Overview of Nitrogen Metabolism and Biosynthesis of Amino Acids CH353 January 22, 2008.
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Transcript of Overview of Nitrogen Metabolism and Biosynthesis of Amino Acids CH353 January 22, 2008.
Overview of Nitrogen Metabolismand Biosynthesis of Amino Acids
CH353 January 22, 2008
The Nitrogen Cycle
• Nitrogen FixationN2 → NH4
+
• NitrificationNH3 → NO2
- → NO3-
• DenitrificationNO2
- & NO3- → N2
• Nitrogen Assimilation NH4
+ → Organic nitrogen
• Deamination
Organic nitrogen → NH4+
Nitrogen Fixation
• Nitrogen Reduction Reaction
N2 + 3 H2 → 2NH3 ΔG′º = -33.5 kJ/mol – Exergonic reaction– High activation energy
– Stability of N2 triple bond D = 930 kJ/mol
• Biological Nitrogen ReductionN2 + 10H+ + 8e- + 16ATP → 2NH4
+ + 16ADP + 16Pi + H2
Nitrogenase Complex
Dinitrogenase reductase• Dimer (Mr 60,000) of same subunits
• One 4Fe-4S between subunits• One ATP/ADP site per subunit• ATP binding enhances reduction
potential: E′º -300 → -420 mV
Dinitrogenase• Tetramer (Mr 240,000):
– 2 copies of 2 different subunits• 2 Mo, 32 Fe, 30 S per tetramer• 8 dinitrogenase reductase dimers each
transfers one e- to dinitrogenase
Nitrogen Assimilation
• Glutamine SynthetaseGlutamate + NH4
+ + ATP → Glutamine + ADP + Pi + H+
• Glutamate Synthase (plants, bacteria)
α-Ketoglutarate + Glutamine + NADPH + H+ → 2 Glutamate + NADP+
• Net Reaction:α-Ketoglutarate + NH4
+ + NADPH + ATP → Glutamate + NADP+ + ADP + Pi
• Glutamate Dehydrogenase
α-Ketoglutarate + NH4+ + NADPH → Glutamate + NADP+
Regulation of Glutamine Synthetase (E. coli)
Covalent Modification
Adenylylation inactivatesglutamine synthase
Adenylyltransferase(AT) regulated by PII
Uridylylation of PII
determines regulation:no UMP - adenylylationUMP – deadenylylation
α-Ketoglutarate & ATPstimulate uridylylation↑ Glutamine synthesis
Glutamine & Piinhibit uridylylation↓ Glutamine synthesis
Regulation of Glutamine Synthetase (E. coli)
Transcription Activation• Uridylylated PII activates gene
encoding glutamine synthetase
Allosteric Inhibition• Glutamine synthetase is inhibited
by alanine, glycine and products of glutamine metabolism
• Binding of each metabolite causes partial inhibition; binding of all completely inhibits enzyme
• Overall effect of 8 inhibitors is more than additive: example of Concerted Inhibition
Biosynthetic Reactions with Glutamine
Amidotransferase reactions• Glutamine is hydrolyzed to
glutamate + NH3 within the enzyme
• Acceptor hydroxyl or ketone often activated with ATP
Glutamine + Aspartate + ATP
↓Glutamate + Asparagine + AMP
+ PPi
Overview of Amino Acid Biosynthesis
3-Phosphoglycerate Serine Glycine Cysteine
Pyruvate Alanine Valine Isoleucine Leucine
α-Ketoglutarate Glutamate Glutamine Proline Arginine
Ribose 5-phosphate Histidine
Erythrose-4-phosphatePhosphoenolpyruvate Tryptophan Phenylalanine Tyrosine
Oxaloacetate Aspartate Asparagine Methionine Lysine Threonine
nonessentialconditionalessential
Key:
Reactions with Pyridoxal Phosphate
• Transamination (aminotransferase) reactions
Glutamate + Pyruvate α-Ketoglutarate + Alanine Glutamate + Oxaloacetate α-Ketoglutarate + Aspartate
Amino Acids from α-Ketoglutarate
Amino Acids from α-Ketoglutarate
α-Ketoglutarate
Glutamate Glutamine
Glutamate -semialdehyde
Arginine
Proline
