Amino Acid Synthesis Flashcards by Connor Schmidt

What is the source of nitrogen for amino acid synthesis?

Atmospheric Nitrogen (N2)

abundant- 80%
Triple Bond is very strong-extrememly unreactive

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Sources of Nitrogen Fixation

1) 60% Diazotrophic (nitrogen fixing) microorganisms
- Rhizobium Bacteria
2) 15% lighting and UV light
3) 25% commercial process
- habers process
- N2 + 3H2-> 2NH3
- requires 300 atm, 500 Degrees F, Iron Catalyst

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Nitrogen Fixation by Rhizobium Bacteria

-Nitrogenase complex

Nitrogenase complex

1) reductase
- Provides 8e- from reduced ferredoxin with high reducing potential
- ATP hydrolysis used to transfer e- to nitrogenase (2ATP/ e-)
2) Nitrogenase
- uses electrons provided by reductase to reduce N2 to NH3+

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Rhizobium Bacteria: Reductase structure

AKA

iron proteins
Fe protein

Dimer of identical subunits

1) 4Fe-4S cluster
- bridges subunits
- transfers E- ONE AT A TIME to Nitrogenase
2) each subunit contains ATP Binding domain (Ploop)

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Rhizobium Bacteria: Nitrogenase Structure

AKA:

Molybdenum-iron protein
MoFe protein

A2B2 Tetramer
1)FeMe Cofactor
-uses electrons from P cluster to reduce N2 to NH3
-2 M-3Fe-3S custers
M=Mo in one cluter
M= Fe in other cluster
*organization unique to nitrogenase
2) P cluster
-stores e- before transfer and use by FeMo

This ENYZME STILL A SUBJECT OF ACTIVE RESEARCH

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Source Of Component for Amino Acid Synthesis?

Nitrogen:
-Glutamate from Ammonia

Carbon Skeleton:

Pyruvate
Acetoacetyl CoA
Acetyl CoA
OAA
Fumurate
Succinyl CoA
A-ketogluterate

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Humans can synthesize the 11 nonessential amino acids. What are they?

Alanine, Arginine, Asparagine, Aspartate
Glutamate, Glycine, Glutamine
Cysteine, Serine, Tyrosine
Proline

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Glutamate Dehydrogenase

Assimilates Ammonium Ion (NH4+) into Glutamate (two step process)
-Transamination of alpha amino group nitrogen of glutamate provides (most) amino acids with alpha amino group

Mechanism:

1) Schiff Base between ammonia and carbonyl of a-ketogluterate
- replace C=O with C=NH2+, and releases H2O
2) Protonated Schiff Base is reduced by transfer of hydride from NADPH

Estabilishes stereochemistry

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Common Feature of amino acid synthesis

Formation of Schiff Base followed by protonation and reduction

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Synthesis of Glutamine

Glutamine Synthetase
Amidation of R group of glutamate produces glutamine

Precursor-Glutamate
Activated by:Phosphorylation-Phosphate added to R group carboxylic acid
-Phosphate is displaced by NH3+ to form Glutamine

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Synthesis of Aspartate

Aspartate Transaminase
-synthesized from a-ketoacids in one step by PYRIDOXAL PHOSPHATE-dependent transaminases

Precursor-OAA
OAA + Glutamate -> Aspartate + a-ketogluterate

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Synthesis of Alanine

Alanine Transaminase
-synthesizes from a-ketoacids in one step by Pyridoxal Phosphate-dependent transmaminases

Precursor: Pyruvate
Pyruvate + Glutamate -> Alanine + a-ketogluterate

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Synthesis of Asparagine

Asparagine Synthetase

Precursor: Aspartate
Activation- Adenylation
-Asparate adenylated to form Acyl Adenylate intermediate
-Glutamine provides NH3+ which displaces AMP to form Asparagine

