ornithine, completing the
urea cycle.
Lysine Biosynthesis
•
Two pathways:
1. a-aminoadipate pathway
2. diaminopimelate pathway
•
Lysine derived from a-ketoglutarate
–
–
•
•
•
•
Reactions 1 through 4 are reminiscent of the first four
reactions in the citric acid cycle
a-ketooadipate
Transamination gives a-aminoadipate
Adenylylation activates the d-COOH for reduction
Reductive amination give saccharopine
Oxidative cleavage yields lysine
Figure 25.24
Lysine biosynthesis in certain fungi
and Euglena: the a-aminoadipic acid
pathway. Reactions 1 through 4 are
reminiscent of the first four reactions
in the citric acid cycle, except that the
product a-ketoadipate has an
additional CH2 unit. Reaction 5 is
catalyzed by a glutamate-dependent
aminotransferase; reaction 6 is the
adenylylation of the d-carboxyl of aaminoadipate to give the 6-adenylyl
derivative. Reductive deadenylylation
by an NADPH-dependent
dehydrogenase in reaction 7 gives aaminoadipic-6-semialdehyde, which in
reaction 8 is coupled with glutamate
via its amino group by a second
NADPH-dependent dehydrogenase.
Oxidative removal of the aketoglutarate moiety by NAD+dependent saccharopine
dehydrogenase in reaction 9 leaves
this amino group as the e-NH3+ of
lysine.
The Aspartate Family
Asp, Asn, Lys, Met, Thr, Ile
• Transamination of Oxaloaceate gives Aspartate
(aspartate aminotransferase)
• Amidation of Asp gives Asparagine ( asparagine
synthetase)
• Met, Thr and Lys are made from Aspartate
• b-Aspartyl semialdehyde and homoserine are
branch points
• Isoleucine, four of its six carbons derived from
Asp (via Thr) and two come from pyruvate
Figure 25.25
Aspartate biosynthesis via transamination of oxaloacetate by glutamate. The enzyme
responsible is PLP-dependent glutamate: aspartate aminotransferase.
Figure 25.26
Asparagine biosynthesis from Asp, Gln, and ATP. b-Aspartyladenylate is an enzymebound intermediate of asparagine synthetase; Asn, Glu, AMP, and PPi are products. (Step
A) Asp + ATP  [b-aspartyladenylate] + PPi. (Step B) [b-Aspartyladenylate] + Gln + H2O
 Asn + Glu + AMP.
Figure 25.27 Biosynthesis of
threonine, methionine, and
lysine, members of the
aspartate family of amino
acids.
b-Aspartyl-semialdehyde is a
common precursor to all three.
It is formed by aspartokinase
(reaction 1) and b-aspartylsemialdehyde dehydrogenase
(reaction 2).
• In E. coli
– Three isozymes of aspartokinase
– Uniquely controlled by one of the three endproducts
Figure 25.27 Biosynthesis of
threonine, methionine, and
lysine, members of the
aspartate family of amino
acids.
b-Aspartyl-semialdehyde is a
common precursor to all three.
It is formed by aspartokinase
(reaction 1) and b-aspartylsemialdehyde dehydrogenase
(reaction 2).
• Role of methionine
– in methylations via S-adenosylmethionine
(SAM; S-AdoMet)
– polyamine biosynthesis
Figure 25.28
The synthesis of Sadenosylmethionine (SAM)
from methionine plus ATP,
and the role of SAM as a
substrate of
methyltransferases in
methyl donor reactions and
in propylamine transfer
reactions, as in the
synthesis of polyamines.
The Pyruvate Family
Ala, Val, Leu, and Ile
• Transamination of pyruvate gives Alanine
• Valine is derived from pyruvate
• Ile synthesis from Thr mimics Val synthesis
from pyruvate (Fig. 25.29)
– Threonine deaminase (also called threonine
dehydratase or serine dehydratase) is sensitive
to Ile
– Ile and val pathway employ the same set of
enzymes
Figure 25.29
Biosynthesis of
valine and isoleucine.
