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Enzyme Kinetics
Fluorescence (%)
10
0 mM
1 mM
2 mM
3 mM
4 mM
5 mM
12.5 mM
8
6
4
2
0
0.001
0.01
0.1
Time (s)
10
100
Foreword
1.2
Equilibria
Fractional equilibration
C HAPTER 1
0.8
0.4
0.0
0.1
100.0
1.2
Fractional Equilibration
10.0
Time (min)
1.0
0.8
0.4
0.0
0.1
1.0
10.0
100.0
Time (min)
S ECTION 1
Reversible Reactions
L EARNING OBJECTIVES
1. Reversible reactions never come to a halt they achieve an equilibrium in which the
forward and reverse reactions are
quantitatively balanced.
2. For any reversible reaction, there is a fixed
relationship between the conentration of
products formed and substrates remaining at
equilibrium.
H2 + I2
Glucose + ATP
k1
Glucose-6-phosphate + ADP
[HI]2
K eq =
[H 2 ][I 2 ]
k-1
K eq =
2HI;
or, in general
pA + qB + rC
xD + yE + zF;
k1
k -1
S ECTION 2
Energy
Example 1
GA
S
G
P
Progress of Reaction
Example 2
Energy
L EARNING O BJECTIVES
GA
P
S
GA
G
Energy
P
G = 0
Progress of Reaction
Progress of Reaction
Forms of Energy
S = S + RT ln[S]
G = P - S
At equilibrium, the rate of P formation is matched
exactly by the rate of S formation. Thus the abilities of
P to form S (to do work) and vice versa are identical.
Hence, at equilibrium,
P = S
Thus,
G = 0
G = H - TS
Thus:
G T
2.
3.
4.
G = -TS
The fall in free energy is due to increased S. The
molecules redistribute to maximize system entropy.
Summary
1. A reaction occurs spontaneously only if G is
negative (P < S).
2. A system is at equilibrium (forward and reverse
reactions are balanced) and no net change occurs
when G = 0 (P = S)
3. The forward reaction cannot occur spontaneously
when G is positive. An input of free energy is
required to drive the reaction.
4. G depends upon the free energy of the products
(final state) minus that of the reactants (initial state).
i.e. G is independent of the reaction mechanism.
5. The irreversible increase in entropy provides a
directional driving force for the reaction.
S ECTION 3
G and equilibria
[C] [D]
G = G o + RTln [A] [B]
G is the standard free energy change
R = gas constant (1.98 cal/mol/degree)1
T = absolute temperature
LEARNING
O BJECTIVES
[A], [B], [C] and [D] are the molar activities of the
reactants under starting conditions.
1one
Defining Keq as
[C] [D]
K eq = [A] [B]
G = -RT ln Keq
P = 1 Atmosphere
pH = 7.0
Rearranging gives us
T = 298K = 25C
Keq = e-G/RT
[C] [D]
0 = G o + RTln [A] [B]
and
[C] [D]
G o =- RTln [A] [B]
=10-G/(2.303 RT)
Substututing R and T (25C) we obtain
Keq =10-G/1.36
when G has units of kcal/mol.
Thus an equilibrium constant of 10 corresponds to a
G of -1.36 kcal/mol (see Table below)
Go
Keq per M
1,000,000
10,000
100
10
1
0.1
0.01
0.0001
0.0000001
kcal/mol
-8.2
-5.5
-2.7
-1.4
0.0
1.4
2.7
5.5
9.5
kJ/mol
-34.2
-22.8
-11.4
-5.7
0.0
5.7
11.4
22.8
39.9
Note: H-bond energies range from 3 to 7 kcal/mol. van der Waals bond energies
are approximately 1 kcal/mol.
10
Thus
A B G1
A C Gtotal
[G6P]
K eq1 = [glucose] [P] = 3.9 x 10 -3 M -1
i
B C G2
Glucose6phosphate + H2O
G = 13.8 kJ/mol
ATP + H2O
ADP + Pi
G = -30.5 kJ/mol
Glucose + Pi
Glucose6phosphate + H2O
2)
ATP + H2O
ADP + Pi
sum:
ATP + glucose
K eq2 =
[ADP] [P]i
5
=
3.9
x
10
M
[ATP]
ADP + glucose6phosphate
11
Summary
1.
At equilibrium, G = 0
2.
3.
4.
5.
J.
Solution
A.
12
14
S ECTION 4
L EARNING O BJECTIVES
Energy
GA
S
P
Progress of Reaction
1. Covalent Catalysis
A nucleophile (electron-rich group with a strong
tendency to donate electrons to an electron-deficient
nucleus) on the enzyme displaces a leaving group on
the substrate. The enzyme-substrate bond is then
hydrolyzed to form product and free enzyme.
2. Acid-base Catalysis
e.g. Lysozyme cleaves the glycosidic bond between
C1 of N-acetylmuramic acid and C4 of Nacteylglucosamine of bacterial cell wall
polysaccharides. Glu35 of lysozyme donates a proton
to the oxygen of the polysaccharide glycosidic bond
thereby hydrolyzing the bond.
Energy
Energy
3. Proximity
X
S
X
X
S
P
Progress of Reaction
P
Progress of Reaction
Summary
1.
1.
2.
3.
4.
2.
3.
4.
5.
