CH.14: Nitrogen Metabolism I

Nitrogen

• Has a major role in the metabolic and structural aspects of all species

• Is found in amino acids, nucleic acids, nucleotides, porphyrins, some lipids, and many other critical biological compounds

Nitrogen fixation

• N₂ is plentiful; extremely stable

• To convert to a useful form requires a large amount of energy

• Animals cannot carry out nitrogen fixation (reduce N₂ to NH₃)

• Some bacteria can reduce N₂ to NH₃

• Plants acquire NH₃ by absorbing NH₃ from soil and by reduction of abosrbed NO₃^-

Nitrogenase complex

• Multienzyme complex for nitrogen fixation

  • Consists of 2 proteins: nitrogenase and nitrogenase reductase that include relatively simple electron-transport system

  • Nitrogenase (Fe-Mo protein)

    • Heterodimer—4 polypeptides subunit with and 2 molybdenum (Mo) between multiple Fe-S clusters

  • Nitrogenase reductase (Fe protein)

    • Dimer of identical subunits

Amino acids

• Essential and Nonessential

  • Amino acids (the constituents of proteins) are one of the main nitrogen-containing groups of compound

  • Serve a variety of functions:

    • Synthesis of proteins

    • Principal source of nitrogen for biosynthetic reactions

    • Nonnitrogen part as a source of energy and a carbon source (biosynthetic reactions)

  • Animals can only synthesize about half of amino acids

Essential amino acids (EAA)

Amino acids that cannot be synthesized in the body and must be provided in the diet

Nonessential amino acids (NAA)

Synthesized in the body from readily available metabolites

Amino acid metabolism

• Dietary protein sources differ widely in the proportion of EAA

• Complete protein (sufficient EAA) is of animal origin

• Plant protein often lack one or more EAA- must be taken in combination

• Amino acids immediately available for metabolic processes are referred to as the amino acid pool (from breakdown of dietary and tissue proteins)

• Amino acids required for the synthesis of proteins and metabolites are continuously synthesized (or interconverted) and degraded

• Nitrogen balance

  • Positive nitrogen balance

  • Negative nitrogen balance

• Once amino acids enter the cells the amino groups are available for synthesis reactions

• The first action in breakdown of amino acids is the removal of the a-amino group to rid it as excess nitrogen and degrading the remaining C-skeleton

• Transaminations

Nitrogen Balance

• Difference between total nitrogen intake and total loss

• Enters the body as food in dietary proteins

• Leaves the body as urea, ammonia, and other products

• Positive nitrogen balance

• Negative nitrogen balance

Positive nitrogen balance

Total nitrogen intake exceeds excreted occurs during growth and repair

Negative nitrogen balance

Total nitrogen intake is less than that excreted occurs in wasting (amino acids of muscle proteins converted to glucose and nitrogen excreted)

Transamination

• The most important reactions in amino acid biosynthesis

• Occurs in all amino acid biosynthesis except lysine and threonine

• Catalyzed by transaminases and require pyridoxal-5-phosphate as a coenzyme

• Reactions occur in 2 stages:

  • The amino group of an amino acid is transferred to the enzyme, forming an aminated enzyme and the corresponding a-keto acid

  • Amino group is transferred to the keto acid acceptor, forming the amino acid product and regenerating the enzyme

• E.g., the a-ketoglutarate / glutamate, oxaloacetate/aspartate, and pyruvate/alanine pairs

Ammonia metabolism

• Ammonia is toxic in high concentrations and must be incorporated into biologically useful compounds

• 2 ways ammonia is directly incorporated into amino acids

  • Reductive amination of a-keto acids

  • Formation of the amide of glutamic acid

Reductive amination of a-keto acids for ammonia metabolism

• Amino acids are synthesized by incorporating NH₄⁺ into a-keto acids

• Occurs primarily in the liver

• Enzymes require either NADPH or NADH

• Reaction freely reversible-excess NH₄⁺ reaction shifts to amino acid synthesis

• When energy is low, amino acid degradation active, providing a-ketoglutarate for the TCA cycle

Formation of amide of glutamic acid for ammonia metabolism

• NH₄⁺ incorporated into cell metabolites by amination of glutamate to glutamine

• Catalyzed by glutamine synthase—high concentration in the rain (sensitive to NH₄⁺)

