Download King Saud University College of Science Biochemistry Dept. Enzyme Mechanism

1
King Saud University
College of Science
Biochemistry Dept.
Enzyme Mechanism
Sample Exam and Answer Key
Q#
1
2
3
4
5
6
Total
Q (pts)
10
10
12
24
24
20
100
Y (pts)
Serial #:.......................Name: .....................................................I. D. #.......................................
1.
(10 pts). A pH-dependent enzyme activity curve is shown in the figure below.
Which of the following pairs of amino acids would be likely candidates as catalytic
functional groups?
a. Glu and Lys
b. Asp and His
c. His and Cys
d. His and Lys
e. His and Arg
6
Vmax
4
2
0
0
2
4
6
8
10
pH
2.
1.
(10 pts). For each of the following multiple choice questions choose the best ONE
Enzymes increase reaction rates
a.
by lowering the energy of the transition state
b.
only if reactions are irreversible
c.
by decreasing the entropy of the enzyme and substrate
d.
by decreasing the standard free energy of activation ()GE^)
2
2.
Enzymes lower energies of transition states
a.
by lowering )GE‡
b.
by increasing )HE‡
c.
by lowering )SE‡
d.
after and only after having bound the substrate
3.
Vmax is
a.
the initial rate of an enzyme-catalyzed reaction
b.
a constant expressing substrate preferences of an enzyme
c.
the rate of an enzyme-catalyzed reaction when substrate concentrations
saturate the active site of the enzyme
d.
the number of substrate molecules converted to product per second
4.
Km is
a.
the dissociation constant of the enzyme-substrate complex in all cases
b.
the substrate concentration at which enzyme is working at half of its maximal
rate
c.
a constant expressing substrate preference of an enzyme
d.
all of the above
5.
In random sequential mechanism
a.
a substrate must bind and be released as product before the second substrate
molecule can bind
b.
each substrate molecule can bind to the enzyme independently
c.
one substrate molecule must bind to the enzyme (but not released) before the
binding of the second
Answer the following questions and support your answer with DIAGRAMS if necessary
3.(12 pts). What an enzyme does?
Answer:
1. Enzyme speeds up the reaction by reducing the activation energy (∆G‡) by allowing
the binding of the transition state into the active site; however, the binding of the
transition state needs to be tighter than the binding of either the substrates or the
products because of the perfect transition state-active site complementarity
2. Enzyme can store energy (binding energy) from the binding of the substrates and use
it later to make catalysis more efficient
4.(24 pts). How can you distinguish between specific and general acid catalysis?
Answer:
Specific acid catalysis:
1. The catalyst speeds up the reaction using H3O+ (hydronium ion) from water is part of
the reaction mechanism
2. The specific acid reaction rate is dependent on pH only, and not on the buffer
concentration
3. The specific acid catalyzed reaction involves the formation of the unstable oxonium
ion intermediate, which then eliminates water from the transition state and it will be
destabilized and slow the reaction as shown in the figure below
3
Specific Acid Catalysis
Electronstatic
interaction
‡
O
O
R1
R
R1
N
R
OH
H
+
N
OH
H
H2O
H
O
Transition state
H
H
H
Hydronium ion
Specific acid
141
General acid catalysis:
1. The enzyme speeds up the reaction using several functional groups from amino acid
residue side chains that can donate protons as acids (e.g., Asp, Glu) is part of the
reaction mechanism
2. The rate of enzymatic reaction is dependent on the buffer concentration, as well as the
appropriate protonation state
3. In enzymatic reaction, the general acid reaction does not form highly unstable species
because the transition state is stabilized by hydrogen bond and the stabilized transition
state will now break down fast to the desired products as shown in the figure below
General Acid Catalysis
General acid
Hydrogen
bond
Eletrostatic
interaction
Enzyme-transition
state intermediate
146
4
5.
(24 pts). Define the following terms:
(a) Effective molarity
(d) Strain or distortion catalysis
Answer:
(a)Effective molarity:
1. It is the ratio of the first order rate constant of intramolecular reaction to the second
order rate constant of intermolecular reaction
2. The intermolecular reaction depends on both the concentration of substrates,
therefore, it proceeds with a second order rate constant (kinter) whereas the
intramolecular reaction depends only on one of substrate concentration, it proceeds
with a first order rate constant (kintra)
3. Dividing kintra/kinter gives the relative rate enhancement of the intramolecular over the
intermolecular catalysis
4. With units of molarity, this ratio can be interpreted as the relative effective
concentration of the intramolecular nucleophile
(b)Strain or distortion catalysis:
1. When substrate bind to the enzyme, it may induces a conformational change in the
active site to fit to a transition state
2. Frequently, in the transition state, the substrate and the enzyme have slightly different
structure (strain or distortion) and increase the reactivity of the substrate
5
6. (10 pts) Complete proposed mechanism of lysozyme?
O
A s p -5 2
S ite D
G lu -3 5
O
O
C H 2O H
O
C H 2O H
O
H
H
OR
H
DH
O
O
H
HO
N HC O C H3
H
OH
H
EH
O
O
A s p -5 2
G lu -3 5
O
O
O
H
S ite E
O
C H 2O H
C H 2O H
O
H
H
H
OR
H
O
N HC O C H3
H
H
O
H
OH
H
H
H
OR
O
H
N HC O C H3
C H 2O H
O
H
H
H
N HC O C H3
O
H
H
OH
N HC O C H3
H 218O
O
A s p -5 2
G lu -3 5
C H 2O H
O
H
H
OR
O
O
O
H
H
O
H
18
N HC O C H3
H
18
O
H
OH
C H 2O H
O
H
O
H
OR
H
A s p -5 2
H
H
N HC O C H3
O
G lu -3 5
O
O
HO
6
Answer:
Eletrostatic interaction
General acid
O
Asp-52
Glu-35
Site D
O
O
CH2OH
O
H
O
CH2OH
H
OR
H
DH
O
O
H
HO
NHCOCH3
H
OH
H
EH
O
O
H
O
Asp-52
Glu-35
O
O
Nucleophilic attack
Site E
O
CH2OH
CH2OH
O
H
H
H
OR
H
O
NHCOCH3
H
O
H
OH
H
O
H
H
H
NHCOCH3
O
H
Transition state intermediate
H
OH
H
H
OR
NHCOCH3
CH2OH
H
O
H
NHCOCH3
H218O
O
Asp-52
Glu-35
CH2OH
O
H
H
OR
O
O
O
H
H
O
H
18
NHCOCH3
Nucleophilic attack
H
O
H
General base
18
OH
CH2OH
O
H
O
H
OR
H
Asp-52
H
H
NHCOCH3
O
Glu-35
O
O
HO
GOOD LUCK (, ASA