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Synthesis of Proline

Multiple Steps
Precursor: Glutamate
Activated- Phosphorylation
-Acyl-Phosphate Int is reduced to Glutamic Y-semialdehyde at the expense of NADPH to NADP+
-Glutamic Y-Semialdehyde dehydrated(No enzymatic reaction-spontaneous) then reduced to Proline at the expense of NADPH to NADP+

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Synthesis of Arginine

Multiple Steps
Precursor: Glutamate
Activated- Phosphorylation
-Acyl Phosphorylate Intermediate is reduced to Glutamic y-semialdehyde at the expense of NADPH to NADP+\
-Glutamate transfers amino group to Glutamic Y-semialdehyde to form Ornithine
-Ornithine enters Urea Cycle-> Arginine

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Synthesis of Serine

Multiple steps
Precursor: 3-Phosphoglycerate
-3-PG oxidized to 3-phosphohydroxypyruvate at the expense of NAD+ to NADH
-Glutamate transfers Amino group to 3-phosphohydroxylpyruvate forming a-ketogluterate + 3-Phosphoserine
-3-phosphoserine is hydrolyzed displacing the Phosphate with OH to produce Serine

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Synthesis of Glycine

2 Pathways

1)Serine hydroxymethyl transferase
-reversing this enzyme can be used to produce serine from glycine
-allows one carbon units to be synthesized from carbohydrates
Precursor- Serine

Serine + THF -> glycine + methylenetetrahydrofolate + H2O

2)Serine Synthase (glycine cleavage enzyme)
Precursor: NH4 + CO2
NH4 + CO2 + N5,N10methyleneTHF + NADH-> GLYCINE + THF + NAD+

THF

tetrahydrofolate=COFACTOR
Carries activated one carbon units
-one carbon units attach to N5 or N10 or both
-one carbon units are interconvertible while attached to THF

Humans unable to synthesize
-obtained from diet or intestinal microorganisms

SAM

S-Adenosylmethionine

Methyl Group donor as part of activated methyl Cycle

Synthesized from Met and ATP
Methyl group is activated due to + charge on sulfur

ALL 3 phosphates are lost
-hydrolyzed into Pi (orthophosphate) and PPi (pyrophosphate)-> further hydrolyzed to 2Pi

Activated Methyl Cycle

Regenerates Methionine from homocysteine

1) Methionine + ATP-> S-adenoysl Methionine (SAM)
2) SAM releases Activated CH3 to form S-adenosyl Homocysteine
3) S-Adenosyl Homocysteine is hydrolyzed to form homocysteine+ adenosine
4) Homocysteine is methylated (-CH3)-> Methionine
- catalyzed by Methionine synthase (aka homocysteine methyl transferase)
- N5-methyl THF-> THF
- requires Methylcobalamin derived from Vit B12

Synthesis of Cysteine

2 steps:
Precursor: Serine and Homocysteine
1)Cystathionine B-Synthase
Homocysteine + Serine-> Cystathionine + H2O
-Ser R group attaches to Sulfur and loses O

2)Cystathioninase
Cystathionine hydrolyzed to NH4+ + a-ketobutyrate + CYSTEINE

Synthesis of Tyrosine

Phenylalanine Hydroxylase (monooxygenase)
Precursor: Phenylalanine
Phe + O2+ tetrahydrobiopterin-> Tyr + H2O + quinoid dihydrobiopterin
-Hydroxylates

-in humans Phe is essential if Phe is not present then tyrosine becomes essential

Synthesis of Amino Acids that are essential in humans are found in?

Plants and bacteria

Plant and Bacteria:

-Phenylalanine, tryptophan, Tyrosine synthesis

Bacteria: substrates

Phophoenolpyruvate from glycolysis
erythrose 4-Phosphate from Pentose phosphate pathway

Intermediates

Shikimate after phosphorylation can be inhibited by Glyphosate (round up)
Chorismate-common intermediate for aromatic aa **

Substrate channeling

Indole is hydrophobic and would diffuse out of cell thus use Substrate channeling: - Substrate goes through channel and is passed from active site to active site until released - speeds up rate of synthesis

Chirality of Amino Acids | -all aminotransferases contain 2 conserved amino acids?

