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NitrogenMetabolism
Dr.KevinAhern
Nitrogen Metabolism
Nitrogen Forms in the Body
Nitrogen Balance Critical
Body Must Make and Break Down Amino Acids
Nitrogen Also Needed for Synthesis of
Nucleotides (ATP, GTP, CTP, UTP, dATP, dCTP, dGTP, dTTP)
Non-Protein Amino Acids
Ornithine
Citrulline
Sarcosine
Other Nitrogen-Containing Compounds
Choline
Vitamins
Carnitine
Ornithine
Citrulline
Sarcosine
Excretion of Nitrogen
Amino Acids Through Transamination Make Nitrogen Mobile
Toxicity of Ammonia means Nitrogen Balance is Critical in the Body
Excretion
Ammonotelic - Excrete Ammonia - Fish
Uricotelic - Excrete Uric Acid - Birds
Ureotelic - Excrete Urea - Most Vertebrates, Some Invertebrates
Produced by Amino
Acid Catabolism
Used in Urea Cycle
Produced by Purine
Catabolism
Produced by
Urea Cycle
Ammonia
Uric Acid
Urea
α-ketoglutarate Family
Transamination to Make Glutamate
Amino Acid X
α-ketoacid #1
α-keto Acid #1 + Amino Acid X
Amino Acid #1 + α-keto Acid X
Amino Acid #2
α-ketoacid X
Glucose-Alanine Cycle
Alanine Metabolism
High Ammonia
Glucose-Alanine Cycle
Important for Removing Ammonia
Transamination in Liver
Creates Glutamate
Breakdown of Glutamate
Yields Amine for Urea Production
Alanine Carries Amine to Liver
Low Ammonia
Urea Cycle
Primarily Occurs in Liver. Also in Kidney
Consists of 4 Cycle Reactions and 1 Feeder Reaction
Feeder Reaction Incorporates 1 Molecule of Ammonia and 1 CO2 Per Turn
Cycle Reaction Provides 1 Amine from an Amino Acid
Output of Cycle is 1 Molecule of Urea Per Turn
The Net Reaction Per Turn of the Cycle is
2 NH3 + CO2 + 3 ATP + H2O → urea + 2 ADP + 4 Pi + AMP
Urea Cycle
Carbamoyl Phosphate Synthetase Reaction
The Source of Ammonium Ion is Glutamine or Glutamate
Requires Action of Glutaminase (Glutamine) or Glutamate Dehydrogenase (Glutamate)
Glutamine + H2O
Glutamate + H2O + NADP+
Glutamate + NH3
α-ketoglutarate + NH3 + NADH + H+
H2O + CO2
HCO3- + NH4+
H2O + CO2
HCO3- + NH4+
2 ATP + HCO3− + NH4+ <=> 2 ADP + Carbamoyl phosphate + P
Ornithine Transcarbamoylase Reaction
Carbamoyl Phosphate
Enzyme Expressed Only in Liver
Most Commonly Deficient Enzyme in Urea Cycle
X-linked Inheritance
In Severe Deficiency, Ammonia Levels Rise
to Lethal Levels if Untreated
Liver Transplant and Low Protein Diet Most
Common Treatments
+
Ornithine
Ornithine Transcarbamoylase
Citrulline
+
Pi
Phosphate
Citrulline Transport to Cytoplasm
Citrulline Movement to Cytoplasm Requires Ornithine-Citrulline Translocase
Antiport - Moves Citrulline Out, Ornithine In
Needed for Both Parts of Urea Cycle
Deficiency of Translocase Mimics Defective Ornithine Transcarbamoylase
Condition at Birth More Serious Than Adult Onset
Argininosuccinate Synthetase
Two Step Reaction
First, AMP Attaches to Amine-rich End of Citrulline
Next, Aspartate Displaces the AMP
The Product is L-argininosuccinate
Reaction is Rate Limiting Step of Cycle
Gene Expression of Enzyme Reduced by Arginine, Increased by Citrulline