The enzymes are (1)
threonine
deaminase, (2)
acetohydroxy acid
synthase, (3)
acetohydroxy acid
isomeroreductase,
(4) dihydroxy acid
dehydratase, and (5)
glutamate-dependent
aminotransferase.
Feedback inhibition
regulates this
pathway: enzyme 1
is isoleucinesensitive, and
enzyme 2 is valinesensitive.
Threonine
deaminase
Acetohydroxy acid
synthase
Acetohydroxy acid
isomeroreductase
Dihydroxy acid
dehydratase
Leucine
Glutamate-dependent
aminotransferase
The Pyruvate Family
Ala, Val, Leu, and Ile
• Transamination of pyruvate gives Alanine
• Valine is derived from pyruvate
• Ile synthesis from Thr mimics Val synthesis
from pyruvate (Fig. 25.29)
• Leu synthesis begins with an a-keto
isovalerate
– Isopropylmalate synthase is sensitive to Leu
Figure 25.30
Biosynthesis of
leucine. The
enzymes are (1) aisopropylmalate
synthase, (2) aisopropylmalate
dehydratase, (3)
isopropylmalate
dehydrogenase, and
(4) leucine
aminotransferase.
Enzyme 1 is
feedback-inhibited by
leucine.
isopropylmalate
synthase
isopropylmalate
dehydratase
isopropylmalate
dehydrogenase
leucine
aminotransferase
3-Phosphoglycerate Family
Ser, Gly, Cys
1. 3-Phosphoglycerate
dehydrogenase diverts 3-PG from
glycolysis to aa paths (3phosphohydroxypyruvate)
2. Transamination by Glu gives 3phosphoserine (3-phosphoserine
aminotransferase)
3. Phosphoserine phosphatase yields
serine
•
Serine hydroxymethylase (PLP) transfers the
b-carbon of Ser to THF to make glycine
Figure 25.32
Biosynthesis of glycine
from serine (a) via serine
hydroxymethyltransferase
and (b) via glycine oxidase.
•
A PLP-dependent enzyme makes Cys
•
A PLP-dependent enzyme makes Cys
Some bacteria
most microorganism and plants
Figure 25.33
Cysteine biosynthesis. (a) Direct sulfhydrylation of serine by H2S.
(b) H2S-dependent sulfhydrylation of O-acetylserine.
Figure 25.34
Sulfate assimilation and the generation
of sulfide for synthesis of organic S
compounds. In reaction 1, ATP
sulfurylase catalyzes the formation of
adenosine-5'-phosphosulfate (APS) +
PPi. In reaction 2, adenosine-5'phosphosulfate 3'-phosphokinase
catalyzes the reaction of adenosine 5'phosphosulfate with a second ATP to
form 3'-phosphoadenosine-5'phosphosulfate (PAPS) + ADP. Both
enzymes are Mg2+-dependent. In
reaction 3, PAPS is reduced to sulfite
(SO32-) in a thioredoxin-dependent
reaction. Thioredoxin is a small (12-kD)
protein that functions in a number of
biological reductions (see Chapter 26).
In reaction 4, sulfite reductase catalyzes
the six-electron reduction of sulfite to
sulfide. NADPH is the electron donor.
Sulfite reductase possesses siroheme as
a prosthetic group, the same heme
found in nitrite reductase (Figure 25.2),
which also catalyzes a six-electron
transfer reaction.
Aromatic Amino Acids
Phe, Tyr, Trp, His
• Chorismate as a branch point in this pathway
(Figs. 25.35)
• Chorismate is synthesized from PEP and
erythrose-4-P
• Via shikimate pathway
• The side chain of chorismate is derived from
a second PEP
Figure 25.35
Some of the aromatic
compounds derived
from chorismate.