17
S ECTION 5
Key to formative
evaluations
Section 1
1. Keq = k1/k-1
2. Keq = [P]eq/[S]eq
3. [S] would fall and [P] would increase so that Keq
remained unchanged.
4. [S] would rise and [P] would fall so that Keq
remained unchanged.
Section 2
1. At equilibrium, G =0 and S = P
2. When the reaction proceeds left to right, G < 0
and S > P
C HAPTER 2
Analysis of
time
dependent
processes
39
38
37
kobs
36
F
35
34
33
32
0
time in seconds
Fraction reacted
This
chapter
time-dependent
Lorem
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dolor sit amet,
processes. We
seek
to:
suspendisse
nulla
pretium,
rhoncus
1.2
0.8
0.6
0.4
0.2
0
10
100
1000
time, msec
10
Glossary
catalysis - the process by which an enzyme or
catalyst accelerates a reaction.
catalyst - an agent that accelerates a chemical
reaction but which is unchanged in amount or
chemistry at the end of the reaction.
chemical equilibrium - a reaction in which forward
and reverse reactions continue to proceed but are
quantitatively balanced
S ECTION 1
Context
Some examples
W.Stuhmer et al.
A
==
RCK1
1 SOpA
RCK3
J6pA
RCK4
4 0
RCK5
A~~~~~~4Op
,,,J@20pA
Fig.
20ms
chann
poten
G/Gm
788
10 AUGUST 2007
VOL 317
SCIENCE
www.sciencemag.org
1.2 T
Vibrational Response
Functions (VRFs) of SelfAssembled Monolayers. B
VRF for a C8 monolayer. C
VRF for a C18 monolayer
From: Wang et al.,
SCIENCE VOL 317, pp
787-790, 2007
Normalized current
CTX-Biotin
1.0
0.5
0.0
Washout
200
-40
Sugar
- Thiol
Sugar of metabolically-labeled
control
Shaker
to voltage+ Thiol
from -60 to 0 mV are shown in Figure 7.
B Chemical
steps
channels with CTXBiotin.
Washout(A) CTXBiotin (10 nM) After TCEP
The
of
size
step
currents at 0 mV varied between
elementary
Initial
Initial
inhibits Shaker-IR K+ currents in CHO-K1 cells treated
0.46
and
1.02
pA
(RCK4)
pA
(RCK5). The single channel
with either 50 M thiol sugar 1 (h) or 0.5% ethanol as a
current - voltage
relations were measured in cell attached
vehicle (s). Only metabolically-labeled
(+thiol sugar)
patchesbywith
normal after
frog
Washout
channels were irreversibly blocked
CTXBiotin
aRinger's solution on the extracellular
side. For
all channels,
simple washout; inhibition was
completely
reversed
by an the current-voltage relation
50 ms
50 ms in
is linear
application
of 1 mM TCEP. Reaction
profiles
were range -20 to 20 mV. However, since
the voltage
monitored with a 200 ms, 40 this
mV is
pulse
every
15s. range for conductance estimation, we
a rather
narrow
measured
the
average
amplitudes at 0 mV membrane
- Thiol Sugar
+ Thiol Sugar
CFrom: Hua
Z, Lvov A, Morinpotential.
TJ, Kobertz
WR.
While
theChemical
RCK1, RCK3 and RCK5 channels have
I/I
I/I
control of metabolically-engineered
voltage-gated
K(+)
rather similar
1.0single-channel current amplitudes, that of the
1.0
channels. Bioorg Med Chem RCK4
Lett 2011.
channel is considerably lower (Table I).
1 nA
1 nA
L EARNING OBJECTIVES
needed to be chemically reversible. Although disulfide bond formation between CTX and thiol-containing sialic acids on the cell surface is an obvious chemoselective and cell friendly reaction, we
chose to label CTX with a bismaleimide that had an internal disulfide bond because maleimides are inherently more stable in water
than MTS reagents. Moreover, this subtle difference would allow
for delivery of a small molecule probe to the modified K+ channel
subunit after cleavage with reductant, which would be useful in
subsequent biochemical, biophysical or imaging experiments. To
simplify the synthesis of the bismaleimide, derivatization of CTX,
and ensure delivery of a molecular probe to a K+ channel subunit,
we set out to
5 synthesize a symmetrical bismaleimide (Scheme 1)
from cystamine dihydrochloride 2 that would allow for the facile
incorporation of a molecular probe in the final step of the synthesis. The amino groups of cystamine 2 were capped with 2 equiv of a
doubly amino-protected, activated ester of L-lysine 3. Selective
deprotection of the Fmoc protecting groups gave the symmetric
diamine 4. Addition of 2 equiv of the NHS-ester of 3-(maleimido)propionic acid and N-Boc deprotection afforded bismaleimide
5, which was subsequently biotinylated with 2 equiv of NHSBiotin to give biotin bismaleimide 6. CTX was then derivatized by
labeling a cysteine mutant of CTX (R19C)32 with 100-fold molar excess of biotin bismaleimide 6 to yield CTXBiotin, which was purified by reverse phase HPLC as we have previously described.17,19
With CTXBiotin in hand, we decided to change both the channel (ShakerIR)33 and the expression system (CHO-K1 cells) to
demonstrate that our approach was versatile and could be used
to label glycosylated ion-conducting subunits. Inactivation-removed Shaker is similar to Q1 in that it is an archetypical volt-
Conductance-voltage relations of
RCK channels. (A) Outward
currents in response to depolarizing
voltage steps. From top to bottom
RCKI, RCK3, RCK4, RCK5. The
traces are responses to 50 ms
voltage steps from -50 to 40 mV in
10 mV intervals.