Enzyme Defects Lead to Citrullinemia - Accumulation of Ammonia
Treated with Low Protein Diet, Arginine Supplementation
1
2
Argininosuccinate Lyase
Important for Production of Arginine
Argininosuccinate Lyase
Source of Fumarate
Deficiency Like That of Other Urea Cycle Enzymes - Ammonia Excess
Bond Cleaved
To Proteins or Remainder
of Urea Cycle
+
Argininosuccinate
Lyase
Argininosuccinic Acid
To Citric Acid Cycle
Arginine
Fumaric Acid
Arginase
Co-expressed with Nitric Oxide Synthase in Smooth Muscle
Increased Arginase Activity Reduces Nitric Oxide Production
Nitric Oxide Relaxes Smooth Muscle and Facilitates Erection of Penis
Deficiency of Arginase Rarest of Urea Cycle Enzymes
Two Forms of Arginase Provide Some Backup When One Deficient
To Mitochondria
Excreted To Complete Cycle Through
Ornithine Citrulline Translocase
Cut
+
+
H2O
Arginase
Arginine
Urea
Ornithine
Urea Cycle
Citrulline
Alternate Means of Producing Citrulline - Nitric Oxide Synthase
Bypasses Mitochondrial Part of Urea Cycle & Produces Nitric Oxide
Arginine is Substrate for Reaction
2 L-arginine + 3 NADPH + 1 H+ + 4 O2
Nitric Oxide Synthase
2 Citrulline +2 Nitric Oxide + 4 H2O + 3 NADP+
Nitric Oxide Important Signaling Molecule in Humans - Vasodilation
Viagra works by enhancing signaling through the nitric oxide pathway in the penis
Nitrites
Nitrite formed by Ionization of Nitrous Acid (HNO2) or Reduction of
Nitrates
Nitrite Used to Cure Meats and Prevent Botulism
Can Be Reduced to Nitric Oxide in Hypoxic Conditions
In Human Diet 80-90% from Reduction of Nitrates in Vegetables
Nitrates in Vegetables From Fertilizers or Plant Stresses
Nitrite Readily Forms Cancer-Causing Nitrosamines in Stomach Acid
Nitrites Oxidize Hemoglobin’s Iron From Ferrous (II) to Ferric(III) State
- Unable to
Carry Oxygen - Can be Serious
Nitrosamines
Nitrosamines Produced by Reaction of Nitrites and Secondary Amines, Such as Proline
Strong Acids (Stomach) or High Temperatures of Frying Favor Production
Found in Processed Meats, Beer, Cigarette Smoke, Chewing Tobacco
Formation Inhibited by Vitamin C
2H+
H2NO2+ → H2O + NO+
Nitrous Acid Ion Nitrosonium Ion
Nitrite
→
NO+ +
H
Secondary
Amine
Nitrosamine
Nitrosamines
Nitrosamines Form DNA Adducts and Cause Cancer in Many Animal Species
Likely Carcinogens In Humans
Evidence for Gastric and Esophageal Cancer Risk
Nitrosamines in Tobacco Form From Nicotine
NNK is Nicotine Derived and Important in Carcinogenesis
NNK in Tobacco and E-cigarettes
NNK Activation by P-450 Activated Signaling Cascades & Uncontrolled Growth
Nicotine-derived nitrosamine ketone (NNK)
(4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone)
Reactive Nitrogen Species
Reactive Nitrogen Species Can Arise from any of the
Molecules Described Here
The Most Potent Reactive Nitrogen Species is Peroxynitrite
Reactive Nitrogen Species
Peroxynitrite is Formed from Nitric Oxide and Superoxide
Peroxynitrite Can Readily React with DNA and Protein, Causing Damage
