Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438 - Introductory Biochemistry - Spring 2013

Lecture Notes:


Nitrogen Fixation

Before we leave the amino acids I want to look at some related processes, the first of which, Nitrogen fixation, is restricted to prokaryotes.

Nitrogen is one of the four most common elements in all living organisms. The problem is that although nitrogen is very common in its elemental form, the N2 molecule is very stable - the triple bond has an energy of 945 kJ/mol (vs. around 350 kJ/mol for single bonds) and is kinetically stable as well, and is thus unavailable to most organisms. Conversion of elemental nitrogen to usable forms, e.g. NH3 or NO3- (nitrogen fixation) occurs to a limited extent via lightning discharges (about 10% of naturally fixed N2) and via a complex process carried out by some bacteria and cyanobacteria.

All biological nitrogen fixing systems have 5 basic requirements:

  1. Nitrogenase (N2 right arrowNH3).
  2. A powerful reductant, such as ferridoxin to provide electrons.
  3. ATP.
  4. An oxygen-free environment.
  5. Regulatory controls.

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

N2 + 8H+ + 8 e- 16 ATP + 16 H2O right arrow 2 NH3 + H2 + 16 ADP + 16 Pi

(Note the obligatory production of H2 by nitrogenase, which increases at low [ATP].)

The electron flow in this process is diagramed as follows:

Nitrogenase Reaction

nitrogenase mechanism diagram


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!)

Structural diagram of the reaction of Glutamate DH

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 Km - that is it can incorporate ammonia into biomolecules at much lower concentrations. Glutamine is made from glutamate and ammonia with energy supplied by ATP:

Structural diagram of the reaction sequence of Glutamine Synthase

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

FYI –Glutamine Synthase Regulation

  • In mammals glutamine synthetases are activated by alpha-ketoglutarate.
  • 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: (text Figure 22-6, p858)
    • 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 (alpha-ketoglutamate and ATP activate [energy is high so grow, but need nitrogen], while glutamine and Pi deactivate).
    • 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 [alpha-kG] increases and [gluNH2] decreases since glutamine synthetase has been at lo activity. The change in [alpha-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 their 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 right arrow AMP + PPi 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.

Biosynthesis of Triacylglycerols and Phospholipids


Triglycerides are synthesized from DHAP (cytosol) or glycerol-3-P (mitosol) and fatty acyl CoA (activated fatty acid) via the pathway outlined in your text. Note that either starting reactant leads first to the monacylglycerol phosphate, Lysophosphatidate:

structural diagram of lysophosphatidate

A second fatty acyl CoA is then added to give Phosphatidic acid, which is then hydrolyzed to the diacylglycerol and a third fatty acyl CoA to give the final product. Note that 2-monoacylglycerol from the intestinal track may also be used to make triglycerides by adding two fatty acyl CoA's.


These phospholipids generally tend to have a saturated fatty acyl group on the C1 position while they mostly have an unsaturated fatty acyl group on the C2 position. The biosynthesis of phosphatidylethanolamine and phosphatidylcholine (lecithin) are outlined in your text. For phosphatidylcholine:

Choline + ATP right arrow Phosphocholine + ADP

Phosphocholine + CTP right arrow CDP-choline + PPi

CDP-choline + Diacylglycerol right arrow Phosphatidylcholine + CMP

Phosphtidylserine is made by exchanging serine (using the side chain -OH group) for ethanolamine on a phosphatidylethanolamine.

The Glyoxylate Cycle

As we've seen fats are only used in biosynthesis for lipids in animals. Plants, on the other hand can use fats for biosynthesis of carbohydrates, amino acids, etc. Of course plants don't generally store lots of energy as fat, except in their mobile forms, such as seeds. Seeds then use this fat, which is a dense form of energy storage, to manufacture the carbohydrate and protein needed to sprout. So how do seeds use fat for biosynthesis?

Plants add two new enzyme activities to the set seen in the TCA Cycle to create a new pathway, the Glyoxylate Cycle or Pathway. The stoichiometry of this pathway is:


2 Acetyl-CoA + NAD+ + FAD right arrow Malate + NADH + H+ + FADH2

The pathway can be represented by a simple cycle with two acetyl-CoA's added with succinate as the product, the Glyoxylate Cycle.

structural diagram of the reactions in the Glyoxalate cycle

In actuality, the pathway is broken up into two parts by being compartmentalised in the mitochodria and a specialized organelle, the Glyoxysome. The two new reactions occur in this organelle:

structural diagram for the Isocitrate lyase reaction

structural diagram for Malate synthase reaction

The acetyl-CoA required for this synthesis is generated in the glyoxysome from fatty acids via a modified beta-oxidation pathway using NAD+ and molecular oxygen (instead of FAD) as oxidants.

Metabolic Integration

Begin with a review of the Stages of Catabolism. Recall the major branch point at pyruvate and the decision point when making acetyl-CoA which is now committed to energy production or lipid biosynthesis. Note also the entry of glucose by facilitated diffusion, which is then committed to the cell by phosphorylation by Hexokinase - the net effect is an active transport, since G-6-P cannot escape the cell.

Fat Biosynthesis

Biosynthesis of fatty acids from glucose requires the integration of a number of pathways. Glucose can ultimately provide carbons, energy equivalents, and reducing equivalents:

"Carbohydrate Stress" and Fasting

As an exercise in the integration of metabolic systems we can look at how the body responds to "carbohydrate stress," the situation occurring when blood glucose levels fall below the normal homeostatic level of about 5 mM. This commonly occurs during a fasting state.

Before following the development of a fasting state and its metabolic consequences, let's set some baselines by noting the potential available fuel in humans as represented by a 'typical' male, as shown in Table 1, below:

Table 1: Average Fuel Storage in a "Normal" 65 kg Man*
Tissue Total amount of fuel Estimated duration of fuel reserve hours (days)
  g kJ Starvation walking running (long distance)
 Liver glycogen 90 1500







 Extracellular glucose 20 320







 Adipose fat 9,000 337,000







 Protein 8,800 150,000







Muscle glycogen 350 6,000







* Assuming 12% of body weight is fat for normal men (normal women are about 26%)

Data from E. A. Newsholme & A. R. Leach (1983) Biochemistry for the Medical Sciences, John Wiley, NY. p 337.

Next let's look at the normal and fasting fuel usage of various tissues in Table 2:

Table 2: Tissue Fuel Usage in Fed and Fasting States in Humans



 used released used released
Liver Glucose (stored as glycogen), Amino acids, Fatty acids Fats, Glucose Amino acids, Lactate, Fatty acids, Glycerol Glucose, Ketone bodies
 Kidney  Glucose   Amino acids, Lactate, Fatty acids, Glycerol   Glucose
 Intestine  Glucose, Aspartate & Glutamate, Asparagine & Glutamine Fatty acids, Amino acids other than asx & glx, Carbohydrates     
 Adipose  Glucose     Fatty acids, glycerol 
 Muscle  Glucose (some stored as glycogen), Branched chain amino acids  Lactate, Alanine & Glutamine  Fatty acids, Ketone bodies, Branched chain amino acids  Amino acids other than branched chain, Lactate
 Brain  Glucose  -  Glucose & Ketone bodies  -

For humans the brain uses about 20% of resting energy, regardless of whether its user is "vegging out" or studying like crazy. In the fed state the brain uses about 4 g/hr of glucose while the anaerobic tissues (e.g. red blood cells) use about 1.5 g/hr. This is a particular problem because the brain is quite restricted in what fuels it can use, while the anaerobic tissues are restricted to glucose alone.



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© R. A. Paselk 2010;

Last modified 6 May 2013