-80
max
Initial
Washout
0.5
max
Initial
After
TCEP
Pharmacology
0.5 of RCK channels
22
A profile of the pharmacological sensitivity of the different
RCK channels to the K+ channel blockers 4-aminopyridine
chann
Hold
inact
low
depe
subs
at 2
Tabl
curr
RCK
sensi
inac
sensi
RCK
bloc
effec
Dis
Comp
RCK
An
funct
chan
ident
pa
prope
sing
shou
chan
func
memb
prel
struc
De
slowl
foun
inact
spina
chann
1988
whic
Fig. I . Time course of 3-0-methylglucose uptake in isolated muscle fibres. Ordinate: ratio
of i n t r a ~ l l u l a ractivity t o extracellula~activity of 3-0-methy1glucose per equixra1ent
volume of bulk external solution, .4bscissa: time in hours. External sugar concentration,
Time course
of 3-0-methylglucose
uptake in isolated
muscle
of Balanus
1 m M Uptake
was measured using rc)nventional
and scintillator
probe (cells
0 )methods.
Kumber of points per conventional determination, five or more. The water content of
nubilis. Ordinate:
ratio of intracellular activity to extracellular activity of 3-0isolated fibres (70%) is shown by the continuous line above the points. The time a t
diame*r, 1352
p m ;k m pAbscissa:
rature.
half-equilibration
is shown by thedmhed
methylglucose
per equivalent
volumeline.
ofMean
bulkfibre
external
solution.
21 OC.
of artificial
lipid bilayers to sugars (Jung & Snell, 1968; Lidgard & Jones,
Ribosomal R N A Stability permeability
and Growth
Conditions
19'75).
At equilibrium, the 3-0-methylglucose space of the fibre is 70 yo,which is in close
contentsof barnacle muscle (71 & 1 7; ; R = 5 ) .
sgreement with estimates of the xra%er
Assuming this water is not bound, these results shour that 3-0-methylglucose is not
accumulated by barnacle muscle and that the transfer of the sugar across the
sarco1emma is mediated by a passive, facilitated process.
EJat ofphloretia on mqzr uptuke. Re18tively low concentrations of phloretin inhibit
C""
Length of chose, h
tion
S ECTION 2
Reaction order
Rate of a reaction
The rate or velocity, v, of a reaction or process
describes how fast it occurs.
The velocity is expressed as a change in
concentration (C) per unit time (t),
dC
v = dt
but may also express the change in a population of
cells with time, the increase or decrease in the
pressure of gas with time or the change in absorption
of light by a colored solution with time.
The order of a reaction
describes how the velocity of the reaction depends
upon the concentration of reactants.
In the (irreversible) isomerization reaction
L EARNING O BJECTIVES
1. Reaction orders
1. Zero-order
2. First order
3. Second order
1. Class 1
2. Class 2 (pseudo first-order)
k1
d [B]
m
=
= k 1 [A]
k
1 [A]
dt
Since m = 1, this reaction is first order with respect to
A and since A is the only independent concentration
variable in the rate equation, the reaction is overall
first-order.
24
The units for this first order reaction are derived from
moles of product formed per second per mole of
reactant or,
M per sec = k 1 M
M per sec
= k 1 = per sec
M
E+S
k1
E$S
d [ES]
1
1
=
k
1 [E] [S] = k 1 [E] [S]
dt
Because m = 1 for both species the reaction is firstorder with respect to E or S but is second order overall
as one single step is involved in the reaction of two
species.
The units of this second order reaction are derived
from moles of product formed per second per mole2 of
reactants or
molarity per sec = k1 (molarity)2
M per sec
= k 1 = per M per sec
M2
N2O5
NO2 + NO3
NO3
NO2 + O
2O
O2
Zero-order kinetics
1The
2NO2
ONOONO
2NO + O2
http://inside.umassmed.edu/uploadedFiles/gsbs/courses/
2012-2013_Core_Course_Files/CoreKinetics.pzf%20-%20for
%20students%20only.zip
and download the GraphPad Prism version 6 software from
Prism 6 Winhttp://cdn.graphpad.com/downloads/prism/6/
InstallPrism6.exe
Prism 6 Windows serial number: GPW6-200512-LEM5-16772
Prism 6 Machttp://cdn.graphpad.com/downloads/prism/6/
InstallPrism6.dmg
Prism 6 Mac serial number: GPM6-200513-LEM5-F3EF2
26
A zero-order reaction
zero-order kinetics
Zero-order reaction
Substrate
Product
80
v (d[P]/dt)
[Substrate] or [Product]
10
100
60
40
20
100
200
[S] M
d [ethanol] d [acetaldehyde]
v ==
= k0
dt
dt
2
0
0
10
TIME
The negative sign is used with reactant - ethanol because its concentration decreases with time. The
concentration of its product, acetaldehyde, increases
with time.
C0
ALDH
+
CH3CHO
CH3CH2OH
0
27
dC
dt = k 0
The units of k0 are molarity per sec. This is a zeroorder reaction because there is no concentration term
in the right hand of the equation.