• Gultamine (neutral, non-toxic molecule) formed is transported to the liver

• At the liver, the amino group attached to the amide side chain is removed by glutaminase (for production of nitrogenous waste)

• NOTE:

  • However:

    • Amide of aspartate (asparagine) is not formed by direct incorporation of NH₄⁺ into aspartate

    • It is by transamination reaction:

      • Aspartate + glutamine + ATP + H₂O → Asparagine + Glutamate + AMP + PPi

Synthesis of nonessential amino acids

• Each member of a class of amino acids is synthesized by a unique pathway

• The carbon skeletons are made from common metabolic intermediates

• Nonessential amino acids are grouped into 6 families—based on their synthetic pathways

  • Glutamate family

  • Serine family

  • Aspartate family

  • Pyruvate family

  • Aromatic family

  • Histidine family

Glutamate family

• a-ketoglutarate, glutamate, glutamine, arginine, and proline

• Common precursor: a-ketoglutarate

• Glutamate—component of proteins, precursor of other amino acids and as excitatory neurotransmitter in CNS

• Glutamine—a precursor for other metabolites (purines, pyrimidines and amino sugars)

Glutamate

Component of proteins, precursor of other amino acids and as excitatory neurotransmiter in CNS

Glutamine

A precursor for other metabolites (purines, pyrimidines and amino sugars)

Serine family

• Serine, glycine, and cysteine

• Common precursor—glycerate-3-phosphate (derived C-skeletons)

• Serine—precursor of ethanolamine and sphingosine

• Glycine—purine, porphyrin and glutathione synthesis

• Cysteine—sulfur metabolism

Serine

Precursor of ethanolamine and sphingosine

Glycine

Purine, porphyrin and glutathione synthesis

Cysteine

Sulfur metabolism

Aspartate family

• Aspartate, asparagine, lysine, methionine, and threonine

• Common precursor—oxaloacetate

• Aspartate—a source of nitrogen (for urea formation), TCA cycle intermediate (fumarate) precursor for nucleotide and other aa synthesis

Aspartate

A source of nitrogen (for urea formation), TCA cycle intermediate (fumarate), precursor for nucleotide and other aa synthesis

Pyruvate family

• Alanine, valine, leucine, and isoleucine

• Common precursor—pyruvate

Aromatic family

• Phenylalanine, tyrosine, and tryptophan

• Common precursor—phosphoenolpyruvate and erythrose-4-phosphate

• Phenylalanine/tyrosine—synthesis of catecholamines (dopamine, epinephrine, and norepinephrine)

• Tryptophan—precursor for synthesis of NAD, NADP, and serotonin

• Aromatic ring is formed by the shikimate pathway with chorismate as a common intermediate

Phenylalanine/Tyrosine

Synthesis of catecholamines (dopamine, epinephrine, norepinephrine)

Tryptophan

Precursor for synthesis of NAD, NADP and serotonin

Histidine family

Nonessential in healthy adults; must be supplied in the diet of infants, starting material—phosphoribosylpyrophosphate

Biosynthesis reactions of amino acids

• Amino acids are precursors of many nitrogen-containing molecules and building blocks for proteins

• Biosynthesis of these molecules involve transfer of carbon groups

• One-carbon metabolism

  • Folic acid

  • S-adenosylmethionine

One-carbon metabolism

• One-carbon groups:

  • Methyl (-CH₃)

  • Methylene (-CH₂-)

  • Formyl (-CHO)

  • Methenyl (-CH=)

• Folic acid and S-adenosylmethionine—most important one-carbon carriers

Folic acid

• (Folate or folacin or vitamin B)

• Converted to tetrahydrofolic acid (THF) in the body

• C-units carried by THF are bound to N⁵ and/or N¹⁰ of the pteridine ring

S-adenosylmethionine

• (SAM)

• Formed from methionine and ATP

• Contains an “activated” methyl thioether group (CH₃ → (can be) → acceptors)

• Major methyl group donor in one-C metabolism (transmethylation reaction)

• Occurs in the synthesis of phospholipids, neurotransmitters and glutathione

Neurotransmitters

• y-Aminobutyric acid (GABA)

• Catecholamines

• Serotonin

y-Aminobutyric acid (GABA)

