BCH2333B INTRODUCTION TO BIOCHEMISTRY MIDTERM 3 17TH MARCH, 2017

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BCH2333B INTRODUCTION TO BIOCHEMISTRY MIDTERM 3 17TH MARCH, 2017

True/False questions (total points: 12)

1.) Enzymes increase the rate of biochemical reactions by stabilizing the transition state and shifting the
chemical equilibrium from reactants to products.
True
False. If false, explain why.

2.) Trypsin hydrolyses the C-terminal peptide bond following lysine and asparagine
True
False. If false, explain why.

3.) The hill coefficient is a measure of the degree of cooperativity in ligand binding to a protein.
True
False. If false, explain why.

4.) Affinity chromatography requires an affinity tag with a high K D to limit non-specific binding of proteins
True
False. If false, explain why.

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BCH2333B INTRODUCTION TO BIOCHEMISTRY MIDTERM 3 17TH MARCH, 2017

Define the following terms (15 points):

MWC model of cooperativity

Cation-exchange chromatography

Dissociation constant (Kd)

Induced fit

Michaelis constant (KM)

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BCH2333B INTRODUCTION TO BIOCHEMISTRY MIDTERM 3 17TH MARCH, 2017

Multiple choice questions. Note that more than one correct answer is possible (12 points):
5.) The hill plot is useful for:
a. Determining if the binding of a ligand by a protein has a positive or negative effect on the ability of
the protein to bind to another ligand
b. Approximating the concentration of ligand required to half saturate the protein
c. Estimating the KD for the initial binding state
d. Determining the number of ligands associated with the slot (n)
e. None of the above

6.) Which of the following two terms contribute the most energy to protein folding:
a. Hydrophobic effect
b. Hydrogen bonding enthalpy
c. Van der Waals enthalpy
d. Electrostatic enthalpy

7.) At pH 10 what will the ratio of ammonia to ammonium be? The pKa of ammonium is 9.24
a. 5.7
b. 0.76
c. 0.17
d. 1.3

8.) When denaturing a protein, which thermodynamic force is primarily affected:

a. Van der waals enthalphy
b. Hydrogen bonding enthalpy
c. Electrostatic bonding enthalpy
d. Hydrophobic effect

9.) When freezing a protein, which thermodynamic force is primarily affected:

a. Van der waals enthalphy
b. Hydrogen bonding enthalpy
c. Electrostatic bonding enthalpy
d. Hydrophobic effect

10.) You are trying to separate the five proteins below by gel filtration chromatography. Which will elute
first from the column?
a. Cytochrome C (12 kDa)
b. RNA polymeraze (99 kDa)
c. Glutamine synthetase (621 kDa)
d. Interferon- (34 kDa)
e. Hemoglobin (62 kDa)

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BCH2333B INTRODUCTION TO BIOCHEMISTRY MIDTERM 3 17TH MARCH, 2017

Short answer questions. (12 points)

1.) What would be the result of an enzyme having a higher binding energy for the substrate compared with
the transition state?

2.) Using thermodynamics, describe how sodium dodecyl sulfate (SDS) favors the unfolded protein state.

3.) When preparing a protein for SDS-PAGE analysis, the native protein must be unfolded. What three steps
are required to convert a native protein to an unfolded protein coated in SDS?

4.) What are the six different classes of enzymes?

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BCH2333B INTRODUCTION TO BIOCHEMISTRY MIDTERM 3 17TH MARCH, 2017

Long answer
Question 1 (10 points): In 1913, Dr. Leonor Michaelis and Dr. Maud Menten developed a general theory of
enzyme action. Given the chemical reaction below, derive the Michaelis-Menten equation. Circle your final
answer and show all steps.

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BCH2333B INTRODUCTION TO BIOCHEMISTRY MIDTERM 3 17TH MARCH, 2017

Question 2 (5 points): If the [S] >>>> KM, the rate of reaction (v) will be ______ order. Justify this observation
using the Michaelis-Menten equation.

Question 3 (5 points). If the [S] <<<< KM, the rate of the reaction (v) will be _______ order. Justify this
observation using the Michaelis-Menten equation.

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BCH2333B INTRODUCTION TO BIOCHEMISTRY MIDTERM 3 17TH MARCH, 2017

Question 4 (8 points). The catalytic efficiency for an enzyme can be described using the specificity constant,
kcat/KM. Assuming the [S] <<< KM, show that the specificity constant is second-order.

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BCH2333B INTRODUCTION TO BIOCHEMISTRY MIDTERM 3 17TH MARCH, 2017

Question 5 (7 points). Below is Lineweaver-Burk plot for a generic enzyme:

Linearize the Michaelis-Menten equation and identify the missing parameters on the graph above. Be sure to
include units for each parameter.

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BCH2333B INTRODUCTION TO BIOCHEMISTRY MIDTERM 3 17TH MARCH, 2017

Question 6 (10 points). Carbon monoxide (CO) poisoning can lead to shortage of breath, severe headaches,
and, at high concentrations, death. The graph indicates the fractional saturation of hemoglobin binding sites
given the partial pressure of oxygen. Note that pO2 in the lungs is approximately 100 Torr and pO2 in resting
tissue is approximately 40 Torr.

a. CO has a higher binding affinity for hemoglobin

compared with oxygen, which shifts the equilibrium of
hemoglobin from T-state to the R-state. On the graph,
label the sigmoidal curve as A (normal O2 binding to
hemoglobin in the absence of CO) and B (O2 binding as
following CO binding to hemoglobin).

b. Explain the impact that CO binding has on hemoglobin affinity of O2 and the ability to deliver oxygen to
resting tissue.

c. Describe O2 binding to hemoglobin using the sequential model of binding.

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BCH2333B INTRODUCTION TO BIOCHEMISTRY MIDTERM 3 17TH MARCH, 2017

Question 7 (7 points). Ion exchange chromatography is commonly used method for the separation of
biomolecules.
a. What are the two types of ion exchange chromatography?

b. In what order would R, V, and E elute from a carboxymethyl column at pH 6. Note that carboxymethyl
is negatively charged at pH 6.0.

c. You are trying to purify a protein using ion exchange chromatography. Unfortunately, your protein
remains on the ion exchange column or it is eluting very slowly. What are the two changes you can
make to try and elute the protein more quickly from your carboxymethyl column?

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BCH2333B INTRODUCTION TO BIOCHEMISTRY MIDTERM 3 17TH MARCH, 2017

Question 8 (8 points). Enolase is a multifunctional protein that has been implicated in autoimmune diseases.
Enolases are involved in the glycolytic pathway and catalyze the dehydration of 2-phosphoglycerate (2-PG) to
form phosphoenolpyruvate using metal ions. Propose a two-step mechanism for the formation of
phosphoenolpyruvate. Be sure to show electrostatic stabilization using the metal ions.

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