Defining C0 as the concentration at zero time and C as
the concentration at any other time, the integrated rate
law is:
C = C0 + k0 t
y = y-intercept + slope * x
This is the equation for a linear relation between the
independent (time) and dependent (concentration)
variables.
We can therefore subject the raw data to linear
regression analysis to obtain C0 (y-intercept) and k0
(the slope).
10
[Substrate] or [Product]
Zero-order reaction
Substrate
Product
8
6
4
2
0
0
10
TIME
y = x-intercept + slope * x
Best-t
values" "
"
Slope" "
"
"
Y-intercept
when
X=0.0"
X-intercept
when
Y=0.0"
Goodness
of
Fit" "
R
squared"
"
"
"
"
"
"
Substrate" "
-1
0
"
"
10
0"
"
10.00""
"
Product"
1
0
0 0""
0" "
"
1.000""
1.000
"
"
"
"
Units
mols/sec
mols
sec
A first-order reaction
1stOrder
dC
dt = k 1 C
[A] or [B]
4
3
[Substrate]
[Product]
The reaction
1
0
0
k1
10
TIME
d [A] d [B]
v =- dt = dt = k 1 [A]
where k1 is the rate constant for this reaction.
The velocity may be expressed in terms of either the
rate of disappearance of reactant (-d[A]/dt) or the rate
of appearance of product (d[P]/dt).
29
Theory
- d [A] = k 1 [A] 0 t
[A] = [A] 0 e -k t
1
ln [A] =- k 1 t + ln [A] 0
Half-life
Defining [A] at t1/2 as [A]0/2
slope x + intercept
ln2 0.693
t 1/2 = k = k
1
1
and because = 1/k1,
t1/2 = 0.693
30
Theory
[B] = [B] {1 - e -k t}
1
Thus we obtain
slope x + intercept
Half-life
Defining [B] at t1/2 as [B]/2
ln2 0.693
t 1/2 = k = k
1
1
and because = 1/k1,
t1/2 = 0.693
31
[A] or [B]
4
3
[Substrate]
[Product]
A second clue comes from the measurement of halftimes. As [Substrate] declines from 5 - 2.5 mM, from
2.5 - 1.25 mM and from 1.25 to 0.625 mM, the time
required for each 50% reduction is unchanged at 1.4
sec.
1st Order
5
0
0
10
TIME
3
2
1.4 sec
1
0
0
10
1.4 sec
1.4 sec
10
TIME
[Substrate]
1
[A]
0.1
0.01
0
[Substrate]
10
TIME
[A] = [A] 0 e -k t
1
2.
3.
4.
33
1stOrder
[Substrate] or [Product]
5
4
[Substrate]
[Product]
2
1
0
0
10
TIME
"
[Substrate]"
[Product]" "
Units
"
"
"
"
"
5.000""
0"
"
0.5000"
1.386
"
2.000""
"
"
"
"
"
0
5.000
0.5000
1.386
2.000
mM
mM
per
sec
sec
sec
"
"
48" "
1.000""
"
"
48
1.000
5. t1/2 = 0.693/k
6. k has units of time-1 (e.g. s-1). There are no
concentration units in k so we need not know
absolute concentrations - only relative
concentrations are needed.
7. k may be obtained by direct curve fitting
procedures using nonlinear regression
8. The full equation for loss of substrate is
[S]t = {[S]0 - [S]} e-(k.t) + [S]
9. The full equation for product formation is
[P]t = [P] (1 - e-(k.t))
10. When a first order reaction is reversible (as most
are), e.g.
k1
k2
Second-order reactions
Fall into 2 categories in which the rate law depends
upon:
2. the product of the concentrations of two dierent
reagents.
Class 1 reactions (A+A P)
[Substrate]
[Product]
3
2
1
0
0
10
TIME
v=k[A]2
[A] or [B]
UUCGAA
v = k [A 2 GCU 2] 2
0.3
[Substrate]
[Product]
[A]0/[A]
0.2
0.1
0.0
-0.1
-0.2
5
TIME
10
1
1
[A] [A] 0 = k t
adding 1/[A]0 to both sides gives
1
1
k
t
=
+
[A] 0
[A]
0
0
10
TIME
[A] 0
[A] = [A] 0 k t + 1
Thus one expects a linear relation between the
reciprocal of the reactant concentration and time.
36
1
2
3
4
5
6
7
8
9
10
[A]0/[A]
10
Increasing
[A]0
0
0
10
TIME
1.5
1.0
kr
0.5
0.0
0
kf
A+A
E+S
10
[A]o
k1
k2
E$S
v=
d [ES]
dt = [E] k 1 [S] - k 2 [ES]
37
R+L
kf
kr
R$L
(note the forward and reverse rate constants have now been called kf
and kr but this name change is purely arbitrary - they could have been
called kon and ko or k1 and k2)
[L]
0.0010
10
5.995
0.0008
3.594
[LR] M
2.154
0.0006
1.292
.774
0.0004
.464
.278
0.0002
.167
.1
0.0000
5
TIME
10
0.0010
10
5.995
0.0008
3.594
2.154
[LR] M
[L]
0.0006
1.292
.774
0.0004
.464
.278
0.0002
.167
.1
0.0000
0.1
10
TIME
kobs vs L
25
20
15
Best-fit values
Slope
Y-intercept when X=0.0
X-intercept when Y=0.0
1/slope
R+L
1.999 0.0001861
0.5012 0.0007352
-0.2507
0.5002
kr
R$L
d [LR]
v = dt = [R] k f [L] - k r [LR]
10
5
0
0
kf
6
[L] M
10
S ECTION 3
3. t1/2 = 0.693/k
4. k has units of time-1 (e.g. s-1).
