1. How does each of your mutations affect the amino acid sequences? Are the mutations missense mutations, silent mutations or nonsense mutations?
   1. Point mutation?
   2. Frameshift-insertion?
   3. Frameshift-deletion

The non mutated Sequence:

1. AUG GAG GUC UUU AAG AGA CAU UUA GAU GUA GCC CUU AGU GAU GUU UAG

Point Mutation:

• AUG GAC GUC UUC AAG AGC CAU UUA GAA GUA GCC CUU AGU GAU GUC UAG
• Translation: Met Asp Val Phe Lys Ser His Leu Glu Val Ala Leu Ser Asp Val Stop.

Frameshift Insertion Mutation:

• AUG UGA GGU UUU UAA GUA GAC AUU UAG AUG UAG CCA CUU AGU GAU GUU UAG
• Translated: Met Stop Gly Leu Stop Val Asp Ila Stop Met Stop Pro Leu Ser Asp Val Stop

Frameshift Deletion Mutation:

• AUG GAG UUU AAG AGA UUA GAU GUA GCC CUU AGU GAU GUU UAG
• Translated: Met Glu Phe Lys Arg Leu Asp Val Ala Leu Ser Asp Val Stop

1. What type of secondary structure would a peptide composed only of the amino acid Lysine form?

a) a random coil at all pH values

b) alpha helix at pH 7

c) alpha helix at pH well above 7

d) alpha helix at pH well below 7

2. During vigorous exercise ATP is hydrolyzed as follows,

ATP----> ADP +Pi + (H+)

what are the consequences of this for the structure-function of hemoglobin?

a) hemoglobin binds O2 stronger as a result of the changing pH

b) lower pH results in deprotonation of His146 which destabilizes the ion pair between monomers

c) lower pH results in protonation of His146 which stabilizes the ion pair between monomers

d) nothing happens to hemoglobin, only myoglobin is affected.

3. If you were to make a mutation to hemoglobin so that it could no longer form tetramers, what would you predict would occur.

a) impossible to predict

b) it would bind oxygen with lower affinity than before

c) it would transport oxygen better

d) hemoglobin would no longer be able to transport oxygen

1.   The following data were obtained from 3 separate enzyme kinetic experiments using 3 different substrates S1, S2 and S3 forming products P1, P2 and P3 respectively. The amount of enzyme in each reaction is 1 µM. Find out the rate and graph the data using a Michaelis-Menton and Lineweaver-Burk plots and determine the values for Km, Vmax, Kcat, and Kcat/Km. Which among the 3 substrate is best substrate for this enzyme and why? (4 points)

[S] (mM)   [P1] (mM) in 60 min   [P2] (mM) in 240 min   [P3] (mM) in 30 min
2.5   0.170   0.126   0.194
2   0.201   0.135   0.213
1.5   0.167   0.129   0.206
1   0.162   0.131   0.203
0.6   0.159   0.135   0.174
0.3   0.148   0.131   0.163
0.15   0.122   0.111   0.122
0.2   0.095   0.117   0.114
0.1   0.075   0.072   0.104
0.05   0.049   0.040   0.071

2.   Use the Michaelis-Menton Equation to calculate the missing values of [S] given below if Vmax = 5 mmol/min. Plot [S] versus V (NOT the reciprocals!). Draw line parallel to the x-axis at Vmax and extend your plotted line to show its approach to Vmax. (2 points)

[S] (mM)   V0 (mmol/min)
10   1.2
[S]1       1.7
[S]2       2.1
[S]3       2.2
[S]4       2.5

3.   Plot the below data and determine the type of inhibition of an enzymatic reaction by inspecting the graph (give an explanation) (2 points)

[S] (mM)   V0 (mM/min)   V0 with Inhibitor present (mM/min)
1   1.3   0.8
2   2.0   1.2
4   2.8   1.7
8   3.6   2.2
12   4.0   2.4

4.   Plot the below data with and without inhibitor (I) and determine the type of inhibition of an enzymatic reaction by inspecting the graph (2 points)

[S] µM   V0 (µmol /min); [I] = 0 nM    V0 (µmol /min); [I] = 25 nM    V0 (µmol /min); [I] = 50 nM
0.4   0.22   0.21   0.20
0.67   0.29   0.26   0.24
1.00   0.32   0.30   0.28
2.00   0.40   0.36   0.32

1. Which of the following is NOT a product of β-oxidation of a 20-carbon fatty acid?

2. Which pathway is NOT active in the liver in the well-fed state?

3. Beriberi is a condition caused by a thiamine deficiency. When analyzing blood samples of an individual with Beriberi, what would you expect to find elevated levels of?

4. Rotenone is a toxin that acts on the electron transport chain. When added to actively respiring mitochondria, the ratio of NADH to NAD⁺ increases while the ratio of FADH₂ to FAD does not change. What component of the ETC does it inhibit?