Cysteine Side Chains are Most Easily Oxidized
Tyrosine Side Chains of Proteins Can Be Nitrosylated
Transition Metals, Such as in Hemoglobin, Myoglobin, and Cytochromes Can Be Oxidized
·NO + O2·−
Nitric Oxide
Superoxide
ONOO−
Peroxynitrite
Amino Acid Metabolism
Introduction
There are 20 Common Amino Acids in Proteins Plus One Rare One
No One Single Pathway for Amino Acid Metabolism
Synthesis Pathways are Grouped According to Common Anabolic Precursors
α-ketoglutarate
Serine
Aspartate
Aromatic
Pyruvate
Histidine
Essential Amino Acids Must Be in Diet
Non-Essential Ones Can be Made by Organism
Essential vs Non-Essential Varies in Humans
α-ketoglutarate Family
Transamination Plays an Important Role
α-keto Acid #1 + Amino Acid X
Amino Acid #1 + α-keto Acid X
α-ketoglutarate Family
Transamination to Make Glutamate
Amino Acid X
α-ketoacid #1
α-keto Acid #1 + Amino Acid X
Amino Acid #1 + α-keto Acid X
Amino Acid #2
α-ketoacid X
α-ketoglutarate Family
Glutamine Synthesis Uses Glutamine Synthetase
α-ketoglutarate Family - Arginine Synthesis
Citrulline + Aspartate
Four Pathways to Make it
ATP
AMP + 2 Pi
Argininosuccinate
NADP+ NADPH + O2
Arginine
Citrulline + Nitric Oxide + H2O
H2O
ADMA
Demythlation
Ornithine + Urea
Deficiency of the Enzyme Arginase Leads to the Genetic Disease of Argininemia Accumulation of Arginine and NH4+ in the Blood
Arginine
ADMA
Serine Family
Two Main Paths Lead to Serine
1. From 3-phosphoglycerate
(Connection to Glycolysis)
3-PG + NAD+
3-phosphohydroxypyruvate
Glutamate
α-ketoglutarate
O-phosphoserine H 2O
Serine + Pi
Serine Family
2. Exchanging Carbon with Glycine and Folates
(Important for Folate Recycling)
Serine + Tetrahydrofolate
Glycine + N5,N10-Methylene Tetrahydrofolate + H2O
Serine Family
Methionine
Cysteine Metabolism
Multiple Ways of Making Cysteine
Primary Means Tied to Methionine
Catabolism
Methionine Adenosyltransferase.
ATP
Pi + PPi
SAM
Transmethylase
Acceptor
CH3-Acceptor
SAH
S-adenosylhomocysteine Hydrolase
High Blood Levels - Cardiovascular Disease,
Stroke Risk
Deficiency Leads to
Homocystinuria
H2O
Adenosine
Homocysteine
Serine
Cystathionine β-synthase
Cystathionine Cystathionase
H2O
NH4+
β-ketobutyrate
Cysteine
Serine Family
Other Cysteine Metabolism
Serine
L-cystine
L-cysteic Acid
Acetyl-CoA
2 NADH + 2H+
H2S
CoA-SH
2 NAD+
Sulfite
O-acetyl-L-serine
H2S
L-cysteine + Acetate
2 Cysteine
L-cysteine
Serine Family
Selenocysteine Metabolism
Sometimes Called 21st Amino Acid
Not Specified Directly in Genetic Code
Uses Stop Codon with Unusual Structure
Synthesized from Serine on tRNA
Serine + tRNA
Non-SER tRNA
SERtRNA
SEL-A
Incorporation Into Proteins
SEL-D
SELtRNA
AspartateFamily
All Family Members Arise from Aspartate
Aspartate Can be Made from One of Them - Asparagine
Numerous Paths Lead to Aspartate
Transamination
Hydrolysis
Urea Cycle
Glutamate + Oxaloacetate
Asparagine + H2O
Argininosuccinate + AMP
Reversal of
Reaction
α-ketoglutarate + Aspartate