5. The half-time (t1/2) and k are invariant of the
starting value of [S].
4. There are two classes of second order reactions.
1. In reactions where two molecules of a
substrate combine to form a product (Class 1
reactions), the reaction is non linear with time
41
S ECTION 4
Formative self-evaluation
questions
1.
What are the units of zero-, first- and secondorder rate constants?
2.
3.
4.
5.
6.
18. How does kobs vary with [S] for a Class 2 secondorder reaction?
7.
8.
9.
S ECTION 5
Key to formative
evaluations
8. t1/2 = 0.693/k
C HAPTER 3
Steady-state
kinetics of
enzymecatalyzed
This chapter considers receptor-ligand
Lorem ipsum
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and dolor
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E
C
I
I
S
E
I
C
kp
E+P
Ki
EI
+
S
Ks
ES + I
C
kp
E+I
+
S
E+P
ESI
Context
120
Vm
100
80
60 0.5 V
m
40
20
0
Km
0
10
20
30
40
50
[S] mM
xlvi
100
v (d[P]/dt)
75
50
25
0
0.01
0.1
10
100
1000
[S] [S]
mM
M
Vm [S]
v = K + [S]
m
This equation is called the Michaelis-Menten equation.
Our challenge in this chapter is to understand why the
phenomenon of enzyme-mediated catalysis is well
approximated by this relationship.
xlvii
S ECTION 1
Michaelis-Menten Kinetics
ES
kr
k2
E+P
k -p
+
(E..S)
2. Noncompetitive
0
E+S
3. Uncompetitive
4. What does Km represent?
kp
EP
k -2
Energy
L EARNING O BJECTIVES
E+S
kf
ES
E+P
Reaction coordinate
48
E+S
kf
kr
ES
kp
E+P
kr = kp = per sec.
2.
49
E+S
ES
kf
[ES]
k r [S]
[E] t =
k
1 + k f [S]
r
d [ES]
dt = k f [E] [S]
The rate of ES breakdown is given by:
[ES]
[S]
=
[E] t
kr
k f + [S]
- d [ES]
dt = k r [ES]
Thus at equilibrium
d [ES] - d [ES]
dt = dt ` k f [E] [S] = k r [ES]
Hence
k
[ES] = k f [E] [S]
r
If we seek to understand how much substrate is
bound (i.e. [ES]) at any given [S] and [E], we can
express [ES] as a fraction of total enzyme [E]t as:
[ES]
[ES]
=
[E] t
[E] + [ES]
per sec
per M per sec = M
When [S] = kr/kf, this means that
[ES]
[S]
[S]
=
=
[E] t
kr
2 [S] = 0.5
k f + [S]
This means that one-half of [Et] = [ES] when [S] = kr/kf
50
E+S
kf
kr
ES
Kd M
0.000001
0.0001
0.01
0.1
1
10
100
10000
10000000
kcal/mol
-8.2
-5.5
-2.7
-1.4
0.0
1.4
2.7
5.5
9.5
kJ/mol
-34.2
-22.8
-11.4
-5.7
0.0
5.7
11.4
22.8
39.9
51
[LR]
M
2.848e-004
3.995e-004
5.262e-004
6.496e-004
7.557e-004
8.377e-004
8.959e-004
9.349e-004
9.599e-004
9.756e-004
0.0010
0.0008
[LR] M
[L]
M"
0.100"
0.167"
0.278"
0.464"
0.774"
1.292"
2.154"
3.594"
5.995"
10.000"
B max [S]
[S] b = K + [S]
d
0.0006
0.0004
0.0002
0.0000
B max = [E] t n
10
[L] M
Bmax = 20 M
Y=Bmax*X/(Kd + X)
Where Bmax is the enzymes capacity to bind S
The result yields:
[LR] M
One site -- Specific binding
Best-fit values
Bmax
0.0010
Kd
0.25
M
M
52
Kd 1
1
1
=
+
[S] b B max [S] B max
[S]
Kd
Kd 1
1
1
=
+
=
+
[S] b B max [S] B max [S] B max [S] B max
This is a linear equation. Plotting 1/[S]b on the y-axis
and 1/[S] on the x axis, we obtain:
slope = Kd/Bmax,
y-intercept = 1/Bmax
[S]
[S]
K d [S]
Kd
1
[S]
=
+
=
+
B max
[S] b B max [S] B max B max
This is a linear equation. Thus plotting [S]/[S]b on the
y-axis and [S] on the x axis, we obtain:
slope = 1/Bmax,
y-intercept = Kd/Bmax
When [S] = -Kd
[S]
Kd
Kd
=
+
[S] b B max B max = 0
x-intercept = 1/-Kd
Lineweaver Burk
x-intercept = -Kd
1/[LR] per M
4000
Best-fit values
Slope