1. Membrane-associated translocators are responsible for importing polypeptides into both mitochondria and peroxisomes. The processes in the two organelles are different because _______________.

   		mitochondria have only one membrane translocator complex and peroxisomes have two
   		polypeptides are unfolded during translocation into mitochondria but not peroxisomes
   		mitochondrial polypeptides bind to cytosolic receptors or chaperones and peroxisomal polypeptides do not
   		peroxisome import requires signal sequences while mitochondrial import does not

2. Match each event in glycolysis with the correct description.

   First ATP investment step Second ATP investment step First step of cleavage stage Substrate-level phosphorylation

   A. Main point of allosteric regulation in the pathway B. Raises the free energy of glucose and traps it in the cell C. Transfers a phosphoryl group from an organic intermediate to ADP D. Produces two 3-carbon molecules that are similar but not identical

What is not true in the description of chloroplast?

Which of the characteristic iS NOT shared in common between mitochondria & chloroplastss?

An enzymatic reaction converts starch to simple sugars using a fungal enzyme that is very pH sensitive and works efficiently ?f and only if the pH of the reaction environment is ?.20. Your statistician, Mr. Biswadeep Pai decides to use a pH meter that is known to give normally distributed replicate measurements. Suppose that ?0 independent replicate measurements of pH yield the following values: {?. ?8, ?. ?7, ?. ?6, ?. ?5, ?. ?1, ?. ?7, ?. ??, ?. ?8, ?. ?9, ?. ??}.

(a) What conclusion can Biswadeep draw at the ? = ?. ?0 level of significance?

(b) What conclusion can Biswadeep draw at the ? = ?. ?5 level of significance?

(c) Is "?f and only if" in the above problem statement a typo?

Many cancer drugs have been developed that inhibit or activate enzyme function.

a. Geftinib is a tyrosine kinase inhibitor that has been shown to specifically target EGFR. EGFR is a receptor tyrosine kinase (RTK) and can become constitutively active in a cancer cell. Before we continue, explain how a RTKs function to transmit a signal (EGF) from the outside of the cell across the membrane to the inside of the cell. Be specific, what changes occur on the outside of the cell when ligand is bound? What changes occur on the inside of the cell that pass that information from the receptor to the first downstream target? I don’t need the entire signal transduction cascade. Rubric (4): correct explanation with the effect of ligand binding by the receptor on outside of the cell (2) and effect of ligand binding on inside of the cell (2).

b. Geftinib has been shown to occupy the ATP binding site of the tyrosine kinase domain of the EGFR, therefore we can hypothesize that it acts as a(n) __________________ inhibitor. (2pts)

c. Let’s think about a specific mutation in the EGFR (L858R).   The Kd for ATP binding to the tyrosine kinase domain of the EGFR (L858R) mutation is 148mM while the Kd of Geftinib for the L858R mutation of the EGFR is 2.4nM. Which binds more tightly (ATP or Geftinib) to EGFR (L858R) and how do these numbers confirm your conclusion in “b”? Rubric (4): correct analysis of binding (2) and correct explanation that relates analysis to conclusion in “b” (2).

d. Researchers determined proteins downstream of activated EGFR that were affected by Geftinib. We know some of these proteins! In the experiment, cancer cells with constitutively active EGFR are treated with or without Geftinib. In these experiments, Raf was downregulated in the cells treated with Geftinib when compared to untreated. Why does this make sense?   Make sure you substantiate your answer with concepts covered this week! Rubric (4): correct correlation is made between specific effect of the drug (1) and obvious application of concepts taught this week is applied to the answer, and they are correct (3).

A colorimetric LDH assay provides a method to measure the enzyme activity of LDH. NADH (MW 633.4 g/mol) absorbs light at 340 nm while NAD+ does not show any absorption at that wavelength. The Beer-Lambert Law relates the absorbance of a solute to its concentration: A = ε*c*L A is absorbance, ε is the molar extinction coefficient, c is concentration, and L is the path length of the solution in the spectrophotometer (usually in cm). In the lab you have available NADH. You dissolve 4.8 mg in 50 ml of buffer and measure an absorbance of 0.2363 at 340 nm in a 0.5 cm cuvette. Subsequently in a 1 cm cuvette, you mix 25 µl of 20 mM NADH, 25 µl pyruvate 0.2 M , 400 µl of TRIS buffer (pH=7.8), and 50 µl of a solution containing lactate dehydrogenase at a concentration of 2 mg/l. You place the cuvette in a spectrophotometer and follow the kinetics of absorbance at 340 nm. You measure a rate of decrease of absorbance (ΔA) of 0.50 min-1 .

a) Determine the absolute amount of NADH consumed after 20 seconds in the reaction (in nmol)

b) Determine the specific activity of LDH as NAD+ produced per LDH in the unit of time (in µmol/mg/min)