(1)
2-keto-3-deoxy-Darabinoheptulosonate-7-P
synthase
(2)
dehydroquinate
synthase (note that
the coenzyme
NAD+ is not altered
in this reaction)
(3)
5-dehydroquinate
dehydratase
(4)
shikimate
dehydrogenase
(5)
shikimate kinase
(6)
3-enolpyruvylshikimate-5phosphate
synthase
(7)
chorismate
synthase.
Figure 25.36
The shikimate pathway leading to the
synthesis of chorismate. The starting
substrates are phosphoenolpyruvate
and erythrose-4-phosphate.
The biosynthesis of phenylalanine,
tyrosine, and tryptophan
• At chorismate, the pathway separates into
three branches, each leading to one of the
aromatic amino acids
• Mammals can synthesize tyr from phe by
phenylalanine hydroxylase (Phenylalanine4-monooxygenase)
Figure 25.37 The biosynthesis of
phenylalanine, tyrosine, and
tryptophan from chorismate.
(1)
chorismate mutase
(2)
prephenate dehydratase
(3)
phenylalanine
aminotransferase
(4)
prephenate dehydrogenase
(5)
tyrosine aminotransferase
(6)
anthranilate synthase
(7)
anthranilate-phosphoribosyl
transferase
(8)
N-(5'-phosphoribosyl)anthranilate isomerase
(9)
indole-3-glycerol phosphate
synthase
(10) tryptophan synthase (asubunit)
(11) tryptophan synthase (bsubunit).
Figure 25.38
The formation of tyrosine from phenylalanine. This reaction is normally the first step in
phenylalanine degradation in most organisms; in mammals, however, it provides a route
for the biosynthesis of Tyr from Phe. (Phenylalanine-4-monooxygenase is also known as
phenylalanine hydroxylase.)
Figure 25.39
Tryptophan synthase is an example
of a "channeling" multienzyme
complex in which indole, the
product of the a-reaction catalyzed
by the a-subunit, passes
intramolecularly to the b-subunit. In
the b-subunit, the hydroxyl of the
substrate L-serine is replaced with
indole via a complicated pyridoxal
phosphate - catalyzed reaction to
produce the final product, Ltryptophan. The schematic figure
shown here is a ribbon diagram of
one a-subunit (blue) and
neighboring b-subunit (the Nterminal domain of the b-subunit is
in orange, C-terminal domain in
red). The tunnel is outlined by the
yellow dot surface and is shown
with several indole molecules
(green) packed in head-to-tail
fashion. The labels "IPP" and "PLP"
point to the active sites of the aand the b-subunits, respectively, in
which a competitive inhibitor (indole
propanol phosphate, IPP) and the coenzyme PLP are
bound. (Adapted from Hyde, C.C., et al., 1988. Threedimensional structure of the tryptophan synthase multienzyme
complex from Salmonella typhimurium. Journal of Biological
Chemistry 263:17857-17871.)
Hitidine biosynthesis
• His synthesis, like that of Trp, shares
metabolic intermediates (PRPP) with purine
biosynthetic pathway
• His operon
• Begin from PRPP and ATP
• The intermediate 5-aminoimidazole-4carboxamide ribonucleotide (AICAR) is a
purine precursor (replenish ATP; Ch 26)
Figure 25.40
The pathway of histidine biosynthesis.
(1) ATP-phosphoribosyl
transferase
(2) pyrophosphohydrolase
(3) phosphoribosyl-AMP
cyclohydrolase
(4) phosphoribosylformimino-5aminoimidazole carboxamide
ribonucleotide isomerase
(5) glutamine amidotransferase
(6) imidazole glycerol-P
dehydratase
(7) L-histidinol phosphate
aminotransferase
(8) histidinol phosphate
phosphatase
(9) histidinol dehydrogenase.
