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310 Homework 4
Problem
1.
An
enzyme
converts
a
substrate
(S)
into
a
product
(P)
through
rapid
and
reversible
formation
of
an
intermediate
complex
between
the
enzyme
and
substrate
(with
equilibrium
dissociation
constant
KM)
and
irreversible
dissociation
of
the
complex
to
form
the
product
and
release
the
enzyme
(with
rate
constant
k2).
Derive
the
rate
expression
of
product
formation
in
the
presence
of
both
a
competitive
inhibitor
(IC,
with
equilibrium
dissociation
constant
KC)
and
an
uncompetitive
inhibitor
(IU,
with
equilibrium
dissociation
constant
KU).
Both
inhibitors
bind
to
the
enzyme
reversibly.
Problem
2.
High-fructose
corn
syrup
(HFSC)
is
a
mixture
of
glucose
and
fructose
commonly
added
to
foods
and
drinks
for
taste
enhancement.
Sucrose
and
fructose
are
monosaccharides
obtained
from
the
hydrolysis
of
sucrose
catalyzed
by
the
enzyme
sucrase.
The
association
and
dissociation
rate
constants
of
sucrose-sucrase
complex
formation
are
109
M-1min-1
and
1.2105min-1
respectively.
The
turnover
number
of
sucrase
is
105
min-1.
0.5
Kg
of
sucrose
is
added
to
a
5L
solution
containing
60
mg
of
sucrase
(MW
180,000
g/mol):
a. How
long
will
it
take
to
degrade
90%
of
sucrose?
b. How
long
will
it
take
to
produce
0.25
Kg
of
fructose?
Problem
3.
An
enzyme
converting
substrate
S
into
product
P
is
immobilized
onto
calcium
alginate
beads
for
continuous
use
in
a
bioreactor.
The
bioreactor
containing
30
nM
of
immobilized
enzyme
is
pretreated
in
a
buffer
solution
at
50C
for
2
hours
to
eliminate
contaminants
from
the
immobilization
reaction.
The
temperature
of
the
bioreactor
is
lowered
to
37C
prior
to
addition
of
the
substrate.
The
rate
constant
of
enzyme
denaturation
at
50C
is
0.05
min1
and
the
activation
energy
is
10
kcal/mol.
The
enzyme
is
stable
at
37C.
The
Michaelis-Menten
constant
is
510-5
M
and
the
turnover
number
is
104
min1.
a. How
much
substrate
needs
to
be
added
to
the
bioreactor
to
achieve
99%
substrate
conversion
in
10
hours?
b. How
long
will
it
take
to
achieve
the
same
conversion
if
the
bioreactor
pretreatment
is
conducted
for
4
hours
instead?
c.
How
long
will
it
take
to
achieve
the
same
conversion
if
the
bioreactor
pretreatment
is
conducted
for
2
hours
but
at
60C?
Problem 4. Consider
an
enzymatic
reaction
in
which
an
enzyme
(E1;
1
M)
is
inhibited
by
its
own
substrate
at
high
substrate
concentration.
The
equilibrium
dissociation
constant
of
enzyme-substrate
complex
formation
at
the
active
site
is
3.210-5
M
and
the
equilibrium
dissociation
constant
of
enzyme-
substrate
complex
formation
at
the
inhibitory
site
is
510-4
M.
The
turnover
number
of
the
enzyme
is
103s-1.
a. Calculate
the
reaction
rate
if
the
substrate
is
80
uM.
b. The
enzyme
E1
was
engineered
to
generate
an
enzyme
variant
(E2)
with
decreased
affinity
at
the
inhibitory
site.
As
a
result,
the
fraction
of
complex
enzyme-substrate
(in
which
the
substrate
is
bound
at
the
inhibitory
site
of
E2)
is
1%
of
that
obtained
with
E1.
Calculate
the
reaction
rate
if
the
substrate
is
80
uM.
c. Compare
enzyme
E1
and
E2:
plot
of
the
reaction
rate
as
a
function
of
substrate
concentration.
d. The
enzyme
E1
was
also
engineered
to
achieve
partial
substrate
conversion
at
the
low
affinity
binding
site
(E3).
As
a
result,
substrate
conversion
occurs
both
at
the
high
affinity
binding
site
(with
turnover
number
103s-1)
and
at
the
low
affinity
binding
site
(with
turnover
number
10
s-1).
What
is
the
reaction
rate
if
the
substrate
is
80
M?
e. Further
engineering
of
enzyme
E1
and
E2
results
in
fully
active
enzymes
E4
and
E5,
respectively,
with
turnover
number
103s-1
for
both
the
high
and
low
affinity
sites.
What
are
the
reaction
rates
of
E4
and
E5
if
the
substrate
is
80
M?
f. Compare
enzyme
E3,
E4
and
E5:
plot
of
the
reaction
rate
as
a
function
of
substrate
concentration.
Problem
5.
The
antibiotic
sulfanilamide
is
similar
in
structure
to
para-aminobenzoic
acid
(PABA),
an
intermediate
in
the
biosynthetic
pathway
for
folic
acid.
Sulfanilamide
can
competitively
inhibit
the
enzyme
that
has
PABA
as
it's
normal
substrate
by
competitively
occupying
the
active
site
of
the
enzyme.
The
equilibrium
dissociation
constant
of
PABA
for
the
enzyme
is
3
M.
You
engineered
a
sulfanilamide
variant
(I1)
with
equilibrium
dissociation
constant
ten
times
lower
than
that
of
PABA.
a. Define
the
fractional
measured
enzyme
effect.
Calculate
the
IC50
of
I1
if
there
is
7
M
PABA
in
the
reaction.
b. You
are
investigating
the
enzyme
mechanism
and
develop
another
inhibitor
(I2)
that
binds
to
the
enzyme
on
a
site
other
than
the
active
site.
I2
binds
to
the
enzyme
with
the
same
affinity
of
I1
but
regardless
of
whether
the
enzyme
is
bound
to
PABA.
You
may
assume
that
I2
presents
the
same
affinity
for
the
unbound
and
PABA-bound
enzyme.
Define
the
fractional
measured
enzyme
effect.
Calculate
the
IC50
of
I2
if
there
is
7
M
PABA
in
the
reaction.