Ornithine
Urea Cycle
Biosynthesis of Proline and Arginine
ornithineaminotransferase
Glutamate α-Ketoglutarate
Amino Acids from 3-Phosphoglycerate
Biosynthesis of Serine and Glycine
Cofactors for One-Carbon Metabolism
One-Carbon Units on Tetrahydrofolate
• tetrahydrofolate (H4 folate) is derived from folic acid
• one-carbons on H4 folate can have 3 redox states
• hydroxymethyl group of serine and formate are primary entry metabolites
• reversible conversions except for production of N 5-methyl H4 folate
The Activated Methyl Cycle
• S-Adenosyl methionine is the methyl donor for nearly all reactions
• Methyl group of methionine is replenished with N5-methyl H4 folate
• Methionine synthase requires coenzyme B12 (from vitamin B12)
Biosynthesis of Cysteine
• Only bacteria and plants can assimilate inorganic sulfur
• In mammals, cysteine is conditionally essential deriving its sulfur from methionine
• Cysteine is biosynthesized from serine and homocysteine, a methionine metabolite
• Sulfhydryl is transferred from homocysteine to serine in two PLP dependent steps with cystathionine as intermediate
Study Problem
• High serum homocysteine level is a risk factor for coronary heart disease and arteriosclerosis
• Some individuals with high homocysteine have variations in the cystathionine β-synthase gene
• A combination of vitamin supplements is recommended for alleviating high homocysteine
• Based upon the pathways for homocysteine synthesis and utilization, which 3 vitamins would you recommend?
Amino Acids from Oxaloacetate and Pyruvate
Amino Acids from Oxaloacetate
Oxaloacetate
Aspartate Asparagine
Pyruvate
Lysine
Aspartate β-semialdehyde
Threonine Methionine
HomoserineDihydropicolinate
Biosynthesis of Lysine, Methione & Threonine
Lysine Methionine & Threonine
Dihydropicolinate
Biosynthesis of Methionine
Biosynthesis of Methionine
Homocysteine Serine Cysteine Homoserine
Cystathionine
H2Ocystathionine β-synthase
H2Ocystathionine
γ-synthase
H2O cystathionine β-lyase
H2Ocystathionine γ-lyase
α-Ketobutyrate Cysteine Pyruvate Homocysteine
Biosynthesis of Cysteine Biosynthesis of Methionine
γ βPLP PLP
PLP PLP
Biosynthesis of Threonine
Amino Acids from Pyruvate
Alanine
Valine Leucine Isoleucine
Pyruvate
Pyruvate
CO2
α-Ketobutyrate
Threonine
acetolactatesynthetase
Acetyl-CoA
CO2
Same enzymes for both valine and isoleucine
Biosynthesis of Isoleucine and Valine
Isoleucine Valine & Leucine
Regulation of Amino Acid Biosynthesis
Multiple Isozymes for• Aspartokinase (A1, A2, A3)
• Homoserine dehydrogenase (B1, B2)
• Threonine dehydratase (C1, C2)
Allosteric regulation of selective isozymes – some unregulated
Sequential feedback inhibition• Same product inhibits its biosynthetic
path at multiple sites
Inhibits first enzyme in pathway
Biosynthesis of Aromatic Amino Acids
Biosynthesis of Aromatic Amino Acids
Phosphoenolpyruvate Erythrose 4-phosphate
Shikimate
Chorismate
Prephenate
CO2CO2
Tyrosine Phenylalanine
Anthranilate
Pyruvate
PRPP
CO2 Serine
Glyceraldehyde 3-phosphate
Tryptophan
Glu Gln
Conversion of Phenylalanine to Tyrosine
• Tyrosine is a conditionally essential amino acid
• It can be synthesized from phenylalanine as part of its catabolic pathway
Biosynthesis of Histidine
PRPP ATP
Histidine
AICARPurineBiosynthesis
Glutamine
Glutamate