Transcript Assessment scenario for metabolic pathway

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?
Enzyme
#1
Precursor
molecule
(starting
molecule)
citrulline
Enzyme
#2
Arginine
Enzyme
#3
ornithine
One possible hypothesis
(ending
molecule)
What are the possible metabolic pathways that
could convert the precursor molecule into
arginine via citrulline and ornithine?
citrulline
Enzyme
#3
Enzyme
#2
Precursor
molecule
(starting
molecule)
Enzyme
#1
ornithine
Arginine
(ending
molecule)
Another possible hypothesis
Two different hypotheses for the
arginine metabolic pathway
Hypothesis A
Precursor
Enzyme 1
Citrulline
Enzyme 2
Ornithine
Enzyme 3 and
Other enzymes
Arginine
Hypothesis B
Precursor
Ornithine
Enzyme 1
Citrulline
Enzyme 2
Arginine
Enzyme 3 and
Other enzymes
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 Neurospora:
•
Missing enzyme 1
•
Missing enzyme 2
•
Missing enzyme 3
•
Normal (wild type)
Experimental description
Each of the Neurospora strains were grown in four
different conditions*:
Growth Medias:
Media with
precursor only
Media with
Ornithine only
Media with
Citrulline only
Media with
Arginine only
*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:
OR
Growth (+)
No growth (-)
Experimental description
Growth Medias:
Media with
precursor only
Media with
Ornithine only
Strains of Neurospora:
•
Missing enzyme 1
•
Missing enzyme 2
•
Missing enzyme 3
•
Normal (wild type)
Media with
Citrulline only
Media with
Arginine only
Possible results:
OR
Growth (+)
No growth (-)
Consider this example…
Metabolite A
Transport
Metabolite A
Enzyme X
Metabolite B
Enzyme Y
Metabolite C
• 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
Consider this example…
Metabolite A
Transport
Metabolite A
X
Enzyme X
Metabolite B
Enzyme Y
Metabolite C
If enzyme X is missing, and only metabolite A
is provided, the organism CANNOT survive
Consider this example…
Metabolite C
Metabolite A
X
Enzyme X
Metabolite B
Enzyme Y
Metabolite C
If enzyme X is missing, and metabolite C is
provided, the organism CAN survive
Consider this example…
Metabolite B
Metabolite A
X
Enzyme X
Metabolite B
Enzyme Y
Metabolite C
If enzyme X is missing, and metabolite B is
provided, the organism CAN survive
• B and C are “downstream” of enzyme X
Experimental description
Growth Medias:
Media with
precursor only
Media with
Ornithine only
Strains of Neurospora:
•
Missing enzyme 1
•
Missing enzyme 2
•
Missing enzyme 3
•
Normal (wild type)
Media with
Citrulline only
Media with
Arginine only
Possible results:
OR
Growth (+)
No growth (-)
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
Precursor
Enzyme 1
Citrulline
Media with
Precursor only
Strain of
mold missing
enzyme 1
Strain of
mold missing
enzyme 2
Strain of
mold missing
enzyme 3
Normal mold
(wild type)
Enzyme 2
Ornithine
Media with
ornithine only
Enzyme 3 and
Other enzymes
Media with
citrulline only
Arginine
Media with
arginine only
Predictions for hypothesis B
Precursor
Ornithine
Enzyme 1
Media with
Precursor only
Strain of
mold missing
enzyme 1
Strain of
mold missing
enzyme 2
Strain of
mold missing
enzyme 3
Normal mold
(wild type)
Citrulline
Enzyme 2
Media with
ornithine only
Arginine
Enzyme 3 and
Other enzymes
Media with
citrulline only
Media with
arginine only
Actual results
Media with
Precursor only
Strain of
mold missing
enzyme 1
Media with
ornithine only
+
Strain of
mold missing
enzyme 2
Media with
citrulline only
+
+
+
+
Strain of
mold missing
enzyme 3
Normal mold
(wild type)
Media with
arginine only
+
+
+
+
+
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
Missing or nonfunctional GLO
enzyme
X
Source: http://www.ajcn.org/cgi/content/abstract/54/6/1203S
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-gulonoy-lactone to L-ascorbic acid (vitamin C).