Amino Acid Metabolism Flashcards
Overview of
Amino Acid Metabolism
- Essential (dietary) vs non-essential (endogenous) AA
- Excess AA catabolized by oxidative degradation.
- During periods of physiological stress proteins degraded to AA and used for energy production.
- There is no storage form of AA solely for energy production.
Essential Amino Acids
HV MILK FTW
- Histidine
- Valine
- Methionine
- Isoleucine
- Leucine
- Lysine
- Phenylalanine
- Threonine
- Tryptophan
Cachexia
The extreme degradation of muscle proteins during periods of physiological stress.
Usually associated with inflammatory diseases.
Results in muscle-wasting and lipolysis.
Favored by increase in glucocorticoids, catecholamines, and cytokines.
Amino Acid
Nitrogen Catabolism
First step in the degradation of AA.
Removal of the α-amino group as NH3 from most L-amino acids accomplished by transamination then oxidative deamination.
(Except for Thr and Lys)
Called Transdeamination
Transamination
- The transfer of the α-amino group from an AA to α-ketoglutarate producing an α-keto acid and glutamate.
- Catalyzed by aminotransferases aka transaminases
- Freely reversible reaction
- Results in the collection of amino groups from different AAs in the form of glutamate with no net loss of nitrogen.
- Glutamate is the only AA that undergoes oxidative deamination to any significant extent.
Mechanism:
- α-NH2 transferred from the AA to the PLP coenzyme generating pyridoxamine phosphate and an α-keto acid.
- Transfer of the α-NH2 from coenzyme to α-ketoglutarate forming glutamate and regenerating PLP.
Aminotransferases
aka
Transaminases
- Each enzyme is specific for one to several amino acids and named for the donor
- Requires a pyridoxal phosphate (PLP) coenzyme
- Vit B6 derivative
- Linked to a lys residue in the active site
- Reaction is freely reversible so can be used to synthesize non-essential AA from α-keto acid precursors.
- Normally intracellular enzymes so can be used as a diagnostic measure of cellular damage
- Example: AST and ALT are abundant in the liver and used as liver function tests
Alanine Aminotransferase
(ALT)
Aspartate Aminotransferase
(AST)
Catalyzes the conversion of aspartate to oxaloacetate.
Used as part of the Malate-Aspartate Shuttle.
Oxidative Deamination
Removal of the amino group from glutamate producing ammonia (NH3) and α-ketoglutarate.
Anaplerotic reaction.
Catalyzed by glutamate dehydrogenase (GDH)
- Mitochondrial enzyme
- Primarily in liver and kidney
- Requires NAD+ for the forward catabolic reaction
- Requires NADPH for the reverse synthetic reaction
- Forward reaction allosterically regulated
-
Activated by ADP
- low energy signal in the mitochondria
- Inhibited by GTP
-
Activated by ADP
- Reverse reaction likely occurs only if [NH3] is high
Deamination of Threonine
“Direct deamination” of threonine produces NH3 and α-ketoglutarate.
Catalyzed by PLP-requiring dehydratase.
Deamination of Lysine
Several enzymatic steps required to generate a dicarboxylic amino acid which an undergo transamination by aminotransferase.
Deamination of D-amino acids
- D-amino acids present in processed foods and microorganisms
- D-ser is an agonist of glutamate neuroreceptor and formed by racemase in the CNS
- Oxidatively deaminated in the liver and kidney peroxisomes by D-amino acid oxidase
- Requires FAD coenzyme
- Ammonia + α-ketoacid + H2O2 produced
Sources of Ammonia
- Produced in AA deamination
- Deamidation of Asn → Asp or Gln → Glu
- Catabolism of nucelotides and other N-containing compounds
- Metabolism of gut bacteria
Properties of Ammonia
- Exists in solution primarily as ammonium ion (NH4+)
- Toxic especially to the brain because can cross the BBB
- Normally present in the blood in very low concentrations
- Hyperammonemia = elevated plasma ammonia levels
- Transported in non-toxic forms:
- Glutamine
- Urea
- Indirectly by Alanine
Glutamine Transport of NH3
Glutamate + NH4+ + ATP → Glutamine
Catalyzed by glutamine synthetase
Cytosolic enzyme primarily in liver, brain, and skeletal muscle.
Upregulated in response to glucocorticoids.
