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1. You are studying a new enzyme isolated from an organism capable of growing at boiling water
temperatures. You obtain data of a wild-type enzyme and a mutant form recently found in a culture of
this same thermophilic organism growing on the bench (a graduate student forgot to put the culture in
the 90°C incubator). You have determined that the two enzymes differ by one amino acid in the
sequence of the respective enzymes within the active site. You name the wild-type enzyme Hot Stuf
and the mutant Not-so Hot Stuf. Below is a table of their activities. Maximum
Hot Stuf 58 µmole/min Not-so Hot
Stuf 0.2 µmole/min Km
M a. Which enzyme has a higher affinity for the substrate?
b. What is the initial velocity of the reaction catalyzed by Hot Stuf at a substrate concentration of
c. Which enzyme shifts Keq, the equilibrium constant, more in the direction of the product?
d. What amino acid change would likely eliminate the Not-so Hot Stuf enzyme from allowing the
bacteria to grow at high temperature? The key amino acid in the wild type enzyme is a
2. Why does the glycolytic pathway occupy a central position in cell metabolism?
3. What is accomplished in the preparatory phase of glycolysis? 4. What is accomplished in the payoff phase of glycolysis?
5. What is the efficiency of recovery, in the form of ATP, of the energy released by glycolysis under
6. Is efficiency of energy recovery higher under the conditions that exist in living cells? Justify your
answer. 7. In the reaction below, what is being oxidized? What is being reduced? 1 8. The C-1 of glucose is radioactively labeled and then enters glycolysis, which carbon of glyceraldehyde3-phosphate will be labeled? What fraction of molecules will carry the radioactive label?
9. The figure below represents a cross section through a membrane and the seven circles represent a ?top
down? view of the seven a helices of bacteriodopsin. What classes of amino acid side chains would
you expect to be projecting from the regions that interact with the membrane lipids, and what side
chains would you expect to find projecting into the pore? This type of orientation is termed what? 10. What are histones? What is their function(s)? Describe the structure shown below. How does it
demonstrate the special characteristics of histones? The figure is taken from the PDB 5CDK. a. What is the sequence of the mRNA that encodes this protein? 2 b. What is the amino acid sequence of this particular protein?
c. What is the pI of this protein and how does this relate to its function?
You may find it helpful to utilize the NCBI website. 11. Which restriction enzyme(s) would you use to cleave the plasmid in order to insert the following cut
pieces of DNA?
GACGTCTTCGAAGGCCTAGGGGCCTCGA What will be the phenotype of the bacteria that are transformed, separately, with the
recombinant plasmids? 3
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