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Amino acid synthesis requires solutions to 3 key biochemical problems
1.
To obtain nitrogen in a more useful form
2.
To generate the correct isomer, stereochemistry is established by
transamination that include PLP
3.
To maintain sufficient amount of 20 aa for protein synthesis, feedback and
allosteric mechanism ensure it.
Nitrogen Fixation: Microorganisms Use ATP and a Powerful Reductant
to Reduce Nitrogen to Ammonia
* Nitrogen fixation: reduction of N2, carried out by some bacteria (none in
higher organism)
- Rhizobium: Diazotrophic (nitrogen-fixing) microorganisms.
* Nitrogenase complex: a reductase which provides e with high reducing
power + a nitrogenase which uses e to reduce N2 to NH3
In principle,
In biological reaction, an input of 2 additional e is required,
* In N2-fixing microorganism, 8 high-potential electrons come from reduced
ferredoxin generated by photosynthesis or oxidative phosphorylation.
At least 16 ATP are hydrolyzed for each N2 reduced.
•Nitrogen fixation: e from ferredoxin to reductase to nitrogenase to reduce N2
to ammonia. ATP hydrolysis within reductase drives conformational changes
necessary for efficient transfer of e.
The Iron-Molybdenum Cofactor of Nitrogenase Binds and Reduce Nitrogen
• Iron protein: a dimer composed of 2 polypeptide chains linked by a 4Fe-4S
cluster/ each monomer; a member of P-loop NTPase family and contains an
ATP-binding site
•Mo-Fe protein: heterodimer composed of 2 alpha and 2 beta, 2 copies of
each of 2 types of cluster (P cluster and FeMo cofactor), Each P cluster: 8 Fe
and 7 SH linked to protein by 6 Cys/ each FeMo cofactor: 1 Mo, 7Fe, 9 SH, 1
homocitrate, linked to protein by 1Cys and 1 His
•Electrons from reductase enter at P-clusters, which are located at alpha-beta
interface.
•Role of P cluster: to store e until used productively to reduce N2 at FeMocofactor
•The FeMo cofactor is the site of nitrogen fixation.
•Nitrogen-reduction site: FeMo cofactor contains an open center that is
likely site of nitrogen binding and reduction.
Ammonium Ion is Assimilated into Amino Acids by Way of Glutamate and
Glutamine
•Glutamate dehydrogenase:
proceeds in 2 steps
1)
Formation of Schiff base between an amine and carbonyl compound is
a key reaction in biosynthesis and degradation.
- Schiff bases are easily protonated.
2) The protonated Schiff base is reduced by transfer of hydride ion from
NADPH to form glutamate: establish the stereochemistry of alpha C (S
configuration) in Glu.
•Establishment of chirality: In active site of glutamate dehydrogenase,
hydride transfer from NADH to specific face of achiral protonated Schiff base
of alpha-ketoglutarate establishes L configuration of Glu
•2nd ammonium ion is incorporated into Glu to form Glm by Glm synthetase:
amidation is driven by hydrolysis of ATP.
•Glutamate synthase in most prokaryotes:
•When NH4+ is limiting, most of the glutamate is made by the sequential
action of glutamine synthetase and glutamate synthase.
•The sum of these reaction:
Amino Acids are Made from Intermediates of the Citric Acid Cycle and
Other Major Pathways
• Their carbon skeletons come from intermediates of
•
glycolysis, the pentose phosphate pathway, or the citric acid cycle.