♦
Plasma Gln levels 25% of circulating AA.
♦
Glutamine → Glutamate
By glutaminase
Mitochondrial enzyme in liver, kidney, and CNS.
Urea Cycle
Overview
- Converts toxic ammonia (NH3) to non-toxic urea.
- Occurs in the liver.
- Urea diffuses into the plasma and excreted by the kidneys as UUN (urinary urea nitrogen)
- Involves 5 enzymatic reactions
- 2 in mitochondrial matrix
- 3 in cytosol
- Consumes 4 equivalents of ATP / urea produced
Urea Cycle
Mechanism
-
HCO3- + NH3 + 2 ATP → Carbamoyl Phosphate
By carbamoyl phosphate synthetase I (CPS I)- Absolute requirement of N-acetylglutamate (NAG) as an allosteric activator.
- Ammonia provides one of the nitrogen atoms of urea
- Carbon dioxide provides the carbon of urea
-
Carbamoyl Phosphate + Ornithine → Citrulline + Pi
By ornithine transcarbamoylase (OTC)**
Citrulline exchanged for ornithine across the mitochondrial matrix by an antiporter.
3. Citrulline + Aspartate + ATP → Argininosuccinate + AMP + PPi
By argininosuccinate synthetase (ASS)
* Asp provides one of the nitrogen atoms of urea
* N in aspartate from glu via transamination of OAA by AST.
4. Argininosuccinate → L-Arginine + Fumarate
By Argininosuccinate lyase
* Cleaved so that only N from Asp remains in the molecule.
* Fumarate formed links UC and TCA cycles
* Can be converted to malate by fumarase then to OAA by malate dehydrogenase.
* NADH produced by malate dehydrogenase compensates for energy cost of UC.
5. L-Arginine → ornithine and urea
By arginase-1
* Enzyme almost exclusive to the liver
* Ornithine transported back into the mitochondria
* Urea exits and excreted
Hepatic Regulation of Ammonia
Glutaminase and GDH complex plus the urea cycle located in the periportal hepatocytes.
Gln → Glu + NH3
NH3 cleared by UC
Gln synthetase in the perivenous hepatocytes.
Ensures any NH3 missed by the UC gets converted to Gln before entering the plasma.
Together results in very little NH3 escaping the liver.
Urea Cycle Regulation
- Changes in substrate concentration
- Activation of CPS I by NAG
- Changes in enzyme concentration
- Increases by 20-30x in prolonged starvation
Urea Cycle Pathologies
- Most disesases are autosomal recessive
- OTC is the only X-linked enzyme of the urea cycle
- Defect causes X-linked OTC deficiency
- Severe hyperammonemia usually seen causing encephalopathy
- Lethargy → Coma → Death
- Can cause orotic aciduria because carbomyl phosphate backs up and is used to make orotic acid in pyrimidine synthesis.
- Treatment goals:
- Promote growth and development
- Prevent hyperammonemia
- Treatment involves:
- Severe protein restriction by providing calories as CHO and fat
- Supplementation with α-keto acid analogs of the essential AA
- Use of antibiotics (because gut bacteria make ammonia)
- Supplementation with arginine in some cases
- Because NAG synthase which produces the NAG required by CPS I is postively effected by Arg.
- Administration of compounds that bind non-essential AA for excretion
CPS I vs CPS II
α-Keto Acid Metabolism
The α-keto acids formed after transdeamination of AA feeds into carbohydrate and lipid metabolism.
Energy intermediate to which an AA is metabolized is determined by its structure.
Leu & Lys are strictly ketogenic.
Phe, Iso, Trp, & Tyr are both ketogenic and glucogenic.
Threonine is very minimally ketogenic but almost exclusively glucogenic.
All the rest are strictly glucogenic.
Branched-chain AA
Catabolism
Leucine, Isoleucine, Valine
Muscle responds to physiological stress by degrading proteins followed by the release & catabolism of BCAA.
- Transamination catalyzed by a single branched-chain amino acid aminotransferase while α-ketoglutarate → glutamate.
- Enzyme almost predominantly in muscle, very low in liver.
- Once α-keto acid formed it can be sent out to the liver which can assist with deamination.
