how do scientists identify the connections in a metabolic pathway? arginine synthesis

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


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


• Normal (wild type)Normal (wild type)


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


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




OR










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


If enzyme X is missing, and only metabolite A is provided, the organism CANNOT survive


Metabolite A

TransportOutside of the cell

Inside of the cell

X


If enzyme X is missing, and metabolite C is provided, the organism CAN survive


Outside of the cell

Inside of the cell

X

Metabolite C


If enzyme X is missing, and metabolite B is provided, the organism CAN survive

• B and C are “downstream” of enzyme X


Outside of the cell

Inside of the cell

X

Metabolite B




OR










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



Normal mold

(wild type)


Other enzymes

OrnithineCitrulline

Predictions for hypothesis B


Other enzymes

Ornithine Citrulline








Normal mold

(wild type)

Actual results








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).

how do scientists identify the connections in a metabolic pathway? arginine synthesis

Documents

different enzyme

arginine pathway arginine

missing enzymes

arginine synthesis slide

cell slide

possible hypothesis

neurospora strains

environment slide