THE UNIVERSITY OF EDINBURGH

CHEMISTRY FOR LIFE SCIENCES 2 (SCBI08003)



Exam Date: Wednesday 18th December 2019 Start Time: 9.30 End Time: 11.30


Please read full instructions before commencing writing



Please answer FOUR questions.

PLEASE ANSWER EACH QUESTION IN A SEPARATE BOOK.



[The bracketed numbers shown against part of a question are only a guide to the likely
allocation of marks in that question.]

Please enter your student examination number on each answer book.

The periodic table and a data sheet are attached to this examination paper.

Unassembled molecular model kits and non-programmable calculators may be used in this
examination.



EXAMINERS

Chairman of the Board of Examiners: Professor S Parsons
External Examiners: Professors M Greaney, M Whittlesey and N Hunt
Senior Internal Examiners: Professors G C Lloyd-Jones, J P Attfield and C A Morrison

This examination will be marked anonymously.







1. Answer all parts.

(a) Metal-containing catalysts are important in both industrial and biological
applications. Summarise the different variables that may be altered to optimise
catalyst function in an industrial setting, and the limitations to these that apply to
enzymes. Explain how these limitations have been overcome by enzymes in living
systems. [4]

(b) Reactive oxygen species such as superoxide and peroxides can be damaging to
biological molecules.

(i) Describe how metalloenzymes protect biological systems from damage by
peroxides. Give any appropriate reactions, and include in your answer
consideration of substrate specificity. [5]

(ii) Give the two reactions that comprise the catalytic cycle of superoxide
dismutase. [2]

(c) Heme is an iron-porphyrin complex and a key component of hemoglobin. One of
the consequences of anemia (iron deficiency) is an increase in levels of zinc-
porphyrin in the blood. With reference to the role of iron in the co-operative binding
and transport of O2, explain why zinc is not able to substitute for iron in hemoglobin
function. [4]

(d) A researcher is attempting to design three small iron-dependent proteins as electron
transfer mediators. The aim is to create proteins with reduction potentials ranging
from approximately 300 mV to +450 mV. For each of the three proteins suggest
which amino-acid side chains could be employed as R1 and R2 in A in order to
‘tune’ the potential of the iron ion. Explain your reasoning. [5]












A













2. Answer all parts.
(a) The enzyme alanine transaminase (ALT) catalyses the reversible interconversion of
alanine A and -ketoglutarate B to pyruvate C and glutamate D. The enzyme is
dependent on the cofactor pyridoxal 5’-phosphate (PLP).



The kinetics of the ALT enzyme from rat liver have been studied at 37 ⁰C and
pH 7.0 under biological conditions using a coupled assay. The concentrations of
each substrate in each individual reaction and the observed initial velocity of
reaction, v0, are shown in the table below.



(i) Use the Michaelis-Menten model and an appropriate graphical method to
determine the apparent vmax and Km for alanine. The Michaelis-Menten
equation is given below: [4]

0
0max
][
][
SK
Sv
v
m 


(ii) Define the term turnover number, kcat. Determine kcat for the ALT enzyme.
[3]

(iii) Explain what is meant by the term coupled assay. Suggest a possible coupled
assay system for the ALT-catalysed reaction. [2]

(b) ALT catalysis proceeds via a ping-pong mechanism and the first step involves
alanine binding and transfer of an amino group to the enzyme active site to produce
pyruvate.

(i) Describe what is meant by the term ping-pong mechanism. Thus, draw a
Cleland diagram for the reaction mechanism of ALT. [4]

(ii) Suggest a role for the PLP cofactor in the ALT catalytic mechanism. [1]




Question 2 continued on next page.

[Alanine]0 / mM 1 2 4 8 16 32
[-ketoglutarate]0 / mM 15 15 15 15 15 15
[ALT]0 / nM 2 2 2 2 2 2
v0 / µMmin
−1 2.5 4.4 7.1 10.1 13.0 15.1



Question 2 continued.

(c) Thermal stability studies have been performed to monitor the denaturation of the
ALT enzyme at different temperatures. The thermodynamic data obtained is shown
below.






(i) Calculate the Gibbs free energy change (G) for denaturation of ALT at 35⁰C
and 55⁰C. [3]

(ii) Determine the percentage of the ALT molecules that are denatured at 55 ⁰C.
[2]

(iii) After removing the PLP cofactor from ALT, G for the denaturation of the
enzyme was found to be −1.9 kJ mol−1 at 45 ⁰C. What does this suggest about
the possible role of PLP in the ALT enzyme? [1]


























T / ⁰C G / kJmol−1 H / kJmol−1 S / Jmol−1K−1
35 ? 75 233
45 0 125 393
55 ? 173 533


3. Answer all parts.

(a) ATP-coupled reactions are used in the biosynthesis of malonyl-CoA and acetyl-
CoA.

