5 - UNIVERSITY OF TORONTO FACULTY OF ARTS AND SCIENCE...

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Unformatted text preview: UNIVERSITY OF TORONTO FACULTY OF ARTS AND SCIENCE DECEMBER 2014 EXAMINATIONS BCH377H Biochemistry Laboratory I Duration 3 hours Tuesday, December 9, 2014 2—5 pm Calculators, notes, and lab reports allowed BCH3 77H Final Exam page 1 You must answer five (5) questions in total. Each one is worth twenty (20) marks for a total of 100 marks. You must answer the first question. You must choose any four (4) of the next five (5) questions. Answer this question. 1. (20 marks) Ken Dill is one of the leading experts on protein folding. In his classic review from 1990 entitled “Dominant Forces in Protein Folding” he said, More than 30 years after Kauzmann ’s insightfitl hypothesis, there is now strong accumulated evidence that hydrophobicity is the dominant force of protein folding, provided that “hydrophobic ” is operationally defined in terms of the transfer of nonpolar amino acids fiom water into a medium that is ‘150 2 4 " AFfom(kcaUmole} ’ ‘ "3 nonpolar and preferably capable of hydrogen bonding. Other V I _ . forces are weaker but can aflect stability. Denaturkflglgfiggmratmn In that same review he published a figure that should be familiar (right). (Note that AFfold is also called AGfold in modern terminology and 1 kcal = 4.2 J.) Explain why proteins become less stable and the free energy of folding increases in the presence of increasing concentrations of denaturant. Answer any four (4) of these five (5) questions. 2. (20 marks) Imagine that you are trying to create a genetically modified carrot that synthesizes a mystery chicken protein (CHK-X) in order to trick vegetarians. You don’t want anyone to detect the presence of this foreign protein in your carrots by doing routine PCR assays to detect recombinant DNA. How would you do this? Should you patent your construct? . (20 marks) What was the pH optimum of trypsin activity? Can you explain this in terms of the normal biological function of the enzyme and the physiological conditions under which it is active? Do you expect there to be a strong correlation between the optimal pH of an enzyme’s activity and the pH of the cell/environment where it is active? (20 marks) What factors do you think will determine the stability (half-life) of proteins in bacterial cells? Discuss examples such as ribosomal proteins, B-galactosidase, and glycolysis enzymes where ribosomal proteins have an extremely long half—life and B-galactosidase has a relatively short half—life. (Glycolysis enzymes are intermediate.) Continued (see next page) ........... .. BCH3 77H Final Exam page 2 Table 5‘! Examples of catalytic constants compare With your data from the MOX lab and the UniProt database? Explain any discrepancies. Enzyme tutu”? Papain 10 5. (20 marks) The kcat values of different enzymes can vary over a wide momma“? ‘02 range (see table). What are the catalytic constants that you found for carwxypepfidas‘i “32 trypsin and alcohol oxidase and how do they compare with the range WW“ 10200 703) and values shown in the table? Are they correlated with the Acetylmfilinesterase 103 difference in KM? Why are the turnover numbers (kw) so different KWSES 103 for different enzymes? ‘ D-ehydrogenases 103 Transaminases 103 Carbonic anhydrase 105 Supercxnde dismutase 105 Cataiase 107 yThe catalytic constants given only alsoer of magn nude. 6. (20 marks) According to Kato et a1: (1976), fit Wetmmfie . the molecular weight of alcohol oxrdase from E m Hansenula polymorpha is 83,000 by SDS “mmmt‘m 3 PAGE (see figure). The molecular weight of :3 E ‘32::me 55mm album the intact enzyme was 669,000 by g 6 -— amine dirnentation e uilibrium How do ou *3 5 .86 q ' y E f‘ umars see interpret those results and how do they r «3 ,Alaolaae Q " 3 2 _._._..___L.__ ...... 0 .1 D .3 0 .5 O .7 O .9 Relative mabxlfly ...
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