Name: 11/11/2020

Student ID:
Instructor: Yunus Emre Türkmen

CHEM 461 Fundamentals of Biochemistry

2020 Fall Semester – Midterm Exam Solutions
(Overall 100 Points, Duration: 2 h)

1. (20 points) In a research article published at ACS Chemical Biology in 2013,

researchers investigated the effect of halogen bonding on aldose reductase
inhibition. Initial studies using compound 1 as the inhibitor molecule suggested the
presence of a halogen bond between the Br atom of 1 and the oxygen of Thr-113
(threonine at the 113th position) of aldose reductase. In order to make a systematic
investigation, the researchers synthesized seven analogues in total (1-7) whose
structures are shown below.

OH OH OH OH

O O O O F
Cl O Br Cl O Br Cl O F Br Cl O F Br
H H H H
N N N N
F
O O F O F O F

1 2 3 4

OH OH OH

O O O
Cl O I Cl O I Cl O F I
H H H
N N N

O O F O F

5 6 7

After the completion of the syntheses of compounds 1-7, their IC50 (half-maximal
inhibitory concentration) values were determined towards aldose reductase. An IC50
value is a measure of an inhibitor’s potency: a lower IC50 value corresponds to
higher potency, while a higher IC50 value means lower potency. Afterwards, X-ray
data obtained when the inhibitors are in the active site of the protein were analyzed.
The distances between the Br or I atom of the inhibitor molecules and the oxygen of
the Thr-113 residue were measured for each co-crystal, and the results are
summarized in the below table.

Compound 1 2 3 4 5 6 7

IC50 (µM) 0.71 0.40 1.19 0.30 1.90 0.19 1.36

X•••O
distance 2.96 2.90 2.91 2.84 3.03 2.95 2.93
(Å)*

* X•••O distance refers to the distance between the threonine oxygen and Br or I (X)
of compounds 1-7 in Angstrom (Å).

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Name: 11/11/2020
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Instructor: Yunus Emre Türkmen

a. Based on the X•••O distance data provided above, do you think compounds 1-7
make halogen bond to the oxygen of Thr-113 residue in aldose reductase? If no,
explain your reasoning. If yes, determine the compounds which make the weakest
and strongest halogen bonds, and provide an explanation.

Van der Waals radii (Å):

Br: 1.85 I: 1.98 O: 1.52

Solution: Sum of the Van der Waals radii for Br and O = 3.37 Å
Sum of the Van der Waals radii for I and O = 3.50 Å

Compound 1 2 3 4 5 6 7

X•••O
distance 2.96 2.90 2.91 2.84 3.03 2.95 2.93
(Å)a

Dd (Å)b 0.41 0.47 0.46 0.53 0.47 0.55 0.57

% Ddc 12.2 13.9 13.6 15.7 13.4 15.7 16.3

a X•••O distance refers to the distance between the threonine oxygen and Br or I (X) of
compounds 1-7 in Angstrom (Å).
b Dd (Å) is the difference between a X•••O distance and the sum of the Van der Waals radii

of X and O.
c % Dd is the ratio of the Dd for an X-O pair to the sum of the Van der Waals radii of X and O.

As can be seen from the above table, the X•••O distances for all compounds (1-7) are
smaller than the sum of their Van der Waals radii, and thus, all compounds make a halogen
bond with the oxygen of Thr-113 residue in aldose reductase.

For the comparison of the strengths of these halogen bonds, we need to consider the
Dd and % Dd values. Based on this analysis, compound 7 is found to make the strongest
halogen bond, while compound 1 makes the weakest halogen bond. In addition, the % Dd
values are observed to correlate well with the number of fluorine atoms in these
compounds.

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Name: 11/11/2020
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Instructor: Yunus Emre Türkmen

b. Do you think there is a correlation between the observed IC50 values and X•••O
distances? Take into account your answer in part (a), and make a detailed analysis.

Solution:

Compound 1 2 3 4 5 6 7

% Dd 12.2 13.9 13.6 15.7 13.4 15.7 16.3

IC50 (µM) 0.71 0.40 1.19 0.30 1.90 0.19 1.36

IC50 values do not exhibit a strong correlation with the X•••O distances. In addition,
the above table shows a comparison of the IC50 values and % Dd values for compounds
1-7. Compounds 3 and 6 appear to be the outliers in these data. This analysis suggests that
factors additional to halogen bonding might be important for the biological activity.

2. (20 points) A mixture of four dipeptides was analyzed by isoelectric focusing as

shown below. The pI values for the dipeptides are shown below each band.

Low pH High pH
(+) (-)

2.8 5.7 7.6 10.9

The list of the four dipeptides is shown below in random order. Determine which
band corresponds to which dipeptide. Explain briefly and/or show briefly your
calculations.

pKa values and the three-letter abbreviations of the 20 amino acids are shown at the
end of this booklet. Note that the left amino acids in these dipeptides represent the
N-terminals. You will also assume that peptide bond formation doesn’t affect the
pKa values of the individual acidic and basic groups.