Lysine that forms the Schiff base with PLP | -Arginine that interacts with alpha-carboxylate group of the ketoacid

Methionine Synthase

AKA homocysteine methyltransferase - requires methylCobalamin derived from Vit B12 - this enzyme exists in some organisms that doesn't require methylcobalamin Homocysteine is methylated by N5,methyl THF-> methionine + THF

PRPP

activated form of ribose

Negative Nitrogen Balance

Deficiency of even one amino acid leads to this More proteins degraded then synthesized, and more nitrogen is excreted then ingested

What do all aminotransferases contain?

- Lysine that forms Schiff base with PLP | - Arginine that interacts with alpha carboxylate group of the ketoacid

Glutathione

Sulfhydryl Buffer and Antioxidant that protects us from Reactive Oxygen Species (ROS) Structure- Tripeptide w/Sulfhydryl ECG -Glutamate R group attaches to Cysteins N displace O- GSH-> GSSG Glutathione reductase -Uses NADPH to reduce GSSG to GSH -Prosthetic group-FAD Glutathione Peroxidase Reduces hydrogen peroxide to water Selenium (Se) replaces Sulfur in R group of cysteine active site -E-Se-(selenolate) reduces peroxide to hydroxyl while being oxidized to to an acid E-S-OH (Seleninic acid) -E-S-OH is then reduced by GSH which is oxidized to GSSG -GSSG then reduced by NADPH to NADP+

Nitric Oxide

Second messenger in signal transduction pathway - synthesized from Arginine, NADPH, and O2 - enzyme is another reason why animals must inhale O2 Nitric Oxide Synthase 1) Arginine ->N-w-hydroxyarginine - O2 (monooxygenase)-> H2O - NADPH to NADP+ 2) N-w-Hydroxyarginine-> Citrulline + NO - O2-> H2O (monooxygenase) - NADPH- to NADP+

Porphyrins | -no mechanism just general info

used to synthesize heme - synthesized from Succinyl CoA and glycine - cyclic compounds that readily bind iron

Where does Heme Synthesis occur? | -Compartmentalization?

Cellular Localization - Mitochondria - Cytoplasm Organ Localization - Liver=variable rate - Erythrocytes producing cells of bone marrow: constant rate

S-aminolevulinate Synthase

1) S-aminolevulinate Synthase Gly + Succinyl CoA -> S-aminolevulinate (ALA or S-aminlevulinic acid) -Amino Group of Glycine attaches to Carbonyl of Succinyl CoA -releases CoA and CO2 -occurs in matrix of mitochondria 8 S-aminoleveulinate is required to synthesize one heme

S-aminolevulinic dehydratase

S-aminolevulinic (x8)-> (x4) Porphobilinogen - occurs in cytosol - two molecule are joined to form porphobilinogen *Lead inhibits causing the anemia associated with lead poisoning

Hydroxymethylbilane synthase

-links 4 porphobilinogen molecules to form linear tetrapyrole

What are the last 3 steps in Porphyrin synthesis

Uroporphyrinogen III synthase - cytosol - cyclized linear tetra pyrrole to form ring Uroporphyrinogen carboxylase - cytosol - produce coproporphyrinogen III has methyl instead of A group Coproporphyrinogen-> Protoporphyrin IX - matrix of mitochondria via multiple steps - Fe added is final step to form HEME

Ferritin

Iron storage protein

Transferrin

Iron transport protein

Ferrochelatase

enhances rate of addition of iron (Fe2+) to protoporphyrin IX -this may occur without enzyme as well

Degradation of Heme

1) Heme-> Biliverdin - O2-> H2O (monooxygnease) - releases Fe3+ - NADPH-> NADP+ 2) Biliverdin-> Bilirubin - Biliverdin reductase - NADPH to NADP+