Biochemistry Ch 24 – Amino Acid Biosynthesis

Amino acid synthesis requires solutions to three key biochemical problems:

 Fixation of nitrogen
o Atmospheric N2 needs to be converted to a form usable by biological systems
o Reduction of N2 to NH3 done by bacteria (will be NH4+ in solution)
o Ammonia is source of nitrogen for all amino acids
o The carbon skeletons are obtained from intermediates of glycolysis, TCA cycle, or pentose
phosphate pathway
 Incorporation into aa’s in a stereospecific manner
o Because all aa’s (except Gly) are chiral, biosynthetic pathways must generate the correct
isomer (only L-aa’s) with high fidelity
o This stereochemistry is established using PLP-associated enzymes
 Synthesis of aa’s only when supplies are low
o Amino acids produced but not needed could be broken down for energy, but it takes a lot of
energy to make them so this would be wasteful
o Avoid this waste of cellular energy by strict feedback and allosteric inhibition

Nitrogen fixation
 Strong triple bond in N2 must be broken; this reduction is energetically favorable but has an extremely
high activation energy barrier
 Haber process – fertilizer manufacturers’ industrial nitrogen fixation
o Converts nitrogen to ammonia: N2 + 3 H2  2 NH3
o Requires high temp & pressure, and catalyst
o Uses approx 1% of earth’s energy consumption
 Biologic process – diazotrophic microorganisms fix 60% of N2
o Converts nitrogen to ammonia: N2 + 8 e- + 8 H+  2 NH3 + H2
o Requires 8 high energy electrons, 2 ATP are hydrolyzed for each electron transfer – these
approx 16 ATP are hydrolyzed for each N2 fixed, not to drive the rxn bc it is already
energetically favorable, but to reduce the activation energy barrier
o Catalyzed by nitrogenase complex
 Two protein-complex
 Reductase – provides electrons
 Nitrogenase – binds N2, accepts e-, reduces it to NH3
 Strictly anaerobic (occurs in root nodules that contain leghemoglobin to bind O 2
tightly) – all O2 must be sequestered to prevent O2 reduction
 Both reductase & nitrogenase in the complex are Fe-S proteins:
 Reductase – 4Fe-4S cluster: transfers electrons one at a time from a donor to
nitrogenase, driven by ATP hydrolysis
 Nitrogenase – electrons from the reductase enter at P clusters which store the
electrons until they can be transferred to FeMo cofactors (contain molybdenum
atom) which are the sites of nitrogen reduction
 Biochemistry Ch 24 – Amino Acid Biosynthesis

Ammonium entry into organic molecules

 The next step, the assimilation of N into biomolecules, is the entry of NH 4+ into amino acids
 Glutamate and glutamine play pivotal roles in this regard:
o Glutamate – source of α-amino groups for most aa’s
o Glutamine – contributes its side-chain nitrogen to many nitrogenous compounds
 Glutamate
o Synthesized from NH4+ and α-ketoglutarate by glutamate dehydrogenase
o Remember glutamate dehydrogenase used NAD to breakdown Glu to make α-ketoglutarate
o In synthetic direction, glutamate dehydrogenase uses NADPH for reductive amination:
α-ketoglutarate + NH4+ + NADPH + H+  glutamate + NADP+ + H2O
 Glutamine
o Another ammonium is incorporated into glutamate to form glutamine
o This amidation of glutamate is carried out by glutamine synthetase, driven by ATP hydrolysis
o ATP phosphorylates glutamate, leaving PO4 as a good leaving group, allowing subsequent
amidation by NH4+ to form glutamine
o Low ammonium conditions:
 Glutamate synthase – allows synthesis of glutamates w/o free NH 4+ by catalyzing the
reductive amination of α-ketoglutarate to glutamate using glutamine as the N donor
 Sequential action of glutamine synthetase and glutamate synthase requires ATP to
capture NH4+ when it is scarce
 Glutamate synthase is less efficient (more energy costly) but it is much more specific
for NH4+ (has high affinity for it in low ammonium conditions)

Other amino acids made from intermediates

 So far we’ve only synthesized Glu and Gln
 The other aa’s have their carbon skeletons derived from intermediates of glycolysis, pentose
phosphate pathway, or TCA cycle
 Amino acids can be grouped into six biosynthetic families based on their precursors: oxaloacetate,
pyruvate, α-ketoglutarate, 3-phosphoglycerate, PEP, Ribose-5-P

Humans cannot synthesize all aa’s

 More complex pathways (usually require 5+ steps) have been dropped through evolution, making us
dependent on diet for 9 essential amino acids
 Even though we can produce 11 of the 20 aa’s, at times aa’s may be required in higher amounts than
we can produce (e.g., growth & development)
 Deficiency leads to negative nitrogen balance (where degradation outweighs synthesis)