Y-intercept when X=0.0
X-intercept when Y=0.0
1/slope
251.0 0.09620
999.2 0.3800
-3.980
0.003983
[LR] M
10000
3000
Best-fit values
Slope
Y-intercept when X=0.0
X-intercept when Y=0.0
999.9 0.03448
250.5 0.1362
-0.2505
8000
slope = Kd/Bmax
2000
y-cept = 1/Bmax
1000
[L]/[LR]
1/[LR] per M
x-cept = -1/Kd
Multiplying by [S]
6000
slope = 1/Bmax
y-cept = Kd/Bmax
x-cept = -Kd
4000
2000
-5
5
1/[L] per M
10
0
10
[L] M
53
3 ) Scatchard method
B max [S]
[S] b = K + [S]
d
[S] b K d + [S]
[S] b K d
B
=
max =
[S]
[S] + [S] b
Dividing by Kd gives
B max [S] b [S] b
K d = [S] + K d
Significance of Bmax
y-intercept = Bmax/Kd
When [S]b = Bmax
[S] b B max B max
[S] = K d - K d = 0
[LR] M
0.004
Best-fit values
Slope
Y-intercept when X=0.0
X-intercept when Y=0.0
1/slope
-3.984 0.002759
0.003986 2.095e-006
0.001000
-0.2510
0.003
0.0005
0.0010
0.001
0.000
0.0000
slope = -1/Kd
y-cept =Bmax/Kd
x-cept = Bmax
0.002
B max = [E] t n
x-intercept = Bmax
[LR]/[L]
0.0015
[LR] M
54
n = Bmax/[E]t = 2
enzyme
Subunit
A
ES
S+E+I
Ki
EI
Subunit Subunit
A
B
1.0
[ligand]b/[Enzyme]t
Subunit B
Subunit A
0.8
Kd
[I]=0
[I]=5
[I]=25
[I]=50
[I]=100
0.6
0.4
0.2
0.0
10
100
[S] M
1000
10000
55
[S] b =
B max [S]
[I]
K d (1 + K ) + [S]
i
Introducing catalysis
Let us now consider the breakdown of ES to
regenerate free enzyme E and release the product, P.
The overall scheme is visualized as
E+S
[I]
K dapp
Kd - 1 = Ki
[I]
Ki = K
dapp
Kd - 1
Thus computation of Kd in the absence of I and Kdapp
at any given [I] permits computation of Ki.
kr
ES
kp
E+P
[I]
K dapp = K d (1 + K )
i
kf
v = kp [ES]
We must again express [ES] in terms of known
quantities
Rate of [ES] formation
+d[ES]/dt = kf [E][S]
Rate of breakdown of [ES]
-d[ES]/dt = kr[ES] + kp [ES]
or
-d[ES]/dt = (kr + kp) [ES]
In the "steady state" the concentrations of
intermediates (e.g. ES) are unchanged, whereas [S] +
[P] can change. If we limit measurements of v to early
stages, [ES] does not change (there is no reverse
reaction)
d[ES]/dt = 0
56
[S]
v
=
kr + kp
k p [E] t
k f + [S]
thus
kf [E] [S] = (kr + kp) [ES]
hence
[E] [S] k r
[E] [S]
[ES] = k + k = k + k
r
p
r
p
kf
The following steps are algebraic tricks:
[E]t = [E] + [ES]
dividing the velocity equation by [E]t we obtain
k p [ES]
v
=
[E] t [E] + [ES]
divide both sides by kp
and defining:
Vm = kp [E]t
and
Km =
kr + kp
kf
Vm [S]
v = K + [S]
m
[ES]
v
=
k p [E] t [E] + [ES]
substituting for [ES]
kf
k r + k p [E] [S]
v
k p [E] t =
k
[E] + k +f k [E] [S]
r
p
canceling [E]
kf
k r + k p [S]
v
k p [E] t =
k
1 + k +f k [S]
r
p
vV
zero-order
kinetics
100
80
Enzyme
Vm [S]
v. K
m
i.e. v increases linearly with [S]
60
rate of reaction M/min
at low [S]
v (d[P]/dt)
80
40
20
0
first-order
kinetics
0
100
60
40
M/min
100.0
25.00
Vm
Km
20
200
[S] M
0
0
20
40
60
[S] M
Const $ x
y = Const 1+ x
2
58
Hanes-Wolf Analysis
The Lineweaver Burk equation is
[S]
Km
1
=
+
v
V m [S] V m [S]
1 Km 1
1
=
+
v
V m [S] V m
Multiplying both sides by [S] gives
thus
1 Km 1
1
=
+
v
V m [S] V m
plotting 1/v vs 1/[S] yields a straight line with slope =
Km/Vm and y-intercept = 1/Vm
0.15
[S] K m
1
=
+
[S]
v
Vm
Vm
0.10
0.8
slope = Km/Vm
0.05
x-cept = -1/Km
y-cept = 1/Vm
-0.1
Thus
0.0
0.1
0.2
1/[S] per M
0.3
0.4
1/v min/ M
Slope
Y-intercept when X=0.0
X-intercept when Y=0.0
1/slope
0.6