His-Gly Leu-Asp Trp-Arg Tyr-Ala

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Name: 11/11/2020
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Instructor: Yunus Emre Türkmen

Solution: The pI values for the dipeptides should be calculated.

His-Gly:

O
H 3N
N CO2H pKa1 = 2.34 pKa2 = 6.00 pKa3 = 9.17
H
CH2
H3A2+ H2A+ HA A-
H N
NH pI = (6.00 + 9.17)/2 = 7.59

H3A2+

Leu-Asp:

OH
O C pKa1 = 1.88 pKa2 = 3.65 pKa3 = 9.60
O CH2
H 3N H3A+ H 2A HA- A2-
N CO2H
H
CH2 pI = (1.88 + 3.65)/2 = 2.77
CHCH3
CH3

H3A+

Trp-Arg:

NH2
H 2N C pKa1 = 2.17 pKa2 = 9.39 pKa3 = 12.48
NH
CH2 H3A2+ H2A+ HA A-
CH2
O CH2 pI = (9.39 + 12.48)/2 = 10.94
H 3N
N CO2H
H
CH2

HN

H3A2+

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Name: 11/11/2020
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Instructor: Yunus Emre Türkmen

Tyr-Ala:

O CH3
H 3N
N CO2H pKa1 = 2.34 pKa2 = 9.11 pKa3 = 10.07
H
CH2
H3A+ H 2A HA- A2-

pI = (2.34 + 9.11)/2 = 5.73

OH

H3A+

Low pH High pH
(+) (-)

2.8 5.7 7.6 10.9

Leu-Asp Tyr-Ala His-Gly Trp-Arg

3) (20 points) Design and provide the details of a synthetic plan for the solid-phase
synthesis of the tripeptide Ile-Lys-Ala. The N terminus of this tripeptide is Ile.

Please show all the steps of the synthesis, and the open molecular structures in
these steps explicitly.

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Name: 11/11/2020
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Instructor: Yunus Emre Türkmen

Solution:

Solid-Phase Synthesis of Ile-Lys-Ala:

O O
TFA
CH3 Base BocHN H 3N
+ Cl Bead O O Bead
Bead
BocHN CO2H CH3 CH3

BocHN CO2H

CH2
CH2 DCC
DMAP
CH2
CH2
NHFmoc
NHFmoc NHFmoc
O TFA O
H H
N N
H 3N O Bead BocHN O Bead
O CH3 O CH3

BocHN CO2H
DCC
H 3C DMAP
CH3

NHFmoc NHFmoc

O O O O
H H
TFA H 3N N
BocHN N N O
N O Bead H Bead
H H 3C O CH3
H 3C O CH3 CH3
CH3

piperidine

NH3
NH3

O O HF
H O O
H 3N N H
N OH H 3N N
H N O Bead
H 3C O CH3 H
CH3 H 3C O CH3
CH3
Ile-Lys-Ala

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Name: 11/11/2020
Student ID:
Instructor: Yunus Emre Türkmen

4) (20 points) a. Let’s assume that, as a researcher, you want to examine the folding
of an enzyme by repeating a classical experiment. For this purpose, you first treat
the native enzyme by 8M urea and excess b-mercaptoethanol. After a certain
amount of time, this protein showed no enzymatic activity.

Then, you removed urea and b-mercaptoethanol from the mixture, and waited for
some time. However, unexpectedly, the protein did not regain its enzymatic activity.
You wondered if there was an experimental error, and therefore, repeated the
experiment. However, you obtained the same result.

How would you explain the results of the first and second stages of this experiment?
Discuss in detail.

Answer:

In the first experiment, 8M urea solution is used to disrupt all non-covalent

interactions, while b-mercaptoethanol is used to break all disulfide bridges. These
lead to complete denaturation of the enzyme and therefore, no enzymatic activity is
observed with this denatured enzyme.

In the classical experiment of Anfinsen with bovine ribonuclease, the enzyme was
observed to regain its catalytic activity after the removal of urea and while b-
mercaptoethanol. However, in the second part of the above experiment, this was not
the case. Even after the removal of urea and while b-mercaptoethanol, you observed
that the enzymatic activity was not regained, which was surprising. This shows that,
this particular enzyme is incapable of attaining its native 3D structure on its own. In
other words, it cannot fold properly without the assistance of another molecule or
other conditions. Some proteins do indeed need other proteins called “chaperones”
to fold properly to their correct 3D geometry.

b. Describe in detail the function of repeating leucine (Leu) residues present in a-

keratin.

Answer:

In a-keratin, every seventh amino acid is a leucine (Leu), which leads to the
formation of “heptad repeats”. Leu has a non-polar, hydrophobic side chain. As a
result, the Leu residues present in the two a-helices of a-keratin have strong,
collective Van der Waals interactions that helps the two helices to be held together.

