Based on what you have learned about the functioning of enzymes and proteins, explain why the pH in the cell has to be buffered.
Draw the structure of the three aromatic amino acids Phenylalanine, Tyrosine,
...
Based on what you have learned about the functioning of enzymes and proteins, explain why the pH in the cell has to be buffered.
Draw the structure of the three aromatic amino acids Phenylalanine, Tyrosine, and Tryptophan. Include the three-letter codes, the single letter codes and the pKa values of the side group when appropriate.
4
3 points What fragments do I get when I cut this peptide with Pepsin and Cyanogen Bromide?
5
4 points Explain the principle of hydrophobic interaction chromatography.
6 The figure shows a two-dimensional gel of a mixture of proteins A, B, and C. The vertical axis represents SDS-PAGE separation run from top to bottom. The horizontal axis represents isoelectric focusing with a pH gradient that runs from left to right (high pH to low pH). Proteins A, B, and C have isoelectric points of 8.2, 6.7, and 5.2, respectively. Complete parts (a) and (b), below.
a
2 points Explain in what order proteins A, B, and C will come down a size exclusion column. Some might be multimeric but assume that each protein contains only one copy of the subunits indicated on the gel
b
2 points Explain in what order proteins A, B, and C will come down a cation exchange column using an increasing salt gradient run at pH 5.
7 For each of these purifications provide a detailed explanation whether you think the protein is pure or not and whether additional steps are needed to confirm purity.
a
2 points
b
2 points
The protein might be pure after step 4 in the case that it is a protein with 3 subunits, but we will not know until more purification steps are performed. A size-exclusion step should be the next step to see if the three bands can be separated or not.
The schematic drawing on the right represents a typical very stable secondary structure found in proteins.
What type of structure is this? α-helix
Explain what properties give this structure its stability.
A student purifies a new protein from Animal A and obtains the sequence. The student compares this sequence with sequences in a
data base and the sequence search provides the sequence alignments below:
The sequences fall in three groups, group I (sequences 1, 2, and 3) with a score at around 500, group II (4-6) with a score at around 300, and group III (7-9) with a score at around 50. For comparison amino acids that are conserved in more than one group are shown in white with a gray background. Amino acids that are conserved in only one group are shown in bold. Amino acids that are not conserved are shown in small caps.
a) What does the value of the score mean?
b) What does it tell you if an amino acid is highly conserved? Which ones are highly conserved (White? Bold? Low caps?)
c) All three groups are similar but not to the same extent. Which groups could be orthologs and which group could be paralogs?
d) What type of enzyme is our unknown protein? Explain how you reach your conclusion.
a) The higher the value the more amino acids are conserved between the unknown protein and the sequence in the table.
b) If an amino acid is highly conserved it probably is very important for the function of the enzyme. Mutation might result in the loss of activity. The white ones are highly conserved.
c) groups I and II are orthologs since they are found in different species but have the same function. group II are the paralogs since these have a different function.
d) The unknown enzyme is a thing-oxidoreductase since it has the most sequence similarity to that type of enzyme in cluding all the highly conserved amino acids.
What is a hydropathy plot? What can we learn from a hydropathy plot?
Describe/draw in detail the ‘catalytic cycle’ of the folding of proteins by the GroEL/GroES complex.
12
4 points Draw the structure of heme c. What makes this molecule suitable to reversibly bind molecular oxygen?
13
4 points Explain in detail the Bohr Effect in hemoglobin
14
8 points a) Give the Michaelis Menten equation and the reactions used to derive this equation.
b) List the important assumptions used by Michaelis and Menten to derive a rate equation for this reaction.
c) The Michaelis-Menten constant, Km, is actually a summary of three terms. What are they?
d) How is Km determined graphically?
a
15
3 points Below are plots (Michaelis AND Lineweaver-Burk) that are used to investigate inhibitor types. For each set of plots assign the inhibitor type and explain (short) your assignment.
16
6 points A large variety of carbohydrates has been discovered with very different properties:
What structural features of these polysaccharides underlie their different physical properties? Explain the biological advantages of their respective properties.
Describe in detail how the sugar code is used to direct white blood cells to sites of inflammation.
18
4 points
What are reactive oxygen species? How are they generated? What role does the pentose phosphate pathway play in the removal of these species?
19
6 points
Draw the glycolysis pathway from glucose to glyceraldehyd-3-phosphate (include all the structures of the intermediates in the pathway, the names of these compounds, other small molecules, and the names of the enzymes catalyzing the respective reactions).
20
5 points
List and explain the five reasons why the hydrolysis of ATP has a large negative ΔG°’
value
Describe in detail how the bifunctional enzyme phosphofructokinase-2/fructose-2,6- bisphophatase and the product fructose-2,6-bisphosphate regulate glycolysis and gluconeogenesis.
Explain in detail the regulation of glycogen synthase
23
4 points
What is the physiological rationale behind coordinated activation of gluconeogenesis and glycogen breakdown in the liver, vs. coordination of glycolysis and glycogen breakdown in muscle? What are the hormones and enzymes central to this process?
24
5 points The figure shows an overview of the different pathways glucose is involved in. Indicate in the figure which of these steps are regulated (9 total), give the enzyme names that catalyze these step and indicate the compounds that are involved in the regulation
[1] hexokinase
- glucose-6-P
[2] phosphofructokinase + fructose-2,6-bisphosphate/AMP
- ATP/citrate
[3] pyruvate kinase + fructose-1,6-BP
- acetyl CoA/ATP/alanine
- cAMP dept. regulation
[4] pyruvate carboxylase + acetyl-CoA
[5] fructose-1,6-bisphosphatase - fructose-2,6-BP/AMP
+ citrate
[6] glucose-6-phosphatase + glucose-6-P
[7] glycogen synthase + insulin
+ glucose-6-P
[8] glycogen phosphorylase + CAMP dept. regulation/ phosphorylation/glucagon/epinephrine
+ AMP
- glucose-6-P/glucose/caffeine
[9] glucose-6-phosphate dehydrogenase - NADPH
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