BIOSC
1000
Sample
Problem
Answers
Lecture
8
1. Explain
why
very
tight
binding
of
a
substrate
to
an
enzyme
is
not
desirable
for
enzyme
catalysis,
whereas
tight
binding
of
the
transition
state
is
desirable.
Tight
binding
of
a
substrate
would
produce
an
enzyme-substrate
complex
that
lies
in
a
thermodynamic
pit
(much
lower
energy
state),
effectively
increasing
the
activation
energy
and
thereby
slowing
down
the
reaction.
Conversely,
tight
binding
of
the
transition
state
lowers
the
energy
of
ES
(Xas
shown
on
your
handout.
Therefore
the
activation
energy
is
decreased,
and
the
rate
of
the
reaction
increases.
2. The
enzyme
hexokinase
catalyzes
the
following
reaction:
Glucose
+
ATP
glucose-6-phosphate
+
ADP
For
this
reaction,
G'
=
-16.7
kJ/mol.
a. Calculate
the
change
in
free
energy
G'
for
this
reaction
under
typical
intracellular
conditions
using
the
following
concentrations:
55
mM
glucose,
5.0
mM
ATP,
1.0
mM
ADP,
and
0.1mM
glucose-6-phosphate.
Assume
the
temperature
is
25C.
[C][D]
G'= G'+RT ln
[A][B]
[glucose 6 phosphate][ADP]
= G'+RT ln
[glucose][ATP]
%
[0.1103 ][1.0 103 ] (
= 16.7kJ /mol + ' 2.48kJ /mol ln
*
[55 103 ][5 103 ] )
&
= 36.3kJ /mol
b. In
the
typical
cell,
is
the
reaction
catalyzed
by
hexokinase
close
to
equilibrium
or
far
from
equilibrium?
Explain.
The
large
negative
value
of
G'
means
the
reaction
is
far
from
equilibrium
in
the
typical
cell.
Therefore
the
reaction
has
a
strong
thermodynamic
drive
to
go
in
the
direction
of
product
formation.
(Remember
that
at
equilibrium
G'
=
0.)
Page 1 of 5
BIOSC
1000
3. Why
is
there
a
high
degree
of
stereospecificity
in
the
interaction
of
enzymes
with
their
substrates?
The
formation
of
an
enzyme-substrate
complex
involves
a
close,
complementary
fitting
of
the
atoms
of
the
amino-acid
side
chains
in
the
active
site
with
the
atoms
of
the
substrate.
Since
stereoisomers
have
different
spatial
arrangements
of
atoms,
only
a
single
stereoisomer
of
a
substrate
usually
fits
into
the
active
site
in
such
a
way
that
it
can
be
acted
on
by
the
enzyme.
4.
Use
the
Michaelis-Menten
equation
to
demonstrate
the
following:
a) v0
becomes
independent
of
[S]
when
[S]
>>
KM
When
[S]
>>
KM,
KM
+
[S]
[S].
Substrate
concentration
has
no
effect
on
velocity,
and
v0
=
Vmax,
as
shown
in
the
upper
right
part
of
the
hyperbolic
curve
of
[S]
vs.
v0.
V [S] Vmax [S]
v 0 = max
= Vmax
K M + [S]
[S]
b) The
reaction
is
first
order
with
respect
to
[S]
when
[S]
<<
KM
When
[S]<<KM,
KM
+
[S]
KM,
and
the
Michaelis-Menten
equation
simplifies
to:
V [S] Vmax [S]
v 0 = max
K M + [S]
KM
Velocity
is
related
to
[S]
by
a
constant
value,
and
the
reaction
is
first
order
with
respect
to
S,
as
shown
in
the
lower
left
part
of
the
hyperbolic
curve
of
[S]
vs.
v0.
c) [S]
=
KM
when
v0
=
Vmax
V
V [S]
v 0 = max = max
2
K M + [S]
K M + [S] = 2[S]
K M = [S]
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BIOSC
1000
5.
An
enzyme
that
follows
Michaelis-Menten
kinetics
has
a
KM
of
1
M.
The
initial
velocity
is
0.1
M/min
at
a
substrate
concentration
of
100
M.