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Name: 11/11/2020
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Instructor: Yunus Emre Türkmen

5. (20 points) (a) You see below the graph that shows the effect of pH on the oxygen
affinity of hemoglobin. The x-axis of this graph is the partial pressure of O2 (1 kPa is
7.50 torr) and the y-axis is the fractional saturation (q).

(i) At a given pO2 value, at what pH does hemoglobin have the highest affinity
towards oxygen?

Answer: Hemoglobin has the highest affinity towards oxygen at pH = 7.6.

(ii) When hemoglobin carries oxygen from lungs to tissues, at what pH will the
oxygen release from the hemoglobin be highest?

Answer: The oxygen release from the hemoglobin will be highest at pH = 7.2.

(b) The level (concentration) of CO2 in tissues affects the oxygen release from
hemoglobin by two mechanisms. Explain these mechanisms in detail.

Answer:
1) The concentration of CO2 affects pH according to the following equilibria:

CO2 + H2O H2CO3 H+ + HCO3-

At lower pH, b1-His146 is protonated forming a salt bridge, which stabilizes the T
state.

2) At high concentrations of CO2, amines can form carbamates which can

participate in the formation of a salt bridge that stabilizes the T state of
hemoglobin.

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Instructor: Yunus Emre Türkmen

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Instructor: Yunus Emre Türkmen

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CHEM 461 Fundamentals of Biochemistry

Fall 2021 - Homework 1 Solutions
(100 points)

1. (20 points) Rank the following cation-p complexes with respect to their cation-p
interaction strengths in the gas phase (Strongest: 1, weakest: 4) and explain briefly.

Li+ K+ Li+ Na+

OCH3
O2N NO2 H3CO
OCH3
Solution: (Correct ranking: 10 points, explanation: 10 points)

Li+ K+ Li+ Na+

OCH3
O2N NO2 H3CO
OCH3
2 4 1 3

Regarding a cation-p interaction, as the p component gets more electron rich, the
interaction gets stronger. In this question, 1,3,5-trimethoxybenzene is the most
electron-rich benzene derivative, whereas 1,4-dinitrobenzene is the most electron
deficient. As for the cation component, Li+ makes the strongest interaction in the gas
phase while K+ makes the weakest among the three cations asked in the problem.

2. (40 points) For the tripeptide Lys-Met-Val, draw the structures of the major ions
or neutral molecules present in solution at the following pH values:

i. 1.24 ii. 2.32 iii. 9.74 iv. 12.45

Note that the far left amino acid in this tripeptide represents the N-terminal, and
that on the far right represents the C-terminal. You will also assume that peptide
bond formation does not affect the pKa values of the individual acidic and basic
groups.

Solution: (Each part is 10 points; 40 points in total)

Name: 10/10/2021
Student ID:
O O O pKa1 = 2.32 O O O
H H H H
H3N CHC N CHC N CHC OH H3N CHC N CHC N CHC O
CH2 CH2 CHCH3 CH2 CH2 CHCH3
CH2 CH2 CH3 CH2 CH2 CH3
CH2 S CH2 S
CH2 CH3 CH2 CH3
NH3 NH3
H 2A +
Fully protonated form of Lys-Met-Val
H3A2+ pKa2 = 8.95

O O O O O O
H H H H
H2N CHC N CHC N CHC O pKa3 = 10.53 H 2 N CHC N CHC N CHC O
CH2 CH2 CHCH3 CH2 CH2 CHCH3
CH2 CH2 CH3 CH2 CH2 CH3
CH2 S CH2 S
CH2 CH3 CH2 CH3
NH2 NH3
A- HA

i. pH = 1.24, major ion/molecule: H3A2+

ii. pH = 2.32, major ion/molecule: 1:1 mixture of H3A2+ and H2A+

iii. pH = 9.74, major ion/molecule: HA

iv. pH = 12.45, major ion/molecule: A-

3. (40 points) The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion

channel protein that responds to the neurotransmitter acetylcholine (structure
shown below). During the course of their studies on nicotinic acetylcholine
receptors, Dougherty and co-workers hypothesized that a Trp (tryptophan) residue
located in the active site of the nAChR might have a significant role in the binding of
acetylcholine to the receptor via participating in a cation-p interaction.

In order to test their hypothesis, Dougherty and co-workers applied site-directed

mutagenesis to prepare a number of proteins to which unnatural Trp derivatives
were incorporated in place of the natural Trp residue at the position a149. The
structures of the side chains of these unnatural Trp derivatives are shown below.
Name: 10/10/2021
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Side Chains of the Trp Derivatives Studied:

H2C H2C H2C H2C

Br F F

N N N N
H H H H
Me O Me F
N Trp 5-Br-Trp 5-F-Trp 5,7-F2-Trp
Me
Me O

acetylcholine
H2C F H2C H2C
F F H3C

F N F N N
H H H
F F
5,6,7-F3-Trp 4,5,6,7-F4-Trp 5-Me-Trp

Afterwards, the researchers determined the EC50 (half-maximal effective

concentration) values for both the natural and mutated proteins (receptors) against
acetylcholine. An EC50 value is a measure of a drug’s or ligand’s potency: a lower
EC50 value corresponds to higher effectiveness, while a higher EC50 value means
lower potency. In the following table, you see the EC50 values for the proteins
investigated in this study.