(i) Explain the role of the ATP and why it is needed in these reactions. [3]

(ii) Many reactions that use ATP also use magnesium ions. Give an explanation
for this observation. [2]

(b) The biosynthesis of fatty acids is carried out by Fatty Acid Synthase (FAS) and is
an iterative process combining a number of steps as shown below.



(i) Step 1 is an example of a Claisen condensation reaction. Draw the structure
of the product C and a curly arrows mechanism for its formation. [4]

(ii) Steps 2 and 4 are both reductions. State the biological reducing agent used by
these enzymes. [1]

(iii) Malonyl/acetyl transferase (MAT) catalyses the reaction of malonyl-CoA
with holo-ACP to give malonyl-ACP (B). Shown below is the active site of
MAT containing malonyl-CoA.



Draw the curly arrow mechanism for the reaction of Ser92 with malonyl-CoA,
and explain the roles of the active site residues in this reaction. [5]


Question 3 continued on next page.


Question 3 continued.

(b) (iv) Thiolactomycin G acts as a reversible inhibitor of a ketosynthase (KS)
subunit from a Type II FAS. It was found that G is competitive with malonyl-
ACP (B) binding. Using the structures of G and the active site of the KS
shown below, explain this observation. [2]



(c) Polyketide synthases (PKS) can use a wide range of acyl-CoA starter and extender
units to make a range of products. The pikromycin PKSs (Pik1 and Pik2) catalyse
formation of products K and L from a synthetic pentaketide chain mimic (J) and
two different extender units (H and I). The PKS subunits are: ketosynthase (KS),
acyltransferase (AT), ketoreductase (KR), acyl carrier protein (ACP) and
thioesterase (TE).




Specific carbon atoms within H and I have been labelled as a and b respectively.
Redraw products K and L and clearly indicate the position of the a and b carbon
atoms in both products. [3]













4. Answer all parts.

(a) The side chain of the amino acid lysine has been shown to be post-translationally
modified (PTM) by (1) biotin, (2) lipoic acid and (3) S-adenosyl methionine
(AdoMet or SAM) as shown below.



(i) Draw the chemical structures of each of the PTM forms of the lysine side-
chain for (1) biotinyl-lysine (2) lipoyl-lysine and (3) methyl-lysine. [6]

(ii) Describe how you could use matrix-assisted laser desorption/ionisation mass
spectrometry (MALDI-MS) to monitor the conversion of the unmodified
lysine residue to the methyl-lysine form. Describe the experimental set up and
what measurements you would make. [4]

(b) Bacterial L-threonine aldolase (TA) is a pyridoxal 5’-phosphate (PLP) dependent
enzyme. Shown below is the PLP:L-Thr external aldimine intermediate formed
between PLP and L-Thr in the active site of TA. [Pi = phosphate]. The TA enzyme
catalyses the conversion of the PLP:L-Thr external aldimine intermediate into a
quinonoid intermediate and acetaldehyde.





Question 4 continued on next page.





Question 4 continued.

(b) (i) Using curly arrows, suggest a chemical mechanism that describes the
conversion of the PLP:L-Thr external aldimine into the quinonoid
intermediate. [3]

(ii) What type of TA active site residue is required to catalyse the conversion of
the PLP:L-Thr external aldimine to the quinonoid intermediate? [1]

(iii) Suggest a method for the detection of the acetaldehyde product. [2]

(c) The purified TA enzyme was studied using UV-Visible spectroscopy and displayed
a spectrum with absorbance maxima at 330 and 425 nm (solid black line) as shown
below.



(i) Upon incubation of TA with D-Thr, a new peak with a maximal absorbance
at 427 nm was observed (marked with an arrow). Suggest what species would
give rise to this absorbance. [2]

(ii) In a separate experiment, upon incubation of TA with L-Thr, a new peak was
observed with a maximal absorbance of 490 nm (marked with an arrow).
Suggest what species would give rise to this absorbance. [2]
















5. Answer all parts.

(a) Shown below is the 1H NMR spectrum of a 160-amino acid residue protein
collected in aqueous buffer at pH 6.5.

(i) Suggest a suitable label and units for the x-axis. [2]

(ii) State which chemical group is responsible for the peaks in region (A). [2]

(iii) Name three amino acid residues likely to give signals in region (B). [2]

(iv) State the origin of the peak in region (C). [2]

(v) The peaks in region (D) are evidence that this protein is folded. Give reasons
to support this conclusion. [2]








Question 5 continued on next page.


5 6 7 8 9 10 4 3 2 1 0
Label ? (Units?)
(A)
(B)
(C)
(D)


Question 5 continued.

(b) Describe or show using a diagram the meanings of the symbols λ, d and θ which
appear in Bragg’s law ( 2 sind  ). [3]

(c) The 008 reflection of an orthorhombic crystal occurs at θ = 4.45° in a diffraction
pattern measured with X-rays with λ = 1.300 Å. What is the value in Å of the c unit
cell length? [2]

(d) Diffraction data were collected on a crystalline protein to a resolution of 2.1 Å using
radiation with  = 1.300 Å.