slope = 1/Vm
y-cept = Km/Vm
x-cept = -Km
0.4
0.2
Slope
Y-intercept when X=0.0
X-intercept when Y=0.0
1/slope
-20
-0.2
20
[S]
[S]/v
0.0100 2.320e-010
0.2500 7.331e-009
-25.00
100.0
40
60
Enzyme Inhibition
Noncompetitive Inhibition
Competitive Inhibition
Schematic
C
I
King-Altman Diagram
E+I
+
S
EI
Ki
E
I
Ki
EI
+
S
Ks
ES + I
C
kp
E+I
+
S
ESI
C
kp
Ks
enzyme
E+P
ES
kp
E+P
E+P
60
Uncompetitive Inhibition
E
+
S
C
+S
Threonine
threonine
Isoleucine
+I
KI
ES + I
C
I
kp
ESI
C
kp
deaminase
E+P
E+P
61
[I]
K m (app) = K m T 1 + K Y
i
Uncompetitive Inhibition
Vm
[I]
T1 +
Y
Ki
Km
K m (app) =
[I]
T1 +
Y
Ki
V m (app) =
62
Noncompetitive Inhibition
Competitive inhibition
100
100
inhibited
v mol/min
v mol/min
80
Control
80
60
40
Control
Michaelis-Menten Perfect fit
Best-fit values
Vmax
100.0
Km
25.00
20
0
0
Control Inhibited
Michaelis-Menten Perfect fit
Best-fit values
Vmax
100.0
50.00
Km
25.00
25.00
60
40
20
inhibited
Perfect fit
0
0
100.0
50.00
50
[S] M
Control
Inhibited
50
[S] M
100
100
Inhibited
Best-fit values
Slope
0.0100 2.100e-010 0.0100 2.572e-010
Y-intercept when X=0.0 0.2500 1.047e-008 0.5000 1.282e-008
X-intercept when Y=0.0 -25.00
-50.00
[S]/v
M.min/mol
control
Inhibited
1.5
[S]/v
M.min/mol
1.0
control
Inhibited
0.5
-25
-50
control
Inhibited
Best-fit values
Slope
0.0100 2.100e-010 0.0200 4.200e-010
Y-intercept when X=0.0 0.2500 1.047e-008 0.5000 2.093e-008
X-intercept when Y=0.0 -25.00
-25.00
50
25
50
75
100
[S] M
100
[S] M
Lineweaver Burk Noncompetitive
control
inhibited
Best-fit values
Slope
0.2500 1.371e-009 0.5000 3.877e-009
Y-intercept when X=0.0 0.0100 2.134e-010 0.0100 6.035e-010
X-intercept when Y=0.0 -0.0400
-0.0200
0.25
0.20
control
inhibited
0.15
1/v
min/mol
1/v
min/mol
0.20
0.10
0.05
0.0
control
inhibited
Best-fit values
Slope
0.2500 1.371e-009 0.5000 3.877e-009
Y-intercept when X=0.0 0.0100 2.134e-010 0.0200 6.035e-010
X-intercept when Y=0.0 -0.0400
-0.0400
control
inhibited
0.15
0.10
0.05
0.1
0.2
1/[S] per M
0.3
0.4
0.0
0.2
1/[S] per M
0.4
63
Uncompetitive Inhibition
Control Inhibited
Michaelis-Menten Perfect fit Perfect fit
Best-fit values
Vmax
100.0
50.00
Km
25.00
12.50
100
v mol/min
80
Km(app)
Vm(app)
Ki
mol/min
None
25
100
Competitive
50
100
10
Noncompetitive
25
50
10
Uncompetitive
12.5
50
10
Inhibition
Control
Inhibited
60
40
20
0
0
50
[S] M
100
[S]/v
M.min/mol
-25
control
Inhibited
Best-fit values
Slope
0.0100 2.100e-010 0.0200 2.749e-010
Y-intercept when X=0.0 0.2500 1.047e-008 0.2500 1.370e-008
X-intercept when Y=0.0 -25.00
-12.50
control
Inhibited
25
50
75
100
[S] M
1/v min/M
Best-fit values
Slope
Y-intercept when X=0.0
X-intercept when Y=0.0
0.10
control
inhibited
0.2500 1.371e-009
0.0100 2.134e-010
-0.0400
0.2500 3.358e-009
0.0200 5.227e-010
-0.08000
Vm
V m (app) - 1
control
inhibited
0.05
0.0
[I]
[I]
Ki = K
m (app)
Km - 1
-0.1
0.15
Determining Ki
0.1
0.2
1/[S] per M
0.3
0.4
64
thus
Noncompetitive
[I]
T
Y
+
1
K
m
[S]
Ki
1 K m(app)
1
=
+
=
+
v
Vm
V m [S]
V m [S]
V m [S]
0.4
1/v min/M
Competitive
Uncompetitive
0.3
hence
Km
1 T Km
1
Y
=
+
[I]
v
K i V m [S] V m [S] + 1
0.2
0.1
-60
-40
-20
K
slope = K V m[S]
i m
20
40
60
[I] M
1 Km
y - intercept = V T [S]
+ 1Y
m
when 1/v = 0
Km
Km
[I] K [S] = T [S]
+ 1Y
i
-Ki(1+[S]/Km)
and
[S] K m
[S]
[I] = - K i K T [S]
+ 1 Y = -Ki T 1 + K Y
m
m
-Ki.