Side Chain EC50 (µM)

Trp 50
5-Br-Trp 88
5-F-Trp 200
5,7-F2-Trp 550
5,6,7-F3-Trp 1400
4,54,6,7-F4-Trp 2700
5-Me-Trp 49

Make a detailed analysis of the data shown above, and discuss whether they
support Dougherty’s hypothesis. In other words, do the above data support the
hypothesis that the Trp residue at position a149 participates in a cation-p
interaction or not?

Answer: (40 points in total for a detailed and correct analysis)

The indole group of the Trp residue at the a149 position nAChR was proposed to
have a cation-p interaction with the ammonium moiety of acetylcholine. According
to this hypothesis, one would expect a lower EC50 value for a stronger cation-p
interaction and a higher EC50 value for a weaker cation-p interaction. Bromine is a
mild electron-withdrawing group making the indole ring slightly more electron-
Name: 10/10/2021
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deficient compared to unsubstituted indole of Trp. From the above data, it is seen
that the EC50 value increased from 50 µM to 88 µM, which is in agreement with the
expectations. Fluorine, on the other hand, is a stronger electron-withdrawing group
than Br, and we see in the data that the number of fluorines has a clear effect on the
EC50 values. For the 4,54,6,7-F4-Trp mutant, the EC50 value was found to be as high
as 2700 µM. Finally, -Me group is a weakly electron-donating group, and the EC50 of
the 5-Me-Trp mutant was observed to be very slightly lower than that of the native
protein. This final result also supports the idea that the effects observed for the 5-
Br-Trp and 5-F-Trp mutants are not a consequence of the sizes of these groups.
Overall, all the data presented in the question support Dougherty’s hypothesis.
Name: 25/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

CHEM 461 Fundamentals of Biochemistry -

Fall 2021 - Homework 2 (100 points)

1. (40 points) Consider the following reactions, which represent enzyme catalysis in
the presence of a reversible competitive inhibitor. In addition to reacting with its
native substrate S, the enzyme can also interact with the inhibitor I to form the EI
complex, reversibly. K is the equilibrium constant for the formation of the EI
complex.

k1 k2
E+S ES E + P
k-1

k3
k3
E+I EI K=
k-3 k−3

Show the derivation of the below rate equation by applying the steady-state
approximation to the reactions given above. Assume that enzyme E and inhibitor E
reaches equilibrium in a fast manner to form the EI complex. [E]T represents the
initial total concentration of the enzyme.

d[P] k1k2 [E]T [S]

V= =
dt (1+ K[I ])(k−1 + k2 ) + k1[S]

Solution:

#[%]
!= = (! × [*+] (4 /012'3)
#'

Steady-State Approximation:

#[*+]
= 0 = (" × [* ] × [+] − (! × [*+] − (#" × [*+]
#'

⇒ (" × [* ] × [+] = [*+] × ((! + (#" ) (6 points)

[* ]$ = [* ] + [*+] + [*9 ] (4 points)

[*9]
:= ⇒ [*9] = : × [* ] × [9 ] (6 /012'3)
[*] × [9]
Name: 25/11/2021
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Instructor: Yunus Emre Türkmen

20 points for the following derivation:

(" × [*] × [+]

[* ]$ = [* ] + + : × [* ] × [9] ⇒ [* ]$
((! + (#" )
(" × [+]
= [* ] × (1 + + : × [9 ])
((! + (#" )

[*]$ × ((! + (#" )

[* ] =
((! + (#" ) + ((" × [+]) + ((! + (#" ) × : × [9]

(" × [*] × [+]

⇒ [*+] =
((! + (#" )
(" × [+] × [*]$ × ((! + (#" )
=
((! + (#" ) × {((! + (#" ) + ((" × [+]) + ((! + (#" ) × : × [9]}

(" × [+] × [*]$

=
{((! + (#" ) + ((" × [+]) + ((! + (#" ) × : × [9]}

#[%] (! × (" × [+] × [*]$

!= = (! × [*+] =
#' ((" × [+]) + (1 + :[9]) × ((! + (#" )

2. (40 points) Catalytic antibodies are antibodies (and thus proteins) that are able to
catalyze certain reactions, similar to enzymes. The research group of Donald Hilvert
at ETH Zürich in Switzerland designed a transition-state (TS) analog with the aim of
developing a catalytic antibody for the reaction shown below.

[Note: When an organism is submitted to a TS analog (as antigen), it produces

antibodies to this TS analog. Since the TS analog can bind efficiently to the active site
of the antibody, this antibody can now serve as a catalyst for the corresponding
reaction.]