- α-keto acids oxidatively decarboxylated and attached to CoA by as single branched-chain α-keto acid dehydrogenase (BCKAD or BCKD). NAD+ → NADH
- TPP, CoA, lipoic acid, NAD, and FAD coenzymes
- BCAA-CoA derivatives undergo FAD-linked dehydrogenations.
- Val & Ile reactions analogous to FA β-oxidation
- Hydration, NAD+-linked dehydrogenation, thiolytic cleavage.
- Forms FADH2
- Leu undergoes dehyrogenation to form methylcrotonyl CoA which undergoes biotin-dependent carboxylation. Forms NADH.
- Val & Ile reactions analogous to FA β-oxidation
- Specific end-products generated
- Leu & Ile → acetyl CoA → ketogenesis
- Ile and Val → propionyl CoA → methylmalonyl CoA by biotin-dependent carboxylation → succinyl CoA by mutase + deoxyadenosyl cobalamin coenzyme (Vit B12)
- Succinyl CoA → OAA → PEP → Pyruvate → Ala
Glucose-Alanine Intertissue Cycle
- Succinyl CoA generated via catabolism of Val & Ile converted to pyruvate then alanine via ALT.
- Alanine transported to the liver where glutamate and pyruvate reform by ALT.
- Pyruvate substrate for gluconeogenesis.
- Glutamate can be deaminated and the NH3 used in the urea cycle.
- Ala is another way of transporting ammonia to the liver.
Glutamine Metabolism
- Glutamine generated by glutamine synthetases.
- Upregulated in response to glucocorticoids.
- Gln enters the blood and is metabolized by the gut, liver, and kidney.
Gut
- Gln deamidated to Glu + NH3 by glutaminase.
- Glu converted to citrulline (similar to urea cycle)
- Citrulline sent out in the blood and picked up by kidney
- Kidney uses citrulline for the synthesis of Arg.
- Has ASS and ASL enzymes
- Arg used for the synthesis of proteins including creatine and nitric oxide
- Glu can also be converted to pyruvate by ALT generating α-ketoglutarate and Ala which are sent out to the blood.
- Ala and α-KG used by liver and kidney for gluconeogenesis.
- Glu converted to citrulline (similar to urea cycle)
Liver and Kidney
- Gln also deaminated to Glu + NH3 by glutaminase.
- Glu can be:
- Transaminated as above
- Oxidatively deaminated by GDH to NH3 + α-KG
- α-KG → OAA → PEP → PEPCK → PEP → Glucose
Hepatic Ammonia Use
- NH3 used in the synthesis of Gln or Urea
- Glu + OAA → α-kg + asp
- Asp used for the urea cycle
Tryptophan Metabolism
Essential AA
Both gluco and ketogenic.
Catabolism
-
Trp first undergoes an oxidative cleavage of the pyrole ring producing N-formylkynurine.
Catalyzed by heme-containing Trp Oxygenase - Hydrolytic cleavage of the formyl group yields kynurenine and formate.
-
Kynurenine can be:
- Catabolized to alanine and acetoacetyl CoA ⇒ primary pathway
- Metabolized to quinolinate ⇒ quantatively minor but physiologically significant pathway
- Quinolinate & dietary niacin can both be converted to NAD/NADP.
Quinolinate Metabolism
Intermediate of tryptophan metabolism
Both quinolinate and dietary niacin (nicotinic acid, NA) can be conerted to NAD and NADP.
NAD/NADP used in redox reactions.
NAD can donate its ADP-ribose moiety to a variety of proteins in ADP-ribosylation.
Pellagra
- Disease of dietary deficiency of niacin (and Trp)
- Prevalent where cornmeal is the dietary staple
- Low in Trp
- Niacin bioavailability is low unless corn soaked in an alkaline solution
- Diagnosed by pattern of urinary AA (neutral/monoaminomonocarboxylic)
- Symptoms include:
- Photosensitivity dermatitis
- Diarrhea
- Intermittent ataxia
Serotonin Synthesis
Made from tryptophan via hydroxylation (THB) and decarboxylation (PLP)
Phenylalanine → Tyrosine
- Phe irreversibly converted to tyr by hydroxylation at C4 by phenylalanine hydroxylase.
