Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 432	Biochemistry	Spring 2002
Lecture Notes:: 30 January	© R. Paselk 2002
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Nitrogen Fixation, cont.

Nitrogen fixation is a biologically expensive process, costing 16 ATP's and 8 electrons per N₂, as seen in the reaction stoichiometry:

The electron flow in this process is diagramed as follows:

Glutamate dehydrogenase. Once ammonia has been formed the nitrogen can be incorporated via one of two major pathways. The first is familiar - Glutamate dehydrogenase found in mitochondria (or bacteria). Note that the equilibria will allow the reaction to go either direction, depending on substrate concentrations. (You may also note the enzyme will accept either NADH or NADPH, the only enzyme known to be non-specific!)

In animals ammonia is quite toxic, so its concentration is kept quite low, and glutamate DH is not a major source of nitrogen incorporation.

Glutamine Synthetase. Glutamine is a major storage form of nitrogen and a source of nitrogen in various synthetic pathways. It has the advantage over glutamate dehydrogenase of a much lower K_m - that is it can incorporate ammonia into biomolecules at much lower concentrations. Glutamine is made from glutamate and ammonia with energy supplied by ATP:

Glutamine's central position in nitrogen metabolism makes control of its biosynthesis essential.

• In mammals glutamine synthetases are activated by a-ketoglutamate.
• In bacteria the control of glutamine synthetase is considerably more complex, as might be expected in an organism where nitrogen incorporation into amino acids, nitrogenous bases, etc. is required for growth. The bacterial enzyme consists of 12 identical subunits arranged in two face-to-face hexagonal rings. Regulation occurs via allosteric effectors and covalent modification:
  • 9 nitrogenous metabolites act as cummulative allosteric feedback inhibitors, each with its own binding site:
    • three amino acid indicators of the cells nitrogen balance (serine, alanine, and glycine - note these are all high turnover intermediates of metabolism).
    • six biomolecules where glutamine is a source of biosynthetic nitrogen: AMP, CTP, carbamoyl phosphate (synthesized by carbamoyl synthetase II - in mammals this is a cytosolic enzyme as opposed to the enzyme used in the Urea cycle), glucosamine-6-P, histadine and tryptophan.
  • E. coli glutamine synthetase activity is decreased by a covalent adenylation via a cascade system which is sensitive to both cell nitrogen and energy availability (a-ketoglutamate and ATP activate [energy is high so grow, but need nitrogen], while glutamine and P_i deactivate). (Figure 20-27 on p 643.)
  • E. coli scenario (modified from TIBS, Jan 1977, p10) Begin with happy bugs in glucose (excess), histadine, ammonia media.
    • Initially taking life easy, they use glutamate DH to incorporate ammonia and contains very little glutamine synthetase, and most of that is in the loactivity adenylateed form. A lack of glutamine synthetase in turn leaves the cells unable to activate the histidine degrading enzymes (glutamine synthetase stimulates transcription of this gene set - cAMP also stimulates transcription and cAMP production is stimulated in turn by a lo [glu])
    • After a while ammonia is depleted (<1mM) and glutamate DH is no longer effective at ammonia uptake, thus [a-kG] increases and [gluNH₂] decreases since glutamine synthetase has been at lo activity. The change in [a-kG]/[gluNH₂] results in the activation of glutamine synthetase (deadenylation by the cascade)
    • As a result the glutamate DH gene is repressed and the histidine genes sytem is activated due to an increase in the [glutamine synthetase]
    • Histidine breaks down to release ammonia and glutamate which can then be used in synthesizing glutamine allowing continued growth.

Degredation of Intracellular Proteins

All cell proteins are turned over, with half-lives commonly ranging from minutes to hours. Generally the proteins used for basic cell operations ("housekeeping proteins, e.g. glycolytic enzymes) have relatively long half-lives, while those involved in adaptation (e.g. gluconeogenesis, ketone body synthesis) have short half-lives. Two well known systems are used by eukaryotic cells to degrade proteins:

1. The lysosomal system.
2. The ubiquitin/proteosome system.

The lysosomal system is generally non-selective in well nourished cells, but becomes more selective with starvation. Thus proteins with the KFERQ sequence (lys-phe-glu-arg-gln) are preferentially degraded, shortening theri half-lives.

The second system is specific to eukaryotes and is based on the degredation of proteins which have had the protein ubiquitin covalently attached to them. Ubiquitin itself is a highly conserved protein (i.e. identical in humans and fruit flys), possibly because it is recognized by a number of other proteins. Ubiquitin is attached to a "condemned" protein in a three stage process:

1. It is conjugated to a thiol group on ubiquitin activating enzyme via the C-terminal carboxyl group to give a thioester bond. As this is an unstable linkage (high energy) ATP energy is required (ATP Æ AMP + PP_i via a two step reaction involving a ubiquitin-adenylate intermediate).
2. The ubiquitin is then transferred to a second protein, ubiquitin-conjugating enzyme.
3. Finally the activated ubiquitin is transferred to the side-chain amino group of a lysine on the target protein by ubiquitin-protein ligase.

The ubiquinated protein is then broken down by the proteosome, a large 28 subunit, 6182 aa, 700 kD, barrel -shapped, protein. The proteosome breaks proteins down to octapeptides, which are released to the cytosol for further degredation. The ubiquitin is recycled, not degraded.

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Pathway Diagrams

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Last modified 30 January 2002