According to this reaction, reactants A and B undergo a Diels-Alder [4+2]

cycloaddition reaction to give C initially. Compound C spontaneously eliminates SO2
gas and is converted to D. This SO2 elimination reaction is both kinetically and
thermodynamically very favored. Finally, compound D forms the final product E by
oxidation with air, spontaneously.
Name: 25/11/2021
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Instructor: Yunus Emre Türkmen

Cl O O O
Diels-Alder S Cl
Cl Cl
O Reaction
S + N Et O
O Cl Cl
Cl N
Cl O
O Et
A B C

-SO2

Cl O Cl O
H
Cl oxidation Cl
N Et N Et
Cl Cl
O H O
Cl Cl
E D

(a) Among the compounds shown below, which one would you select and use as a
transition-state analog for the above reaction? Explain your reasoning briefly.

O
Cl Cl Cl
Br
Cl Cl Me O Cl Cl
Br Cl
O O
S O N Et
O Cl Cl Cl Cl
Br N Cl N
Me O O
Br O Et Cl Et

1 2 3 4

Solution:

Compound 2 is the ideal transition-state analog for this reaction. (10 points) The
initial Diels-Alder reaction is the rate-determining step, and it is an endergonic
reaction. Therefore, according to the Hammond-Leffler postulate, the structure of
the transition-state resembles the structure of C. The structure of compound 2 is the
one closest to the structure of C. (10 points)

(b) Product inhibition can be defined as a type of enzyme inhibition where the
product of a reaction binds to the enzyme tightly and inhibits its activity. Would you
expect a product inhibition to be present in the above reaction catalyzed by the
catalytic antibody? Explain your reasoning.
Name: 25/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

Solution: No, I would not expect a product inhibition for this process. (10 points)
The structure of the final product E is flat and therefore, very different than the 3D-
shape of C and the transition-state analog. (10 points)

3. (20 points) Hemoglobin functions as an oxygen carrier protein whereas the main
function of myoglobin is oxygen storage in muscle cells. Discuss the structural
features of these two proteins, and explain in detail the reason and the mechanism
of their different functions.

Answer: Myoglobin does not exhibit cooperative behavior for oxygen binding, and it
has a very high affinity towards oxygen. Therefore, it releases oxygen only when the
partial pressure of oxygen is very low, and there is need for oxygen. (10 points) On
the other hand, hemoglobin exhibits cooperative behavior, and its oxygen binding
curve is S-shaped. This means that, it can have a high oxygen binding in lungs, and it
can also release a high amount of oxygen in tissues. This makes hemoglobin an
effective oxygen carrier protein. (10 points)
Name: 22/12/2021
Student ID:
Instructor: Yunus Emre Türkmen

CHEM 461 Fundamentals of Biochemistry

Fall 2021 - Homework 3 Solutions (100 points)

1. (20 points) Which of the following conformations of glucose (shown below) is

thermodynamically the most stable? Explain briefly.

HO HO
OH HO HOH2C
OH HO
O HO O
HO O OH
HO
OH
OH OH OH

A B C

Solution: Correct answer: 10 points; Explanation: 10 points

Conformation C is the most stable, because 1) chair conformation is more stable

than boat conformation; 2) all substituents are equatorial (in A they are all axial).

2. (40 points) Determine whether each of the following sugars is

i. Ketose or aldose
ii. triose, tetrose, pentose or hexose
iii. furanose or pyranose (if applicable)
iv. with an a or b configuration at the anomeric carbon.

Note: Please answer all i-iv for all four sugars (a-d).

OH
OH OH
a. H b. O
HO O HO
OH
OH OH H
OH OH

CHO
HO H HO OH
c. OH
d. O
H OH
H OH
CH2OH
CH2OH OH

Solution: Correct answers for each compound is 10 points; in total 40 points.

Name: 22/12/2021
Student ID:
Instructor: Yunus Emre Türkmen

a. Aldose; hexose; furanose; a

b. Aldose; hexose; pyranose; b
c. Aldose; pentose; not applicable; not applicable
d. Ketose; hexose; furanose; b

3. (40 points) A transfer RNA, whose anticodon is complementary to the UGU codon
in mRNA, is enzymatically conjugated to a 14C-labeled cysteine. The cysteine unit is
then chemically modified to alanine (with the use of Raney nickel, which removes
the sulfur atom of cysteine). The altered aminoacyl-tRNA is added to a protein
synthesizing system containing normal components except for this tRNA. The mRNA
added to this mixture contains the following sequence:

5’-UUUUGCCAUGUUUGUGCU-3’

What is the sequence of the corresponding radiolabeled peptide? Indicate the amino
acid residue in this peptide that is labeled with 14C.

Solution: Each amino acid in the peptide is 5 points (6x5 = 30 points); 10 points for
the radiolabeled amino acid.

