IGEM:IMPERIAL/2009/Encapsulation/PKU

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Contents

Phenylketonuria - PKU

Summary


Solution Enzyme(s) involved Cofactor? Viable in small intestine? References Feasible
Phenylalanine hydroxylase. We would need to make the E. Coli secrete a modified PAH enzyme that is less susceptible to proteolysis. Phenylalanine Hydroxylase BH4, this is what the only drug for phenylketonuria (Kuvan) targets. Kuvan is a functional analog of this co-factor thus only targeting a subset of PKU patients The enzyme can be rendered protease resistant via full phosphorylation and operates at pH similar to that of the small intestine.[1] Paper Doable, not the easiest of solutions
Phenylalanine ammonia-lyase Phenylalanine ammonia-lyase None The enzyme was shielded against proteases either by secreting a protease inhibitor (aprotinin) or by retaining it into the transfected E. Coli cells. Problems with these two approaches are -respectively- that we would inhibit normal digestion of peptides with proteases and that we would need to keep the modified bacterium alive. Too many drawbacks

Absorption in gut

Phenylalanine is absorbed as an amino acid in our gut. [2] This is because the hydrolysis of peptides involving phenylalanine is fast enough. It takes 175 minutes or approximately 3 hours for all phenylalanine related peptides to be digested, after which, phenylalanine will be present as an amino acid to be absorbed. [3] For this experiment, volunteers are fed a milk-protein meal.

Image:mpmeal.jpg [4]

Image:phenylabsorbstats.jpg

Great book on digestion [1]

Metabolism of Phenylalanine

Phenylalanine is fully catabolised to CO2 and water, after its hydroxylation to tyrosine. This is the rate limiting step in the whole process. The complex system requires PAH, tetrahydrobiopterin co-enzyme (BH4) and several enzymes for the regeneration of BH4. The characteristics of phenylalanine metabolism have been modelled[5][6].

Conversion to tyrosine

Image:pku_meta1.jpg


Image:pku_meta2.jpg

Phenylalanine is converted to tyrosine by phenylalanine hydroxylase. Tyrosine synthesis in the body depends on the availability of phenylalanine. When phenylalanine intake is limited, tyrosine can become a conditionally indispensable amino acid.

Image:tyro_meta2.jpg

The carboxyl carbon of the tyrosine is released as CO2 when tyrosine degrades. The remainder of the molecule is converted to fumarate or acetoacetate, which enter the tricarboxylic acid cycle.

[2] [3]

BH4

PAH PhenylAlanine Hydroxylase

This is the enzyme that catalyses the reaction from phenylalanine to tyrosine. It is naturally produced by the liver. However, it was expressed in E. Coli and shown to be functional at pH 7.0[1]. Furthermore, phosphorylation by the C-subunit kinase rendered the enzyme resistant to the Xa protease (fig 1 below) which is a member of the serine protease family[1]. The serine protease family includes some of the most abundant proteases present in the human gastro-intestinal tract such as chemotrypsin-like, signal peptidases (More Info on Serine Proteases).
Therefore we think that by fully phosphorylating the PAH enzyme during its production process, we can protect it against the protease-rich environment of the small intestine.
The PAH enzyme is activated by L-Phenylalanine[1] and thus is expected to be activated in the environment of the small intestine where food is naturally degraded into amino acids. These amino acids are subsequently absorbed by the gut. Fig. 1 Protection of PAH enzyme by full-phosphorylation

References

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  1. Error fetching PMID 8573072: [PAH1]
  2. Error fetching PMID 1413009: [absorption]
  3. Error fetching PMID 5424261: [absorption1]
  4. Error fetching PMID 5424259: [absorption2]
  5. Error fetching PMID 10077654: [MetabolismModel]
  6. Error fetching PMID 1503063: [MetabolismModel2]
All Medline abstracts: PubMed HubMed
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