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

SarcosineCitrullineOrnithine

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

Ammonia Uric Acid Urea

Produced by Amino Acid Catabolism

Used in Urea CycleProduced by Purine

CatabolismProduced by Urea Cycle

α-ketoglutarate Family

Transamination to Make Glutamate

α-ketoacid #1

Amino Acid #2

α-keto Acid #1 + Amino Acid X

Amino Acid #1 + α-keto Acid X

Amino Acid X

α-ketoacid X

Glucose-Alanine Cycle

Alanine Metabolism

Glucose-Alanine Cycle Important for Removing Ammonia

High Ammonia Low Ammonia

Transamination in Liver Creates Glutamate

Alanine Carries Amine to Liver

Breakdown of Glutamate Yields Amine for Urea Production

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

2 ATP + HCO3− + NH4+ <=> 2 ADP + Carbamoyl phosphate + P

The Source of Ammonium Ion is Glutamine or Glutamate Requires Action of Glutaminase (Glutamine) or Glutamate Dehydrogenase (Glutamate)

Glutamine + H2O

Glutamate + NH3

Glutamate + H2O + NADP+

α-ketoglutarate + NH3 + NADH + H+

H2O + CO2

HCO3- + NH4+

H2O + CO2

HCO3- + NH4+

Ornithine Transcarbamoylase Reaction

+

Carbamoyl Phosphate

Ornithine

+Pi

Citrulline

Phosphate

Ornithine Transcarbamoylase

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

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

12

Argininosuccinate Lyase

+

Argininosuccinic Acid Arginine Fumaric Acid

Bond Cleaved

Important for Production of Arginine Source of Fumarate Deficiency Like That of Other Urea Cycle Enzymes - Ammonia Excess

Argininosuccinate Lyase

Argininosuccinate Lyase

To Proteins or Remainder of Urea Cycle To Citric Acid Cycle

Arginase

+ H2O +Arginine Urea Ornithine

CutExcreted

To Mitochondria To Complete Cycle Through

Ornithine Citrulline Translocase

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

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

Nitric Oxide Important Signaling Molecule in Humans - Vasodilation

2 L-arginine + 3 NADPH + 1 H+ + 4 O2

2 Citrulline +2 Nitric Oxide + 4 H2O + 3 NADP+

Nitric Oxide Synthase

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

Nitrosamine

NO+ + →H

Secondary Amine

Nitrite

H2NO2+ → H2O + NO+

Nitrosonium Ion

2H+

Nitrous Acid Ion

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 SpeciesPeroxynitrite 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·− ONOO−

Nitric OxideSuperoxide

Peroxynitrite

Amino Acid Metabolism

IntroductionThere 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 Roleα-keto Acid #1 + Amino Acid X

Amino Acid #1 + α-keto Acid X

α-ketoglutarate Family

Transamination to Make Glutamate

α-ketoacid #1

Amino Acid #2

α-keto Acid #1 + Amino Acid X

Amino Acid #1 + α-keto Acid X

Amino Acid X

α-ketoacid X

α-ketoglutarate Family

Glutamine Synthesis Uses Glutamine Synthetase

α-ketoglutarate Family - Arginine Synthesis

Four Pathways to Make it

Deficiency of the Enzyme Arginase Leads to the Genetic Disease of Argininemia - Accumulation of Arginine and NH4+ in the Blood

ADMAArginine

Arginine

Citrulline + AspartateATP

ArgininosuccinateAMP + 2 Pi

Ornithine + Urea

H2O

Citrulline + Nitric Oxide + H2O

NADP+ NADPH + O2

DemythlationADMA

Serine Family

3-PG + NAD+

3-phosphohydroxypyruvate

O-phosphoserine

Serine + Pi

H2O

Two Main Paths Lead to Serine

1. From 3-phosphoglycerate (Connection to Glycolysis)

Glutamateα-ketoglutarate

Serine Family

2. Exchanging Carbon with Glycine and Folates (Important for Folate Recycling)

Serine + Tetrahydrofolate

Glycine + N5,N10-Methylene Tetrahydrofolate + H2O

Serine Family

Cysteine Metabolism Multiple Ways of Making Cysteine Primary Means Tied to Methionine Catabolism

Methionine

SAM

SAH

Homocysteine

Cystathionine

Cysteine

Serine

ATPPi + PPi

AcceptorCH3-Acceptor

Adenosine H2O

β-ketobutyrate

Transmethylase

S-adenosylhomocysteine Hydrolase

Cystathionine β-synthase

Methionine Adenosyltransferase.