Sequence of the peptide: Phe-Cys-His-Val-Ala-Ala

The radiolabeled amino acid is the alanine shown above in bold.

Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

CHEM 461 Fundamentals of Biochemistry

2021 Fall Semester – Midterm 1 Solutions
(Overall 100 Points, Duration: 2 h)

1. (20 points) A researcher purified a nonapeptide and wanted to determine its

amino acid sequence. First, they treated the nonapeptide with 6 M HCl at 110 °C for
one day and determined the amino acid composition of this nonapeptide in random
order as follows: (Arg, Trp2, Met, Leu, Ala2, Cys, Lys).

The C terminus of this nonapeptide was determined by using the enzyme

carboxypeptidase A, and found to be Leu.

In another experiment, the researcher treated the nonapeptide with cyanogen

bromide and isolated two peptide fragments. The amino acid compositions of these
fragments were found to be (Leu, Ala, Lys, Trp, Arg, Cys) and (Trp, Ala, Met).

When the nonapeptide was reacted with o-iodosobenzoate, three peptide fragments
were isolated with the following amino acid compositions: (Arg, Trp, Ala, Met),
(Trp, Ala) and (Leu, Lys, Cys).

In a final experiment, the nonapeptide was treated with the enzyme trypsin, and
three shorter fragments were isolated. The amino acid compositions of these
shorter fragments were determined as (Cys, Leu), (Ala2, Met, Arg, Trp) and (Lys,
Trp).

Based on the results of these experiments, determine the amino acid sequence of
this nonapeptide using the information given in the table at the end of this booklet.

Solution:

From N-terminal to C-terminal: Ala-Trp-Met-Ala-Arg-Trp-Lys-Cys-Leu

1
Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

2. (20 points) a) The unfolding of the a helix of a polypeptide to a randomly coiled

conformation is accompanied by a large decrease in a property called specific
rotation, which is a measure of a solution’s capacity to rotate circularly polarized
light. Polyglutamate, a polypeptide made up of only L-Glu residues, has the a-helical
conformation at pH 3. When the pH is increased to 7, there is a large decrease in the
specific rotation of the solution. Similarly, polylysine (L-Lys residues) is an a helix at
pH 10, but when the pH is lowered to 7, the specific rotation also decreases, as can
be seen in the graph shown below.

Provide an explanation for the effect of the pH changes on the conformations of

poly(Glu) and poly(Lys).

Answer: 10 points for part (a).

Glutamate has -CH2CH2CO2- group in its side chain, which is present in its
protonated -CO2H state at low pH values. Since it is uncharged in this state, poly(Glu)
can adopt a helical conformation (a helix). However, at high pH, the side chain is in
the deprotonated -CO2- state. As such, poly(Glu) is highly negatively charged at high
pH values. Such negatively charged groups can act as hydrogen bond acceptors and
break the a helix resulting in a random conformation.

A similar situation is present in the case of poly(Lys). The side chain of lysine has
the -CH2CH2CH2CH2NH3+ group which is in its protonated form at low pH values.
Therefore, this highly positively charged polypeptide (poly(Lys)) adopts a random
conformation. On the other hand, at high pH values, the amine group of lysine is in
its deprotonated form (-NH2) which makes it neutral. As such, poly(Lys) is now
stable in helical conformation forming an a helix.

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Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

b) The molecular mass of a protein purified by gel-filtration chromatography was

determined to be 60 kDa (kiloDaltons). Chromatography in the presence of 6 M urea
gave a 30-kDa species. When the chromatography of the same protein was repeated
in the presence of 6 M urea and 10 mM b-mercaptoethanol, a single molecular
species of 15 kDa was obtained.

Describe the structure of this protein. What are the functions of urea and b-
mercaptoethanol in these experiments?

Answer: 10 points for part (b).

Urea disrupts all non-covalent interactions leading to the denaturation of the

protein. b-Mercaptoethanol breaks disulfide bonds by reducing them to -SH groups.

6 M urea
S S 2 S
S S S 30 kDa

60 kDa
SH
6 M urea
4 15 kDa
10 mM 𝛽-mercaptoethanol

3) (20 points) In an imaginary drug-discovery program, a researcher identified

compound 1 as a potential candidate to bind to and inhibit enzyme A, with an IC50
value of 8.6 µM.

In order to understand the molecular origin of this binding, the researcher co-
crystallized enzyme A with compound 1 and obtained the 3D structure of this
enzyme-1 complex using X-Ray crystallography. In this structure, there is a serine
residue whose side chain oxygen is in close proximity with the –Br (bromine) of
compound 1, and there is a tryptophan residue whose indole ring is on top of the
aromatic (benzene) ring of 1. Therefore, the researcher suspected halogen bonding
and aromatic p-p interaction as two potential candidates responsible for the
biological activity of compound 1.