Amino acid synthesis inhibitors as herbicides
(inhibitor of acetohydroxy acid synthase)
(inhibitor of 3-enolpyruvyl-shikimate-5phosphate synthase)
(fig 25.36)
(inhibitor of imidazol glycerol-P dehydrtase)
(fig 25.40)
(fig 25.29)
(inhibitor of glutamine synthetase)
25.5 – Degradation of Amino Acids
The 20 amino acids are degraded to produce
(mostly) TCA intermediates
• Energy requirement
– 90% from oxidation of carbohydrates and fats
– 10% from oxidation of amino acids
• The primary physiological purpose of amino
acids is to serve as building blocks for protein
synthesis
• The classifications of amino acids in Figure
25.41
• Glucogenic and ketogenic
Figure 25.41
Metabolic degradation of the common
amino acids. The 20 common amino
acids can be classified according to
their degradation products.
Those that give rise to precursors
for glucose synthesis, such as
a-ketoglutarate,
succinyl-CoA,
fumarate,
oxaloacetate, and
pyruvate, are termed glucogenic
(shown in pink).
Those degraded to acetyl-CoA or
acetoacetate are called
ketogenic (shown in blue)
because they can be converted to
fatty acids or ketone bodies.
Some amino acids are both
glucogenic and ketogenic.
C-3 family (pyruvate):
Ala, Ser, Cys, Gly, Thr, Trp
C-4 family (oxaloaceate & fumarate):
Oxaloaceate: Asp, Asn
Fumarate: Asp, Phe, Tyr
C-5 family (a-ketoglutarate):
Glu, Gln, Arg, Pro, His
Succinyl-CoA:
Ile, Met, Val
Acetyl-CoA & acetoacetate
Ile, Leu, Thr, Trp
Leu, Lys, Phe, Tyr
C-3 family:
Ala, Ser, Cys,
Gly, Thr, Trp
Figure 25.42
Formation of pyruvate from
alanine, serine, cysteine,
glycine, tryptophan, or
threonine.
ADL page 847
The serine dehydratase reaction mechanism-an example of a PLP-dependent belimination reaction.
Figure 25.43
The degradation of
the C-5 family of
amino acids leads to
a-ketoglutarate via
glutamate. The
histidine carbons,
numbered 1 through
5, become carbons 1
through 5 of
glutamate, as
indicated.
Figure 25.44
Valine, isoleucine, and
methionine are converted via
propionyl-CoA to succinyl-CoA
for entry into the citric acid
cycle.
The shaded carbon atoms of
the three amino acids give rise
to propionyl-CoA.
All three amino acids lose their
a-carboxyl group as CO2.
Methionine first becomes Sadenosylmethionine, then
homocysteine (see Figure
25.28). The terminal two
carbons of isoleucine become
acetyl-CoA.
Figure 25.45
Leucine is degraded to acetyl-CoA and acetoacetate via b-hydroxy-b-methylglutaryl-CoA,
which is also the intermediate in ketone body formation from fatty acids (see Chapter 23).
Figure 25.46
Lysine degradation via the saccharopine, a-ketoadipate pathway culminates in the
formation of acetoacetyl-CoA.
Figure 25.47
Phenylalanine and tyrosine degradation. (1) Transamination of Tyr gives phydroxyphenylpyruvate, which (2) is oxidized to homogentisate by p-hydroxy-phenylpyruvate
dioxygenase in an ascorbic acid (vitamin C) - dependent reaction.
(3) The ring opening of homogentisate by homogentisate dioxygenase gives 4-maleylacetoacetate. (4) 4-Maleylacetoacetate isomerase gives 4-fumarylacetoacetate, which (5) is
hydrolyzed by fumarylacetoacetase.
Hereditary defects
Maple syrup urine disease
– After the initial step (deamination) to produce aketo acids
– The defect in oxidative decarboxylation of Ile,
Leu, and Val (25.44)
Phenylketonuria
– The defect in phenylalanine hydoxylase (25.38)
– Accumulation of phenylpyruvate
Alkaptouria
– Homogentisate dioxygenase (25.47)
Nitrogen excretion
Ammonotelic:
– Ammonia
– Aquatic animals
Ureotelic:
– Urea
– Terrestrial vetebrates
Uricotelic:
– Uric acid
– Birds and reptiles