Generation of Glu, Asp, & Ala from α-ketoacids

 Three α-ketoacids – α-ketoglutarate, oxaloacetate, and pyruvate – can be converted into amino acids
in one step through the addition of an amino group
 These reactions are carried out by PLP-dependent transaminases. PLP holds the aa precursors and
intermediates in a chiral-specific conformation to ensure generation of only L-amino acids
 Glutamate – produced by reductive amination of α-ketoglutarate
o We just discussed this mechanism, via glutamate dehydrogenase with NADPH
o Glu is the most abundant aa; it plays central role in aa synthesis as amine donor:
 Aspartate – produced by transamination of oxaloacetate
o Oxaloacetate + Glu  Asp + α-ketoglutarate
 Alanine – produced by transamination of pyruvate
o Pyruvate + Glu  Ala + α-ketoglutarate
 Biochemistry Ch 24 – Amino Acid Biosynthesis

Generation of Gln & Asn by amidation

 Glutamine – produced by amidation of glutamate
o We already discussed this mechanism, via glutamine synthetase
 Asparagine – produced by amidation of aspartate
o Requires an acyl-adenylated intermediate; driven by ATP hydrolysis
o Similar to formation of Gln, but the source of the sidechain amine for Asn is not NH4+, but
rather the sidechain of glutamine  it does not require free ammonium in the cytosol for its
synthesis. This donation of amine from Gln is a common pathway for aa biosynthesis.

Generation of Pro & Arg from glutamate

 Glutamate can be reduced to a semialdehyde (via ATP hydrolysis and NAPDH) with two fates:
o Transamination to ornithine which will enter urea cycle to produce arginine
o Cyclization & reduction to proline

Generation of Ser, Gly, & Cys from 3-PG

 3-phosphoglycerate, a glycolytic intermediate, can be oxidized and transaminated to yield serine
 Serine can then be modified: subtract 1C to yield glycine; add 1C & sulfur to yield cysteine
 Generation of Gly & Cys uses one-carbon carriers: tetrahydrofolate & S-adenosylmethione (SAM):
o Tetrahydrofolate
 Carrier of one carbon in multiple oxidation states (CH 3, CH2, CHO, etc but not CO2 –
remember it is carried by biotin)
 The carbon carried by THF is bonded to its N-5 and/or N-10 nitrogen atom
 THF consists of pteridine ring, aminobenzoate, and 1-4 glutamates
 Mammals can synthesize these compounds but cannot make the linkage, so they
obtain THF in diet via dihydrotolate (Vit B9)
 Carbon groups can be interconverted between oxidation states while bound to THF,
making them very useful donors in a variety of reactions
 Serine + THF  Glycine + methylenetetrahydrofolate
o The side chain β-methylene group of serine is transferred to THF with loss of hydroxyl group
o Result is glycine, with THF now methylene-THF, the methylene group can undergo various
conversions between oxidation states for variety of biosyntheses
 THF can carry a methyl group but its transfer potential is not sufficiently high for most biosynthetic
methylations. The activated methyl donor is usually S-adenosylmethionine:
o S-adenosylmethionine (SAM)
 Synthesized by transfer of an adenosyl group from ATP to methionine
 This adenylation of methionine is unusual: the triphosphate group of ATP is split to PP i
and Pi, the pyrophosphate is subsequently hydrolyzed to 2 P i
 Methyl transfer from SAM to an acceptor (e.g., aa Activated methyl cycle
precursor) produces homocysteine
 Methionine can then be regenerated by the transfer of a
methyl group from methyl-THF to homocysteine
 The high transfer ability is created by coupling
methionine to ATP to yield SAM
 Biochemistry Ch 24 – Amino Acid Biosynthesis
 SAM is used for methy-additions to small molecules, and also for DNA methylation
 Serine + Homocysteine  Cysteine
o Homocysteine (from the activated methyl cycle) is also source of sulfur for the synthesis of
cysteine from serine
o This conversion requires two enzymes homologous to aminotransferases, that both require
PLP to ensure L-chirality

Essential amino acid synthesis

 We discussed the production of the most of the 11 aa’s that can be produced by humans, the others
are synthesized in prokaryotes and plants
 The essential amino acids have more complicated pathways and thus require more complex
regulation to maintain an appropriate balance of aa’s in the cell
 This difference in basic intermediary metabolism (the dependence on pathways we lack) has been
used as target for inhibitors/poisons:
o Glyphosphate (Roundup) inhibits the aromatic aa pathway present in plants and bacteria but
not present in humans