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Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

O O IC50 (µM)
S
N CH3 1: X = Br 8.6
O N
2: X = I 33

3: X = Cl 2.8

X CF3 4: X = F 0.23

In order to determine which non-covalent interaction is the main interaction

responsible for this bioactivity, the researcher synthesized compounds 2-4 and
determined the corresponding IC50 values, which are shown above.

In another experiment, the researcher made a single amino acid mutation on

enzyme A and mutated the tryptophan residue to phenylalanine. The IC50 value of
compound A using this mutated enzyme was found to be >50 µM.

Finally, in the crystal structure of enzyme A-compound 1 complex, the distance

between the serine side chain oxygen and bromine of 1 was found to be 3.66 Å. The
Van der Waals radii of bromine and oxygen are 1.90 and 1.52 Å, respectively.

Based on these observations and experiments, which non-covalent interaction,

halogen bonding or aromatic p-p interaction, do you think is responsible for the
binding of compound 1 to enzyme A? Explain your reasoning in detail.

Note: IC50 refers to the half maximal inhibitory concentration.

Solution: 20 points in total.

i) If there were a halogen bond between the serine oxygen of enzyme A and
the drug candidate, then the IC50 would be expected to decrease when the
halogen is changed from F®Cl®Br®I. However, the data show an
opposite trend. Therefore, the IC50 values do not support halogen
bonding. On the other hand, with the increasing electronegativity of X
(I®Br®Cl®F) the electron density on the benzene ring decreases, which
is expected to lead to an increase in the strength of a potential aromatic p-
p interaction with the tryptophan residue. As such, the IC50 values
support an aromatic p-p interaction.

ii) The aromatic ring of phenylalanine (benzene) is less electron rich than
the aromatic ring of tryptophan (indole). Therefore, the p-p interaction
would be weaker in the case of phenylalanine compared to tryptophan.
This is in accordance with the observed IC50 value (>50 µM).

4
Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

iii) The sum of the Van der Waals radii of bromine and oxygen is 3.42 Å,
which is smaller than the actual distance between Br of compound 1 and
serine oxygen of enzyme A. This finding does not support the presence of
a halogen bonding.

4) (20 points) Design and provide the details of a synthetic plan for the solid-phase
synthesis of the tripeptide Ala-Tyr-Phe. The N terminus of this tripeptide is Ala.
Please show all the steps of the synthesis explicitly.

One of the protecting groups used for phenols is the t-Bu (tertiary butyl) group. tBu-
protected phenols can be deprotected under acidic conditions. TFA (trifluroacetic
acid) is a commonly used acid for this purpose.

Protection
OH with t-Bu group Ot-Bu TFA OH

R R Deprotection R

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Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

Solution: 20 points in total.

O O
Ph piperidine
Base Fmoc-HN H 2N
+ Cl Bead O O Bead
Bead
Fmoc-HN CO2H Ph
Ph
N-Fmoc-Phe

Fmoc-HN CO2H

DCC
DMAP
t-BuO
t-BuO

O t-BuO
H
N piperidine
H 2N O Bead
O O
H
Ph N
Fmoc-HN O Bead
O
Fmoc-HN CO2H Ph
DCC
CH3 DMAP

HO
t-BuO

O O
O O H
H TFA Fmoc-HN N
Fmoc-HN N N O Bead
N O Bead H
H CH3 O
CH3 O Ph
Ph

piperidine

HO HO

O O O
H HF H
+H
3N N CO2- H 2N N
N N O Bead
H H
CH3 O CH3 O
Ph Ph

Ala-Tyr-Phe

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Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

5. (20 points) The structure of Asperversiamide A, a natural product with a cyclic

oligopeptide structure, is shown below. This natural product was first isolated in
2019 from a coral-derived fungus Aspergillus versicolor, which was collected from
the South China Sea. Asperversiamide A was found to have a potent inhibitory
activity against infectious Mycobacterium marinum.

a) Circle and identify (name) all amino acid residues present in Asperversiamide A.
Determine whether each amino acid residue has a (D) or (L) configuration
stereochemically. Stereochemistries of (D) and (L) amino acids with a general
structure are shown below.

b) Please show all peptide bonds with an arrow in the below structure.

H HO O
N
O CH3
N
NH H
O
NH HN
O
NH HN
HO H O
N
O
O

Asperversiamide A

R H H R
+H
3N CO2- +H
3N CO2-

L-amino acid D-amino acid

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Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

Solution:

a) 14 points in total.

D-Trp L-Ser
D-Ala
H HO O
N
O CH3
N
NH H
OH
NH HN
O
NH HN D-Val
HO H O
N
D-Ser O
OH

D-Val L-Phe

Asperversiamide A

b) 6 points in total.