What
is
the
initial
velocity
when
[S]
is
equal
to:
When
[S]
=
100M,
[S]
>>
KM,
so
v0
=
Vmax
=
0.1
M/min.
a) 1
mM
For
any
substrate
concentration
greater
than
100
M,
v0
=
Vmax
=
0.1
M/min.
b) 1
M
When
[S]
=
KM,
v0
=
Vmax/2,
or
0.05
M/min.
c) 2
M
Since
KM
and
Vmax
are
known,
the
Michaelis-Menten
equation
can
be
used
to
calculate
v0
at
any
substrate
concentration.
For
[S]
=
2
M:
V [S] (0.1M /min)(2M ) 0.2
v 0 = max
=
=
M /min = 0.067M /min
K M + [S]
1M + 2M
3
6.
Imagine
you
are
studying
the
activities
of
2
enzymes
that
catalyze
the
same
reaction
(different
enzymes
that
catalyze
the
same
reaction
are
known
as
isozymes).
You
perform
a
kinetic
analysis
and
find
the
o
versus
[S]
shown
below.
a)
Which
enzyme
has
a
higher
affinity
for
the
substrate?
Why?
B,
because
the
substrate
concentration
at
maximal
velocity
(Km)
is
lower
than
the
same
point
for
A.
b)
Which
enzyme
turns
over
substrate
to
product
more
efficiently?
Why?
A
appears
to
turn
over
substrate
more
efficiently
because
it
has
a
higher
Vmax,
but
we
cant
be
more
precise
in
this
determination
because
we
dont
know
if
we
have
the
same
amount
of
enzyme
in
both
reactions
Page 3 of 5
BIOSC
1000
c)
Which
enzyme
would
be
more
active
under
in
vivo
conditions
of
substrate
concentration?
To
determine
optimal
activity
under
physiologic
conditions
we
would
need
to
calculate
the
catalytic
efficiency,
kcat/Km.
Enzyme
A
has
a
higher
Vmax
but
also
a
higher
Km
than
enzyme
B,
so
without
any
other
information
we
really
cant
choose
between
these
enzymes
7.
Our
most
common
painkilling
medications
(aspirin,
acetaminophen,
and
ibuprofen)
are
irreversible
inhibitors
of
enzyme
activity
why?
Once
an
irreversible
inhibitor
binds,
enzyme
activity
is
essential
permanently
decreased;
the
only
way
to
restore
enzyme
activity
to
normal
levels
is
to
synthesize
new
enzyme
proteins.
Thus,
irreversible
inhibition
provides
for
a
long-lasting
effect
of
the
drug.
Aspirin
can
exert
its
effects
up
to
19
hours.
8. Imagine that you are working for a big pharmaceutical company trying to identify the
next generation of painkillers. NSAIDs (non-steroidal anti-inflammatory drugs) like
acetaminophen (Tylenol) and ibuprofen (Advil) work by inhibiting cyclooxgenase
(COX) enzymes. The COX enzyme family converts the precursor fatty acid, arachidonic
acid, to PGG2, the immediate precursor of a variety of prostaglandins. Some
prostaglandins trigger fever, inflammation and its associated pain whereas others can be
tissue protective. As a drug designer, you want your new chemical to be selective and
effective.
Your kinetic analysis involves measuring the rate of the conversion of arachidonic acid
(AA) to PGG2 by 0.1 M COX-1 in the absence and presence of various test compounds,
as summarized in the following table.
Table 1: Rate of PGG2 formation in the presence and absence of test compounds
[AA]
Rate of
PPG2 formation
PPG2
(M)
formation of
in the presence
formation in
the presence
PGG2 (M/sec)
of 10 g/ml
pitt3791
of 10 g/ml
pitt3793
(M/sec)
(M/sec)
0.05
23.5
16.67
8.85
0.1
32.2
25.25
11.63
0.15
36.9
30.49
12.82
0.25
41.8
37.04
14.29
0.35
44.0
38.91
14.92
Page 4 of 5
BIOSC
1000
a.) Use the Lineweaver-Burk analysis to calculate the KM and Vmax for the enzyme in the
presence of these two inhibitors.
b). What type of inhibitors are they?
For this question (a and b) you need to plot the data. [AA] is substrate and the rate
is v0 so you can use this info to make double reciprocal plots (Lineweaver-Burke).
You can then determine the KM, Vmax and type of inhibitor from the plot. You will
need to plot this on graph paper (or computer) to get good answers. Just sketching it
will not work.
Page 5 of 5