Cystathionase

Deficiency Leads to Homocystinuria

High Blood Levels - Cardiovascular Disease, Stroke Risk

H2ONH4+

Serine Family

O-acetyl-L-serine

Acetyl-CoACoA-SH

L-cysteine + Acetate

H2S2 Cysteine

2 NADH + 2H+

2 NAD+

Serine L-cystine L-cysteic Acid

L-cysteine

H2SSulfite

Other Cysteine Metabolism

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 SERtRNA

Non-SER tRNA

SEL-A SEL-DSELtRNA

Incorporation Into Proteins

AspartateFamily

All Family Members Arise from Aspartate Aspartate Can be Made from One of Them - Asparagine Numerous Paths Lead to Aspartate

Asparagine + H2OGlutamate + Oxaloacetate Argininosuccinate + AMP

α-ketoglutarate + Aspartate Aspartate + NH4+ Aspartate + Citrullyl-AMP

Transamination Hydrolysis

Reversal ofReaction

Toxic

Urea Cycle

Aspartate Family

Asparagine Metabolism

Aspartate + Glutamate + ATP

Asparagine + α-ketoglutarate + AMP + PPi

Energetically Costly Essentially Not Reversible

Asparagine Synthetase

Synthesis Breakdown

Asparagine + H2O

Aspartate + NH4+

Toxic

Asparaginase

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

Serotonin Neurotransmitter Causes Vasoconstriction Enhances Memory/Learning, Contributor to Happy Feelings

Niacin Vitamin B3 Nicotinamide Derived From it - Part of NAD+/NADH & NADP+/NADPH Deficiency Leads to Pellagra

Auxins Indole-3-Acetic Acid Most Important Stimulate Cell Division and Rooting in Plants

Melatonin

Serotonin

Indole-3-Acetic Acid

Niacin

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

CO2

L-Norepinephrine

L-Norepinephrine

L-Ephinephrine

Aromatic FamilyTyrosine 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

Tyrosine is a Precursor of Thyroid Hormones

Secretion of Thyroglobulin

Export from Cell

Iodide Export& Oxidation

Iodination

Transport Into Cell

Thyroglobulin Breakdown

Transport Into Blood

Aromatic Family

Tyrosine Metabolism

Thyroid Hormones

T3 (Triiodothyronine) T4 (Thyroxine)

Deiodinases

All are Se-Containing Enzymes

More Active Form More Abundant Form

Aromatic Family

Tyrosine Metabolism

Melanin - Oxidized and Polymerized Tyrosine Benzoquinone Portion of Coenzyme Q

Tyrosine Unit

Further Polymerization

From Tyrosine

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

p-hydroxyphenylpyruvate

Homogentisate

4-Maleylacetoacetate

4-Fumarylacetoacetate

Fumarate + Acetoacetate

Tyrosine TransaminaseType II

p-hydroxyphenylpyruvate DioxygenaseType III

AlcaptonuriaAlcaptonuria

4-fumarylacetoacetaseType I

Pyruvate FamilyAlanine Metabolism

Most Easily Produced from Pyruvate - Transamination Byproduct of Catabolism of Valine, Leucine, and Isoleucine Glucose-Alanine Cycle

Alanine Transaminase

Glutamate + Pyruvate

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-β-methylvalerate (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

Starting Material for VAL & LEUMakes Starting Material for ILE

Used by All Three

High ILE Favors VAL & LEU

High VAL Favors ILE

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

GlycogenicAlanine, Cysteine, Glycine, Serine Asparagine, Aspartate, Arginine Histidine, Proline, Glutamine Glutamate, Methionine, Valine

KetogenicLysine, Leucine

BothThreonine,Tryptophan, Tyrosine, Phenylalanine, Isoleucine

Three Categories 1. Glycogenic - Broken Down to Glycolysis/Gluconeogenesis Intermediates 2. Ketogenic - Broken Down to Acetyl-CoA 3. Both - Makes Intermediates in Both Pathways

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