Peptide bonds (shown by x):

H HO O
N
O CH3
x N
NH H
x x O
NH HN
O x
x
NH HN
HO Hx O
x
N
O
O

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Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

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Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

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Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

11
CHEM 461 – Fundamentals of
Biochemistry
Sample Midterm Exam
Solu?ons

Instructor: Yunus Emre Türkmen

Bilkent University
Department of Chemistry
 Solu?on:

• Tyrosine: phenol, HBD and weak HBA • Asparagine and glutamine:

• Tryptophan: indole, HBD amide, HBA and HBD
• Methionine: thioether, weak HBA • Serine and threonine:
• Lysine: ammonium, protonated form: HBD alcohol, HBA and HBD
• Arginine: guanidinium, protonated form: HBD • Cysteine: thiol, weak HBA and HBD
• His?dine: imidazole, HBA and HBD • Aspartate and glutamate:
carboxylate, deprotonated form: HBA
Solu?on: Phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp) and hisCdine (His) are
capable of parCcipaCng in π-π interacCons.
Strength of π-π interacCons with the electron-deficient hexafluorobenzene:

Trp > Tyr > Phe > His

--------------------------------------------------------------------------------------------------------------------

Solu?on: Lysine (Lys) and arginine (Arg) can parCcipate as caCon components
in caCon-π interacCons. HisCdine is parCally protonated at physiological pH (pH =
7.4), and can parCcipate to some extent in caCon-π interacCons.
Solu?on: Proline is the only amino acid having a secondary amine group in its
structure, and therefore, it is the ideal candidate for use as an organocatalyst in the
above transformaCon.
Solu?on: Glycine is the smallest amino acid, and due to this reason, it is present
Repeatedly in the interior region of collagen.
 Solu?on: CO2H pK1 = 2.19 CO2- pK2 = 4.25 CO2- pK3 = 9.67 CO2-
+H N +H N C H H2N C H
+H N C H
3 3 C H 3

CH2 CH2 CH2 CH2

CH2 CH2 CH2 CH2
CO2H CO2H CO2- CO2-

H3A+ H2A HA- A2-

fully protonated form

of glutamate pI = (2.19+4.25)/2 = 3.22
--------------------------------------------------------------------------------------------------------------------
Solu?on:
CO2H pK1 = 1.96 CO2- pK2 = 8.18 CO2- pK3 = 10.28 CO2-
+H N +H N C H H2N C H
+H N C H
3 3 C H 3

CH2 CH2 CH2 CH2

SH SH S- S-

H3A+ H2A HA- A2-

fully protonated form

of cysteine pI = (1.96+8.18)/2 = 5.07
 Solu?on:
pK1 = 1.82 pK2 = 6.00 pK2 = 9.04 pK2 = 12.48
H4A3+ H3A2+ H2A+ HA A-

H O H O H pI = (9.04+12.48)/2 = 10.76
H H
+H N C C N C C N C CO2H
3
CH2 CH2 H2C
CH2 CH NH
H C CH3 fully protonated form
CH2 3
N of Arg-Leu-His
NH
+H N C
H H4A3+
2
NH2

--------------------------------------------------------------------------------------------------------------------
Solu?on:
pK1 = 2.02 pK2 = 3.65 pK2 = 9.60 pK2 = 10.53
H4A2+ H3A+ H2A HA- A2-

H O H O H pI = (3.65+9.60)/2 = 6.63
H H
+H N C C N C C N C CO H
3 2
CH2 CH2 CH2
CO2H CH2
O NH2 fully protonated form
CH2
of Asp-Lys-Asn
CH2
H4A2+
NH3+
 Solu?on:
Interior region of the ion channel: The hydrophilic region of
AmB – the region enclosed by the green rectangle – will face
the interior region of the ion channel to create a polar,
hydrophilic environment. The interacCons of the polar
funcConal groups with the ions can be described as ion-dipole
and ion-ion (ion pair) interacCons. More specifically, alcohol
groups can coordinate to Na+ and K+ caCons, and can make H-
bonds to Cl- anions. Ester group can coordinate to Na+ and K+
caCons. The carboxylate (CO2-) group will have an ion-pair with
Na+ and K+, while the ammonium (NH3+) will have an ion-pair
with Cl-.

Exterior region of the ion channel: The hydrophobic polyene

region of AmB – the region enclosed by the red circle – will
face the exterior of the ion channel so that it will have a
favorable interacCon with the hydrophobic lipid bilayer via
Van der Waals interac?ons.
Solu?on: Interac?on strengths: C > D > B > E > A
 1

O 1: Proline
2 N 2: Alanine
H3C O 3: N-Methylalanine
O O
O NH O HO
O N O
H
N HN
H3C N N
H O
CH3 O
3
Enopeptin B

O 6
N 5 4: Proline
5: Serine
H3C O 6: Phenylalanine
O OH
O N O HO
O N O
H
N HN
non-peptide bond between proline H3C N N
carbonyl and serine -OH. H O
CH3 OH

P5
4
P1 Enopeptin B
O
N
P2 O an amide bond, but not
H3C between two amino acids
O O
O NH O HO
O N O
H
N HN
H3C N N
H O
CH3 O

P3 P4