How do scientists identify the connections in a metabolic
pathway?
Arginine synthesis
What are the possible metabolic pathways that could convert the precursor molecule into
arginine via citrulline and ornithine?
Precursor molecule
Arginine
citrulline
ornithine
Enzyme#1
Enzyme#2
Enzyme#3(starting
molecule)
(endingmolecule)
One possible hypothesis
What are the possible metabolic pathways that could convert the precursor molecule into
arginine via citrulline and ornithine?
Precursor molecule
Arginine
citrulline
ornithine
Enzyme#1
Enzyme#2
Enzyme#3
(startingmolecule)
(endingmolecule)
Another possible hypothesis
Hypothesis A
Hypothesis B
Precursor ArginineEnzyme 1 Enzyme 2 Enzyme 3 and
Other enzymes
OrnithineCitrulline
Precursor ArginineEnzyme 1 Enzyme 2 Enzyme 3 and
Other enzymes
Ornithine Citrulline
Two different hypotheses for the arginine metabolic pathway
Determining the arginine pathway
• Arginine is an amino acid required for Neurospora (bread mold) to survive
• Normal (wild type) Neurospora can make arginine using other metabolites and enzymes in a metabolic pathway
• Strains of Neurospora which are missing enzymes in the metabolic pathway can obtain arginine and other metabolites from their growth media and environment
Experimental description
Four strains of Neurospora (bread mold) were used:
• The wild type strain has all 3 functional enzymes in the arginine pathway
• The other strains are each missing a different enzyme in the arginine pathway
Strains of Strains of NeurosporaNeurospora::• Missing enzyme 1Missing enzyme 1
• Missing enzyme 2Missing enzyme 2
• Missing enzyme 3Missing enzyme 3
• Normal (wild type)Normal (wild type)
Experimental description
Each of the Neurospora strains were grown in four different conditions*:
Media with Media with precursor onlyprecursor only
Media with Media with Ornithine onlyOrnithine only
Media with Media with Citrulline onlyCitrulline only
Media with Media with Arginine onlyArginine only
Growth Medias:Growth Medias:
*All of the media had the same sugars and salts but no additional amino acids
Experimental description
Neurospora cultures will grow if arginine is provided in the media or if they are able to make arginine from other molecules in the media
Possible results:Possible results:
Growth (+)Growth (+) No growth (-)No growth (-)
OR
Experimental description
Possible results:Possible results:
Growth (+)Growth (+) No growth (-)No growth (-)
OR
Strains of Strains of NeurosporaNeurospora::• Missing enzyme 1Missing enzyme 1
• Missing enzyme 2Missing enzyme 2
• Missing enzyme 3Missing enzyme 3
• Normal (wild type)Normal (wild type)
Media with Media with precursor onlyprecursor only
Media with Media with Ornithine onlyOrnithine only
Media with Media with Citrulline onlyCitrulline only
Media with Media with Arginine onlyArginine only
Growth Medias:Growth Medias:
Consider this example…
• Metabolite C is required for survival
• Metabolite A is obtained from the environment and transported into the cell
• A metabolic pathway converts A into C
Metabolite A Metabolite B Metabolite CEnzyme X Enzyme Y
Metabolite A
TransportOutside of the cell
Inside of the cell
Consider this example…
If enzyme X is missing, and only metabolite A is provided, the organism CANNOT survive
Metabolite A Metabolite B Metabolite CEnzyme X Enzyme Y
Metabolite A
TransportOutside of the cell
Inside of the cell
X
Consider this example…
If enzyme X is missing, and metabolite C is provided, the organism CAN survive
Metabolite A Metabolite B Metabolite CEnzyme X Enzyme Y
Outside of the cell
Inside of the cell
X
Metabolite C
Consider this example…
If enzyme X is missing, and metabolite B is provided, the organism CAN survive
• B and C are “downstream” of enzyme X
Metabolite A Metabolite B Metabolite CEnzyme X Enzyme Y
Outside of the cell
Inside of the cell
X
Metabolite B
Experimental description
Possible results:Possible results:
Growth (+)Growth (+) No growth (-)No growth (-)
OR
Strains of Strains of NeurosporaNeurospora::• Missing enzyme 1Missing enzyme 1
• Missing enzyme 2Missing enzyme 2
• Missing enzyme 3Missing enzyme 3
• Normal (wild type)Normal (wild type)
Media with Media with precursor onlyprecursor only
Media with Media with Ornithine onlyOrnithine only
Media with Media with Citrulline onlyCitrulline only
Media with Media with Arginine onlyArginine only
Growth Medias:Growth Medias:
HYPOTHESES/PREDICTIONS
• Make predictions for results that would support hypothesis A and predictions for results supporting hypothesis B.
• On your charts, use a + to indicate growth and a – to indicate no growth
Predictions for hypothesis A
Media with Precursor only
Media with ornithine only
Media with citrulline only
Media with arginine only
Strain of mold missing enzyme 1
Strain of mold missing enzyme 2
Strain of mold missing enzyme 3
Normal mold
(wild type)
Precursor ArginineEnzyme 1 Enzyme 2 Enzyme 3 and
Other enzymes
OrnithineCitrulline
Predictions for hypothesis B
Precursor ArginineEnzyme 1 Enzyme 2 Enzyme 3 and
Other enzymes
Ornithine Citrulline
Media with Precursor only
Media with ornithine only
Media with citrulline only
Media with arginine only
Strain of mold missing enzyme 1
Strain of mold missing enzyme 2
Strain of mold missing enzyme 3
Normal mold
(wild type)
Actual results
Media with Precursor only
Media with ornithine only
Media with citrulline only
Media with arginine only
Strain of mold missing enzyme 1
Strain of mold missing enzyme 2
Strain of mold missing enzyme 3
Normal mold
(wild type)
+ + +
+ +
+
+ + + +
Conclusion
1) Explain which hypothesis is supported by the experimental results.
2) How do you know that the other hypothesis was not correct? (Explain which specific data refute the alternative hypothesis.)
3) Other than arginine, what molecule could you give to the Neurospora strain missing enzyme #2 in order for this strain to survive?
Application to Disease
• Vitamin C is required for several processes in the body
• Most animals have a metabolic pathway allowing them to synthesize vitamin C from simpler metabolites
• Humans are not able to make vitamin C because they do not have all the enzymes in the metabolic pathway for vitamin C.
Metabolic Pathway of Vitamin C Biosynthesis in Animals
X
Source: http://www.ajcn.org/cgi/content/abstract/54/6/1203S
Missing or non-functional GLO
enzyme
Treatment for Vitamin C Deficiency
How are humans able to live without having a metabolic pathway to make their own vitamin C?
• Vitamin C must be obtained by eating foods with this molecule
• People who do not eat enough vitamin C suffer from a condition called scurvy
• Citrus fruits like oranges, grapefruit, and limes have a high vitamin C content
Additional fun notes!
• Primates, guinea pigs, the red-vented bulbul (an Asian bird), the Indian fruit-eating bat, rainbow trout and Coho salmon are the only animal species that cannot make vitamin C.
• These animals must obtain vitamin C from food in their diet.
• Like humans, guinea pigs also lack the enzyme gulonolactone oxidase (GLO), which is an enzyme necessary to convert L-gulono-y-lactone to L-ascorbic acid (vitamin C).