Aspartate + NH4+
Toxic
Aspartate + Citrullyl-AMP
Aspartate Family
Asparagine Metabolism
Synthesis
Breakdown
Aspartate + Glutamate + ATP
Asparagine + H2O
Asparagine Synthetase
Asparagine + α-ketoglutarate + AMP + PPi
Energetically Costly
Essentially Not Reversible
Asparaginase
Aspartate + NH4+
Toxic
Aromatic Family Outline
Tryptophan
Melatonin
Serotonin
Niacin
Auxins
Phenylalanine
Phenylketonuria
Tyrosine
Catecholamines
Thyroid Hormones
Melanin
Aromatic Family
Tryptophan, Phenylalanine, and Tyrosine
Each Derived from Phosphoenolpyruvate and Erythrose-4-phosphate
Synthesis Pathways Complex
Each Involves Shikimic Acid and Chorismic Acid
Phenylalanine and Tyrosine Pathways Overlap
Hormones and Neurotransmitters Made from Each
Aromatic Family
Tryptophan
Interesting Regulation of Synthesis in Bacteria
Attenuation - All 5 Genes on One Operon
When Tryptophan High, Transcription of Operon Aborts Early
When Tryptophan Low, Transcription of Operon Continues Through All Genes
Molecules Made from Tryptophan
Melatonin
Circadian Rhythm Sensing
Affects Mood, Sleep, Blood Pressure
Production Affected by Blue Light
Melatonin
Serotonin
Serotonin
Neurotransmitter
Causes Vasoconstriction
Enhances Memory/Learning, Contributor to Happy Feelings
Niacin
Niacin
Vitamin B3
Nicotinamide Derived From it - Part of NAD+/NADH & NADP+/NADPH
Deficiency Leads to Pellagra
Auxins
Indole-3-Acetic Acid Most Important
Indole-3-Acetic Acid
Stimulate Cell Division and Rooting in Plants
Aromatic Family
Phenylalanine (PHE)
An Essential Amino Acid and Precursor of Tyrosine
PHE Hydroxylase Catalyzes Formation of Tyrosine from PHE
Deficiency of the Enzyme PHE Hydroxylase Causes Phenylketonuria
High PHE Levels Cause Damage to Brain
Treatable by Reducing PHE Levels
Nutrasweet Contains PHE
Aromatic Family
Tyrosine (TYR)
Not Essential if PHE Present
Precursor of Catecholamines - L-Dopa, L-Dopamine, Norepinephrine, and Epinephrine
Donates Electrons to Reduce Chlorophyll in Photosystem II
Forms Radical in Ribonucleotide Reductase
L-Norepinephrine
CO2
L-Norepinephrine
L-Ephinephrine
Aromatic Family
Tyrosine Metabolism
L-Dopa
Precursor to Dopamine
Crosses Blood-Brain Barrier
Used to Treat Parkinson’s Disease
Dopamine
Neurotransmitter
Inhibits Norepinephrine Release in Blood Vessels - Acts as Vasodilator
Reduces Insulin Production in Pancreas
Deficiency Causes Parkinson’s Disease
Links to Schizophrenia and ADHD
Norepinephrine
Hormone and Neurotransmitter
Works Through Noradrenergic Receptors
Fight or Flight Response
Increases Heart Rate and Blood Pressure
Epinephrine (Adrenalin)
Hormone
Actions Similar to Norepinephrine
Fight or Flight Response
Increases Heart Rate and Blood Pressure
Aromatic Family
Secretion of Thyroglobulin
Tyrosine is a Precursor of Thyroid Hormones
Export from Cell
Iodide Export
& Oxidation
Iodination
Thyroglobulin Breakdown
Transport Into Cell
Transport Into Blood
Aromatic Family
Tyrosine Metabolism
Thyroid Hormones
All are Se-Containing Enzymes
