MBIO 2710 / Topic 10: Nitrogen Catabolism

What are 9 different oxidation states of Nitrogen?

> +5 = NO₃^- (nitrate)

> +4 = [NO₂]

> +3 = NO₂^- (nitrite)

> +2 = [NO]

> +1 =N₂O₂^-2 (hyponitrite)

> 0 = N₂

> -1 = N₂H₂ (diimide)

> -2 = H₂NOH (hydroxyl amine)

> -3 = NH₃ (ammonia)

Why can’t N≡N be used in metabolism?

sp bond is extremely strong and stable → energetically expensive and difficult to break → enzymes can’t do it direcrtly

What are the two reasons why NO₃^- so close to O₂? What does this mean in the general scheme of energy release?

> Both are strong oxidizing agents.

> Energy gap between donors and acceptors is similar to that w/ O₂.

> This means that NO₂^- can be used as a final e^- acceptor in ETC.

Why does biological nitrogen fixation require less energy than non-biological fixation?

> Non-biological: large energy input to break sp bond (N≡N) of N₂ (high T & pressure)

> Biological: enzymes (e.g. nitrogenase) lower activation energy → occurs at 25°C & 1 atm

How many electron transfers does it take to reduce nitrogen gas to ammonia?

3 × 2e^- transfers.

Why does the nitrogen reduction stage have to be anaerobic?

O₂ reactive w/ metal clusters of nitrogenase → irreversibly inactivates nitrogenase.

What can be done w/ NH₃ once it is produced in nitrogen fixation?

> Incorporated into organic form via transamination.

> Excreted into soil → NH₃ to nitrate by bacteria via nitrification.

What can be done w/ NO₃^- once produced via nitrification?

> Energy released in nitrification used by bacteria for growth.

> NO₃^- = not volatile & non-toxic → most abundant form of nitrogen accumulated in soil to be re-reduced to NH₃ for soil.

How do bacteria use NO₃^- in respiration & nitrogen metabolism?

> NO₃^- used as a terminal e^- acceptor in ETC.

> After ETC, nitrate reduced stepwise to either N₂ (denitrification; to make energy) or NH₃ (nitrate reduction; for biosynthesis).

What is the difference in the toleration of high NH₃ levels between prokaryotes and eukaryotes via glu deH₂ase, and what this means for eukaryotes>

> In prokaryotes, high NH₃ levels favour the forward reaction.

> In eukaryotes, GDH works the opp direction b/c eukaryotic GDH has a higher K_M and thus lower aff → less efficient at high NH₃ assimilation.

> Eukaryotes then rely on the glutamine synthetase-glutamate synthase cycle for ammonia incorporation.

How does glutamate synthase (GOGAT) maintain balance in the GS-GOGAT cycle under low NH₃ conditions?

> GS converts glu → gln by adding NH_3.

> GOGAT regenerates glu from gln + α-ketoglutarate

> Prevents glu depletion while allowing NH₃ assimilation, especially when NH₃ levels are low.

hat does “isoform” mean, and how does it relate to carbamoyl phosphate synthetase (CPS) I and II?

> Isoforms = diff ver of prot w/ distinct fxns and coords.

> CPS I + CPS II = isoforms = CPS I mitochondrial using NH₃, but CPS II cytoplasmic, using gln nitrogen.

Why do cells salvage purine bases when nucleic acids break down instead of making purines de novo?

> Salvaging purine bases recycles them into nts efficiently.

> De novo purine synthesis = energy-intensive & complex.

> Salvage saves ATp and resources by reusing existing bases.

What does partial deficiency in PRTase do?

Overproduction of uric acid and sodium urate = relatively insoluble in water + precipitates in cartilage & kidneys w/ painful side fx → gout.

What does allopurinol do to bypass sodium urate?

> Competitively inhibits xanthine oxidase.

> Reduces uric acid production → free A, G, hypoxanthine, and xanthine to be excreted.

> Bypasses insoluble sodium urate → prevents crystal buildup & gout.

What does full deficiency in PRTase do?

> Salvage pathway = primary route for nt synth in brain & nerve cells.

> Genetic defect in both copies of gene → full deficiency → Lesch-Nyhan syndrome.

> Enzyme missing = Brain and nerve cells develop abnormally → tendency to self-mutilate increases.

What is DHFR’s role in anti-cancer therapy?

> DHFR regenerates THF = essential for dTMP synth.

> Inhibiting DHFR → ↓ dTMP → ↓ DNA synthesis

> Rapidly dividing cancer cells need DNA → DHFR inhibition slows their growth

> Normal cells less affected due to slower division.