(i) What was the maximum value of θ for which data were collected in this
experiment? [1]

(ii) Would these data be suitable for investigating the orientations of the amino
acid side chains in the region of the active site of the protein? Justify your
answer. [2]

(iii) Explain why most protein crystals diffract X-rays weakly at atomic
resolution. [2]

























University of Edinburgh
School of Chemistry

Chemical and Physical Constants
Gas constant R = 8.314 J K–1 mol–1
= 0.08206 litre atm K–1 mol–1
Faraday constant F = 96485 C mol–1
Avogadro constant NA = 6.022 × 10
23 mol–1
Mass of a hydrogen atom (1
1
H) mH = 1.674 × 10
–27 kg
Atomic mass unit mu = 1.661 × 10
–27 kg
Rest mass of an electron me = 9.109 × 10
–31 kg
Charge on an electron e = F / NA = 1.602 × 10
–19 C
Boltzmann constant kB = R / NA = 1.381 × 10
–23 J K–1
Planck constant h = 6.626 × 10–34 J s
 = 1.055 × 10–34 J s
Speed of light in a vacuum c = 2.998 × 108 m s–1
Permittivity of free space 0 = 8.854 × 10–12 C2 J–1 m–1
Standard acceleration due to gravity g = 9.807 m s–2
Molar volume of ideal gas
at P = 1 atm and T = 298.15 K Vm = 24.47 litres


Conversion factors
1 thermochemical calorie (cal) = 4.184 J
1 standard atmosphere (atm) = 1.013 × 105 N m–2
1 bar = 105 N m–2
1 torr (= 1 mm Hg) = 1/760 atm = 133.3 N m–2
1 electron volt (eV) = 1.602 × 10–19 J  96485 J mol–1  8.066 × 105 m–1  8066 cm–1
1 Ångström unit (Å) = 10–10 m

Abbreviations
Cp = cyclopentadienyl
Ph = Me = CH3
Et = CH2CH3
i-Pr = CH(CH3)2
t-Bu = C(CH3)3
Ac =
C
O
CH3


Periodic Table of the Elements

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
1
H
1.008
2
He
4.003
3
Li
6.941
4
Be
9.012
5
B
10.811
6
C
12.011
7
N
14.007
8
O
15.999
9
F
18.998
10
Ne
20.180
11
Na
22.99
12
Mg
24.31
13
Al
26.98
14
Si
28.09
15
P
30.97
16
S
32.07
17
Cl
35.45
18
Ar
39.95
19
K
39.10
20
Ca
40.08
21
Sc
44.96
22
Ti
47.88
23
V
50.94
24
Cr
52.00
25
Mn
54.93
26
Fe
55.85
27
Co
58.93
28
Ni
58.69
29
Cu
63.55
30
Zn
65.39
31
Ga
69.72
32
Ge
72.61
33
As
74.92
34
Se
78.96
35
Br
79.90
36
Kr
83.80
37
Rb
85.47
38
Sr
87.62
39
Y
88.91
40
Zr
91.22
41
Nb
92.91
42
Mo
95.94
43
Tc
(98)
44
Ru
101.07
45
Rh
102.91
46
Pd
106.42
47
Ag
107.87
48
Cd
112.41
49
In
114.82
50
Sn
118.71
51
Sb
121.75
52
Te
127.60
53
I
126.90
54
Xe
131.29
55
Cs
132.91
56
Ba
137.33
57
La
138.91
72
Hf
178.49
73
Ta
180.95
74
W
183.85
75
Re
186.21
76
Os
190.23
77
Ir
192.22
78
Pt
195.08
79
Au
196.97
80
Hg
200.59
81
Tl
204.38
82
Pb
207.2
83
Bi
208.98
84
Po
(209)
85
At
(210)
86
Rn
(222)
87
Fr
(223)
88
Ra
226.03
89
Ac
227.03


58
Ce
140.12
59
Pr
140.91
60
Nd
144.24
61
Pm
(145)
62
Sm
150.36
63
Eu
151.97
64
Gd
157.25
65
Tb
158.93
66
Dy
162.50
67
Ho
164.93
68
Er
167.26
69
Tm
168.93
70
Yb
173.04
71
Lu
174.97

90
Th
232.04
91
Pa
231.04
92
U
238.03
93
Np
237.05
94
Pu
(244)
95
Am
(243)
96
Cm
(247)
97
Bk
(247)
98
Cf
(251)
99
Es
(252)
100
Fm
(257)
101
Md
(258)
102
No
(259)
103
Lr
(260)


(For radioactive elements that do not occur in nature, the mass number of the most stable isotope is given in parentheses)


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