Deiodinases
T3 (Triiodothyronine)
More Active Form
T4 (Thyroxine)
More Abundant Form
Aromatic Family
Tyrosine Metabolism
Melanin - Oxidized and Polymerized Tyrosine
Benzoquinone Portion of Coenzyme Q
Tyrosine Unit
From Tyrosine
Further Polymerization
Aromatic Family
Tyrosine Metabolism & Disease
Tyrosinemia - Problems with Tyrosine
Catabolism
Type I
Type II
Type III
Alcaptonuria - Black Urine Disease
Treatments
Restricted TYR/PHE Diet
Liver Transplant
Tyrosine
Tyrosine Transaminase
Type II
p-hydroxyphenylpyruvate
p-hydroxyphenylpyruvate Dioxygenase
Type III
Homogentisate
Alcaptonuria
Alcaptonuria
4-Maleylacetoacetate
4-Fumarylacetoacetate
Type I
4-fumarylacetoacetase
Fumarate + Acetoacetate
Pyruvate Family
Alanine Metabolism
Most Easily Produced from Pyruvate - Transamination
Byproduct of Catabolism of Valine, Leucine, and Isoleucine
Glucose-Alanine Cycle
Glutamate + Pyruvate
Alanine Transaminase
α-ketoglutarate + Alanine
Pyruvate Family
Leucine (LEU) /Valine (VAL) /Isoleucine (ILE) Metabolism
Branched Chain Amino Acids (BCAAs)
Several Common Steps
Start with Decarboxylation and Attachment of Two Carbon Piece to TPP
Valine and Leucine Pathways Involve Attachment of Two Carbon Piece to Pyruvate
Isoleucine Pathway Involves Attaching Two Carbon Piece to α-ketobutyrate
Penultimate Products - α-ketoisocaproate (LEU), α-ketoisovalerate (VAL), and α-keto-βmethylvalerate (ILE) Each is Transaminated to Make Final Amino Acid
Isoleucine
Leucine
Valine
Pyruvate Family
Leucine (LEU) /Valine (VAL) /Isoleucine (ILE) Metabolism
Synthesis Feedback Regulated Through Threonine Deaminase
Starting Material for ILE
Makes Starting
Material for ILE
Starting Material for VAL & LEU
Used by
All Three
High VAL
Favors ILE
High ILE
Favors VAL & LEU
Pyruvate Family
Histidine (HIS) Metabolism
Most Complex of All the Amino Acids
Overlaps Nucleotide Metabolism with Ribose-5-Phosphate & PRPP
10 Steps in Pathway
Second Enzyme of Pathway (ATP-phosphoribosyltransferase) Feedback Inhibited by Histidine
Histidine
Amino Acid Catabolism
Three Categories
1. Glycogenic - Broken Down to Glycolysis/
Gluconeogenesis Intermediates
2. Ketogenic - Broken Down to Acetyl-CoA
3. Both - Makes Intermediates in Both
Pathways
Glycogenic
Alanine, Cysteine, Glycine, Serine
Asparagine, Aspartate, Arginine
Histidine, Proline, Glutamine
Glutamate, Methionine, Valine
Ketogenic
Lysine, Leucine
Both
Threonine,Tryptophan, Tyrosine,
Phenylalanine, Isoleucine
AminoAcidCatabolism
Most Diseases of Amino Acid Metabolism Arise from Problems with Catabolism
Alcaptonuria - Phenylalanine and Tyrosine
Methylmalonic Acidemia - Methionine, Threonine, Isoleucine and Valine
Maple Syrup Urine Disease - Valine, Leucine, Isoleucine
Homocystinuria - Methionine
Tyrosinemia - Tyrosine
Argininemia - Arginine
Hypermethioninemia - Methionine
Hyperlysinemia - Lysine
Glycine Encephalopathy - Glycine
Propionic Acidemia - Methionine, Threonine, Isoleucine and Valine
Hyperprolinemia - Proline