Regulation of aa synthesis
 Control of aa synthesis is two-fold:
o Long-term – amounts of enzymes are controlled by transcriptionally regulated synthesis vs.
proteasomal degradation
o More immediate – control of activity via some form of feedback inhibition, reversible because
enzyme isn’t wasted
 Inhibition is usually focused on the committed step, the first irreversible step in a multi-enzyme
pathway, where the final product of the pathway (Z below) often inhibits the enzyme that catalyzes
the committed step (AB). This is usually the main, if not the only, regulation of the pathway.

o e.g., serine synthesis in E. Coli

 Committed step in serine synthesis is oxidation of 3-PG by 3-PG-dehydrogenase
 3-PG-dehydrogenase enzyme is inhibited by serine: it is a tetramer with two dimeric
regulatory domains, each of which binds 2 serines
 Binding of serine to regulatory site partially inactivates the enzyme by lowering its V max
 An enzyme bound to 4 serines is essentially inactive
 Thus, if serine is abundant in the cell, 3-PG-DHase is inhibited so 3-PG is not wasted
 But, serine is used for synthesis of glycine and cysteine; so if Gly & Cys pathways are
uninhibited they will prevent the accumulation of serine
 Complex regulation of branched pathways
o Pathways that generate 2 or more aa’s from a common committed step require multiple
feedback mechanisms
o e.g., hydroxyethyl-TPP can react with pyruvate to generate Leu & Val, or
with α-ketobutyrate to make Ile
 one determinant of which path it follows is the concentration of
pyruvate & α-ketobutyrate
 α-ketobutyrate synthesis from Thr is via threonine deaminase
which is inhibited by Ile, but stimulated by Val (bc if Ile is high we
don’t need threonine deaminase working to produce α-
ketobutyrate which is used to make Ile)
 Biochemistry Ch 24 – Amino Acid Biosynthesis
 Regulation of ratio of branched chain amino acids is to balance the production of Ile vs
Val/Leu (because it is difficult to modify the amount of pyruvate)
 Enzyme multiplicity
o Two or more enzymes catalyze the same committed step
o Each enzyme is feedback inhibited by products of the each reaction

 Cumulative feedback inhibition

o Each product of a multi-product pathway can act independently to partially inhibit a common
step in the reaction
o e.g., glutamine synthetase in E. Coli
 Gln is the donor of amines for many different products (several aa’s, carbamoyl
phosphate, nucleotide bases, etc)
 Glutamine synthetase can be partially inhibited by many of the final products
 But no single product can produce the full inhibitory effect
 Enzymatic cascade modulates enzyme activity
o e.g., glutamine synthetase
 activity is controlled by reversible covalent modification by adenylation via attachment
of AMP (adenylated enzyme is less active)
 Coupling protein – adenylyl transferase that couples AMP to glutamine synthetase and
is controlled by regulatory protein (P) in two states, active & deactive:
 PA causes adenylation, partly inactivating glutamine synthetase
 PD has opposite effect, removing the AMP from glutamine synthetase
 Note- this is adenylation, not phosphorylation, but regulation is similar to
kinase/phosphatase activity
 High α-ketoglutarate stimulates PD
 High glutamine stimulates PA (we want glutamine synthetase adenylated and inactive
bc glutamine levels are adequate)
 This cascade amplifies the signals and increases the potential for regulation

Amino acids can be used for many biomolecules

 Besides their obvious role as building blocks of proteins, specific aa’s are the starting material for
synthesis of various molecules (e.g., purines & pyrimidines, reactive end of sphingosine, various
signaling molecules and hormones, and nicotinamide unit of NAD +)
 Glutathione
o Tripeptide found in high concentration in cells (contains Glu, Cys, Gly)
o It is a highly distinctive amino acid derivative with several important roles
o It cycles between a reduced thiol form (GSH) and an oxidized disulfide dimer form (GSSG)
o GSSG can be reduced back to GSH via glutathione reductase and NADPH
o The ratio of GSH is approx 500x that of GSSG, thus preserving a reducing environment
 Biochemistry Ch 24 – Amino Acid Biosynthesis
o GSH plays key role in detoxification by reacting with peroxides, catalyzed by glutathione
peroxidase
 Nitric oxide
o Short lived cellular messenger produced by nitric oxide synthase from arginine, NADPH, and O2
o NO works by binding to and activating guanylate cyclase, an important enzyme in signal
transduction
o NO causes a variety of cellular responses, ranging from mitochondrial biogenesis (growth &
division) to local modulation of blood pressure
 Porphyrins
o Complex ring compounds that form hemes & chlorophylls
o They are synthesized from succinyl CoA and glycine, forming a cyclized compound bound to an
iron ion
o Heme degradation is clinically important process at wound sites and in liver:
 Heme + 2 O2 + NADPH releases the iron and transforms the porphyrin to billiverdin
(green compound) which is reduced to billirubin (red-brown compound)
 This chromatic change can be seen in a severe bruise
 Jaundice – billirubin released into circulation (indication of liver malfunction), giving
skin a yellowish color