- Liver enzyme
- Requires tetrahydrobiopterin (THB, BH4) coenzyme
- Hydroxylases that work on aromatic AA use THB instead of PLP
Phenylketonuria
(PKU)
- Caused by a deficiency in phenylalanine hydroxylase
- Most common disease of enzyme deficiency in AA metabolism
- Autosomal recessive
- Causes hyperphenylalaminemia (HPA)
- As a consequence of enzyme deficiency normally minor pathways become amplified:
- Causes phenylpyruvate to appear in the urine
- Gives urine a characteristic “mousey” odor
- Can cause reduced pigmentation because melanin synthesized from Tyr
- Symptoms include:
- Microcephaly
- Severe intellectual disability
- Drastically shortened life expectancy
- Diagnosis
- First disease to be part of newborn screening ⇒ Guthrie Test
- Neonatal screening now available
- New Tandem Mass Spec testing
- Treatment
- Diet low in Phe coupled with tyr supplementation
- Aspartame (Asp-phe-methylester)
- Multifactorial
- Diet low in Phe coupled with tyr supplementation
- Offspring of women with PKU can have high incidence of microcephaly, mental retardation, and major cardiac anomalies due to intrauterine exposure to high Phe which acts as a teratogen.
Tyrosine Catabolism
Methionine Catabolism
Met and Cys are both glucogenic.
Met is an essential AA.
-
Met consenses with ATP forming S-_adenosylmethionine_ (SAM or AdoMet).
- Rare in that only adenosine remains in the molecule and Pi + PPi hydrolyzed driving rxn.
- CH3 group in SAM is attached to a sulfur and is readily transferred by methyl transferases to a variety of acceptors including phospholipids, proteins, nucleic acids, and biogenic amines.
- After donating the methyl group SAM becomes S-adenosylhomocysteine (SAH or AdoCys) which gets hydrolyzed to adenosine and homocysteine.
-
Homocysteine (hcy) has 2 possible fates:
-
Transulfuration to form cysteine via 2 step process both requiring PLP
- Hcy condenses with ser to form cystathionine catalyzed by cystathionine synthase
- Cystathionine is hydrolyzed to cysteine, α-ketobutyrate, and NH3
-
Remethylated to Met
- Catalyzed by Methionine synthase (aka homocysteine methyl transferase)
- Only 1 of 2 reactions that require Vit B12.
- ONLY reaction where N5-methyl THF carrries and donates a methyl group.
- Vit B12 acts as recipient then donates methyl to Hcy for Met.
- If B12 deficient THF trapped in the non-utilizable N5-methyl THF form ⇒ folate trap
-
Transulfuration to form cysteine via 2 step process both requiring PLP
One-carbon Carriers
SAM and THF (cosubstrates)
Biotin (prosthetic group)
Carry and donate activated one-carbon units
Tetrahydrofolate
(THF)
- Biologically active form of the water-soluble vitamin folic acid (folate)
- Formed from folic acid in a two-step NADPH-requiring reduction reaction
- Catalyzed by enzyme of the intestinal mucosa dihydrofolate reductase
- THF can get methylated at N10
- Typically Ser is the donor but Gly and His also sources
- Rxn requires PLP
- Once bound to THF the one-carbon unit can be oxidized or reduced
- methyl-THF most reduced
- formyl-THF most oxidized
- Can form a bridge between N5 and N10
⇒ N5,N10-methylene THF- Used for dUMP → dTMP
- Can be reduced to N5-methyl THF
- Major form in blood bound to albumin
- In tissues N5-methyl THF gets demethylated to THF as Hcy → Met
Folate Deficiency
- Most common vitamin deficiency in the US
- Can lead to macrocytic anemia
- Folate supplementation in the periconceptual period to significantly reduce NTD in fetus
- If Vit B12 deficient then THF trapped in the 5-methyl THF form
Biotin
- Water-soluble B-complex vitamin
- Prosthetic group of most enzymes involved in carboxylation reactions
- Acetyl CoA carboxylase
- Propionyl CoA carboxylase
- Pyruvate carboxylase
- Methylcrotonyl CoA carboxylase
- Attaches via Lys residue
- Apocarboxylase is biotinated by holocarboxylase synthetase
- Degradation of holoenzymes produces biocytin (biotin-lys) which is cleaved by biotinidase to recycle biotin
Deficiency in either enzyme causes multiple carboxylase deficiency.
Clinical signs are neurological and cutaneous.
Treated with oral biotin.
Disorders of AA Metabolism
Not for memorization.