• Inhibitor of the central nervous system

• Glutamate → (glutamate decarboxylase) → y-Aminobutyric acid + CO₂

  • Enzyme requires pyridoxal phosphate)

Catecholamines

• Dopamine (D)

• Norepinephrine (NE)

• Epinephrine (E)

• NE and E—also released by adrenal medulla and peripheral nervous system—regarded as a hormone because they regulate metabolism

Serotonin

• Inhibitor of CNS

• Implicated in eating disorders, mood swings, temperature regulation, pain perception, and sleep disorders

• 5-hydroxytryptophan decarboxylase—pyridoxal phosphate requiring enzyme

Nucleotides

• Perform a variety of functions:

  • Coenzyme components—NADH, FADH₂ and CoASH contain nucleotide components

  • Nucleic components—building blocks for DNA and RNA synthesis

  • Energy source—ATP represents the major form of stored energy in the cell

  • Allosteric control—ATP, ADP, and other nucleotides serve as allosteric modulators in several pathways

  • Group donors—UDP-glucose, CDP-choline, and S-adenosylmethionine are nucleotide derivatives that serve as carriers of groups to be transferred to other molecules in various reactions

  • Signal transmission—cAMP serves as second messengers in transmission of extracellular signals intercellularly

Nucleotide metabolism

• Purine and pyrimidine nucleotides can be synthesized in de novo and salvage pathway

• Purine nucleotides

• Biosynthesis of AMP and GMP

Purine nucleotides

• De novo synthesis begins with

  • a-D-ribose-5-phosphate → (several steps) → inosine-5’-monophosphate (IMP)

• Precursors to base components of IMP (hypoxanthine) include:

  • Glutamine

  • Glycine

  • CO₂

  • Aspartate

  • N¹⁰-formyl THF

Biosynthesis of AMP and GMP

• IMP is converted to either AMP or GMP

• AMP formation requires GTP and GMP formation requires ATP

• IMP → AMP (adenylate)

  • An amino group (from aspartate) replaces a keto group on IMP

  • Adenylosuccinate formed eliminates fumarate to form AMP

• IMP → GMP (guanylate)

  • Dehydrogenation reaction to form xanthosine monophosphate (XMP)

  • An amino group (from glutamine) replaces a keto group on XMP

• Nucleotide triphosphate—most common nucleotide used in metabolism

  • AMP + ATP → (adenylate kinase) → 2 ADP

  • NMP + ATP ← (nucleoside monophosphate kinase) → NDP + ADP

  • N₁DP + N₂TP ← (nucleoside diphosphate kinase) → N₁TP + N₂DP

    • (N₁ and N₂ are purine or pyrimidine nucleotides)

Salvage pathway

• De novo synthesis of nucleotides is an energy-requiring process

• In the purine salvage pathway, purine bases obtained from normal turnover of cellular nucleic acids or from diet are converted into nucleotides

• Lesch-Nyhan syndrome

Lesch-Nyhan Syndrome

• Caused by a deficiency of HGPRTase

• Hypoxanthine and guanine are degraded to uric acid instead of being used for salvage

• Showed that the salvage pathway is more than just an energy-saving measure

Pyrimidine nucleotides

• Synthesis—simpler than the purine pathway and it consumes fewer ATP molecules

• Donor groups for the pyrimidine ring

• The pyrimidine ring is synthesized first and then attached to the ribose phosphate

• Animals do not appear to have a significant pyrimidine salvage pathway analogous to the purine salvage pathway

Deoxyribonucleotides

• All nucleotides discussed so far are ribonucleotides (components of RNA)

• Components of DNA are 2’-deoxyribonucleotides

• Synthesis of 2’-deoxyribonucleotides

Synthesis of 2’-deoxyribonucleotides

• Deoxyribonucleotides are formed by the reduction of ribonucleotides

• All 4 ribonucleotides diphosphates are converted by a single enzyme

  • Ribonucleotide reductase

• The final step is to phosphorylate the diphosphates:

  • dNDP + ATP ← (nucleoside diphosphate kinase) → dNTP + ADP

• Same enzyme phosphorylates ribonucleotide diphosphates

Salvage pathway

• De novo synthesis of nucleotides is an energy-requiring process

• In the purine salvage pathway, purine bases are obtained from normal turnover of cellular nucleic acids or from diet are converted into nucleotides