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