How do scientists identify the connections in a metabolic pathway?

1. How do scientists identify the connections in a metabolic pathway? Arginine synthesis

2. What are the possible metabolic pathways that could convert the precursor molecule into arginine via citrulline and ornithine? citrulline Enzyme #1 Enzyme #2 Precursor molecule Arginine Enzyme #3 (ending molecule) (starting molecule) ornithine One possible hypothesis

3. 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 Arginine (ending molecule) Enzyme #1 (starting molecule) ornithine Another possible hypothesis

4. Precursor Citrulline Ornithine Arginine Enzyme 1 Enzyme 2 Enzyme 3 and Other enzymes Precursor Ornithine Citrulline Arginine Enzyme 1 Enzyme 2 Enzyme 3 and Other enzymes Two different hypotheses for the arginine metabolic pathway Hypothesis A Hypothesis B

5. 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

6. 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

7. Each of the Neurospora strains were grown in four different conditions*: Growth Medias: Media with Arginine only Media with Citrulline only Media with precursor only Media with Ornithine only Experimental description *All of the media had the same sugars and salts but no additional amino acids

8. 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

9. Growth Medias: Media with Arginine only Media with Citrulline only Media with precursor only Media with Ornithine only • Strains of Neurospora: • Missing enzyme 1 • Missing enzyme 2 • Missing enzyme 3 • Normal (wild type) Possible results: OR Growth (+) No growth (-) Experimental description

10. Consider this example… Metabolite A • 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 Outside of the cell Transport Inside of the cell Metabolite A Metabolite B Metabolite C Enzyme X Enzyme Y

11. Consider this example… Metabolite A If enzyme X is missing, and only metaboliteA is provided, the organism CANNOT survive Outside of the cell Transport Inside of the cell X Metabolite A Metabolite B Metabolite C Enzyme X Enzyme Y

12. Consider this example… Metabolite C If enzyme X is missing, and metabolite C is provided, the organism CANsurvive Outside of the cell Inside of the cell X Metabolite A Metabolite B Metabolite C Enzyme X Enzyme Y

13. Consider this example… Metabolite B If enzyme X is missing, and metabolite B is provided, the organism CANsurvive • B and C are “downstream” of enzyme X Outside of the cell Inside of the cell X Metabolite A Metabolite B Metabolite C Enzyme X Enzyme Y

14. Growth Medias: Media with Arginine only Media with Citrulline only Media with precursor only Media with Ornithine only • Strains of Neurospora: • Missing enzyme 1 • Missing enzyme 2 • Missing enzyme 3 • Normal (wild type) Possible results: OR Growth (+) No growth (-) Experimental description

15. 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

16. Precursor Citrulline Ornithine Arginine Enzyme 1 Enzyme 2 Enzyme 3 and Other enzymes Predictions for hypothesis A

17. Precursor Ornithine Citrulline Arginine Enzyme 1 Enzyme 2 Enzyme 3 and Other enzymes Predictions for hypothesis B

18. Actual results

19. 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?

20. 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.

21. Metabolic Pathway of Vitamin C Biosynthesis in Animals Missing or non-functional GLO enzyme X Source: http://www.ajcn.org/cgi/content/abstract/54/6/1203S

22. 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

23. 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).