Give a brief explanation for each of your answers. a. Which protein is silk? b. Which protein is wool? c. Which protein is collagen?

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1 1. Three proteins were extracted from strange organisms brought back to earth by space travelers to a distant planet. The three proteins were analyzed by x-ray crystallography and were found to be structurally quite "earth-like". One protein is similar to wool and is predominantly α- helix. One protein is similar to silk and is predominantly β-sheet. The third protein is a collagen triple helix. Unfortunately, when faithful lab assistant Klutz centrifuged the three tubes containing what remained of the three proteins, the labels came off the tubes. You now do not know which tube contains what protein! And the "National Enquirer" is interviewing you tomorrow. C'est terrible!! There is not enough time or material left to repeat the crystallographic analysis. However, an undergraduate in the lab who is taking a biochemistry course is able to identify the proteins quickly by using HPLC to perform an amino acid composition analysis of each protein. The % composition of each amino acid is shown in the table. The three "unknown" proteins are A, B, and C. Give a brief explanation for each of your answers. a. Which protein is silk? b. Which protein is wool? Hypro = hydroxyproline c. Which protein is collagen?

2 2. Retinopathy associated with the disease diabetes mellitus causes blindness. It has been observed that in retinal cells of afflicted individuals, the intracellular concentration of inositol is higher than in normal individuals. Evidence suggests that the abnormally high concentration of inositol may directly relate to the development of blindness. Scientists studying the problem have demonstrated that transport of inositol into the retinal cells is defective, and is the cause of the abnormally high intracellular concentration of inositol. Kinetic analysis of inositol transport in afflicted individuals resulted in the following observations: i)the K M is not affected. That is, the K M for inositol in the afflicted individuals is the same as is observed in normal individuals. ii) The V MAX however, is twice as large in the afflicted individuals as it is in normal individuals. Based on this data, explain how too much inositol is transported into the retinal cells of the patients with diabetic retinopathy. Specifically, what is the matter with the inositol transport system? Why is the K M the same? Why is the V MAX so high?

3 3. Recently, mutations in the human gene encoding the enzyme superoxide dismutase (SOD1) were found to be the cause of the degenerative disorder of motor neurons called ALS or Lou Gehrig's disease. The authors of the paper identified 11 different missense mutations in the SOD1 gene. For several reasons, the authors suggest that, rather than decreasing enzyme activity, the mutations might actually increase SOD1 enzyme activity. In an editorial commentary on the article, the following statement is made, a statement that contradicts the above suggestion: "The native SOD1 operates at a diffusion-limited rate, so any mutation...must be expected to impair function." Explain your answers. i) Based on your understanding of Michaelis-Menten kinetics, explain what is meant by "a diffusion-limited rate". ii) Why does the author of the commentary think the mutations in SOD1 would be "expected to impair function"? iii) Would you expect the affinity of SOD1 for its substrate to be low or high? iv) Would you expect the K M of such a perfect enzyme to be low or high?

4 4. About what charge would you expect the following peptide to have at ph 4, ph 7, ph9, and ph 12? SHOW YOUR WORK and use Table 2.4 in Stryer. ph 4: ph 7: ph 9: ph 12:

5 5. What is the order of elution when a mixture of lys, cys and asp, in a buffer at ph12, is bound to an anion exchange column, and then eluted with buffers of decreasing ph? (The anion exchange resin is positively charged). Explain your answer and use Table 2.4 in Stryer.

6 6. i) Calculate the G ' for the reaction: gly-trp + H 2 O gly + trp from the following standard free energies of formation ( G f )at 25 C. gly = kcal/mol trp = kcal/mol H 2 O= kcal/mol gly-trp = kcal/mol ii) Calculate the amount of free energy that must be expended in order to synthesize 10g of the peptide. The average molecular weight of an amino acid residue is 110. iii) How many moles of ATP must be hydrolyzed according to the equation: ATP + H 2 O ADP + P i G ' = -8 kcal/mol in order to provide the energy for the synthesis of the 10g of peptide?

7 7. The following reaction takes place in a hypothetical cell at 37 C: R-O-P R-OH + P i G' = kcal/mol The intracellular levels of the phosphorylated metabolite, R-O-P, and its hydrolysis products, R-OH and P i are at the following concentrations: R-O-P = 2 x 10-2 M R-OH = 4 x 10-5 M P i = 5 x 10-2 M i) Calculate the G ' for this hydrolysis reaction. ii) Calculate the G ' for the coupled reaction assuming the G ' for the formation of ATP is 7.3 kcal/mol: R-O-P + ADP R-OH + ATP iii) Would the coupled reaction lead spontaneously to the synthesis of ATP? Explain.

8 8. Explain why the K M of most enzymes changes with ph.

9 9. For the enzyme aspartate transcarbamylase, succinate acts as a competitive inhibitor of one of the two substrates, the amino acid aspartate. The dependence of velocity on [asp] is shown in graph (a). *Assume the second substrate is present in excess in these experiments and can be ignored* i) Explain the curve shown in graph (a). ii) In the experiment shown in graph (b), [asp] is held constant at the low level indicated by the arrow in graph (a). Increasing amounts of succinate are added. Succinate cannot participate as a substrate in the reaction. Explain the curve shown in graph (b).