-Ki(1+Km/[S])
65
V m (app) [S]
v = K + [S]
m
thus
thus
[I]
[I]
T1 +
Y T1 +
Y
K
m
[S]
Ki
Ki
Km
1
+ V
v = V m(app) [S] + V m(app) [S] =
V m [S]
m
hence
and
[I] 1 K m
1 T
Y
T
Y
=
+
1
v
K i V m [S] + 1
1
1 1 T Km
1 T Km
Y
Y
=
+
+
[I]
1
v
K i V m [S]
V m [S] + 1
V m(app) [S]
v= K
m(app) + [S]
1 Km
1 Km
[I] V T [S]
+ 1 Y = - K i V T [S]
+ 1Y
m
m
thus
[I] = - K i
K m(app)
[S]
Km
1
=
+
=
v V m(app) [S] V m(app) [S] V m [S] +
hence
T1 +
[I]
Y
Ki
Vm
1
1 T Km
1
Y
=
+
+
1
[I]
v V m [S]
K i Vm
and
[I] - K m
T
Y
K i = [S] + 1
Km
x - cept = [I] = - K i T [S]
+ 1Y
and
Ki =
[I]
T Km + 1 Y
[S]
66
67
kr + kp
kf
E+S
ks
k-s
ES
k1
EP
k2
kp
E+P
k-p
k 1 k -p
k cat = k + k + k
-p
2
1
However, for the purposes of the discussion that
follows we shall assume that kcat = kp
68
V m [S]
v. K
m
ke =
where
70
S ECTION 2
B max [S]
[S] b = K + [S]
d
4. What is the relationship between [E]t and Bmax?
B max = [E] t n
1. What assumptions did we make in order to derive
the Michaelis-menten equation for an enzyme
catalyzed reaction?
1. The reverse reaction (P S) is negligible.
2. Assume only a single central complex (ES)
exists. i.e. ES breaks down directly to E + P.
3. [S] >> [E]. The instantaneous interaction of S
and E to form ES does not significantly aect
free [S].
Ligand Binding
E+S
kf
kr
ES
1. Kd = -1/slope
4. Kd = 1/Keq
2. Bmax = x-intercept
[I]
Ki = K
dapp
Kd - 1
Catalysis
Vm [S]
v = K + [S]
m
11. How is Vm related to [E]t?
1. Vm = [E]t kcat
12. What is Km?
1. Km is that [S] where v = Vm/2
13. How is Km related to the rate constants describing
the reaction?
1. The answer is dierent for each specific kinetic
model.
2. For our reaction, Km = (kp+kr)/kf
14. How can we compute Km and Vm from
measurements of v at varying [S]?
1. Nonlinear regression analysis of plots of v vs [S]
using the Michaelis-menten equation to obtain
best estimates of Km and Vm.
2. Linear transformation of the v vs [S] data:
1.Lineweaver-Burk (1/v vs 1/[S]) gives
1. Km= -1/x-intercept
2. Vm = 1/y-intercept
3. Hanes-Wolf ([S]/v vs [S]) gives
1. Km = -x-intercept
2. Vm = 1/slope
15. How do inhibitors aect substrate/velocity plots?
1. Competitive inhibitors
1. Leave Vm unchanged
2. Increase Kmapp
72
2. Noncompetitive inhibitors
1. Decrease Vmapp
2. Leave Km unchanged
3. Uncompetitive inhibitors
1. Decrease Vmapp
2. Decrease Kmapp
16. How do we compute Ki for an inhibitor?
1. By measuring Km and Vm for a reaction in the
absence of an inhibitor and Kmapp and Vmapp in
the presence of the inhibitor.
1. For competitive inhibition, Ki is calculated as
[I]
Ki = K
m (app)
Km - 1
2. For noncompetitive and uncompetitive
inhibition, Ki is calculated as
Ki =
[S]
x - cept = - K i T 1 + K Y
m
[I]
Vm
V m (app) - 1
Km
x - cept = - K i T [S]
+ 1Y
17. When is an enzyme kinetically perfect?
1. kcat/Km for a kinetically perfect enzyme 108
M-1.s-1
18. What does kinetic perfection really mean?
1. It means that the enzyme is so catalytically
active that when the ES complex forms,
substrate is immediately converted to product
or to an intermediate species thereby
preventing the ES complex from dissociating
back to E and S.
19. There are a great many equations in this chapter.
Do I need to know them all?
1. No. They are provided to show how our
understanding of this field develops from basic
concepts. You should, however, try to
understand the derivations of the many
solutions presented.
73
E+S
kf
kr
ES
Vm [S]
v = K + [S]
m
74
S ECTION 3
9.
Formative self-evaluation
questions
2.
What is Kd?
3.
4.
5.
6.
7.
8.
76
S ECTION 4
Key to formative
evaluations
B max [S]
[S] b = K + [S]
d
2. Kd is that [S] at which [S]b is Bmax/2. It is also
known as the dissociation constant of the ES
complex.
3. Kd obtained from Michaelis-Menten and from
pseudo-first-order analsis of binding kinetics
should be identical. The advantage of the latter
analysis is that you also obtain kf and kr. The
advantage of the former analysis is that you do not
have to perform complex time-course studies.
1. Kd = -1/x-intercept
2. Bmax = 1/y-intercept
2. Hanes-Wolf ([S]/[S]b vs [S]) gives
1. Kd = -x-intercept
2. Bmax = 1/slope
3. Scatchard ([S]b/[S] vs [S]b) gives
1. Kd = -1/slope
2. Bmax = x-intercept
6. Curve-fitting to the Michaelis-Menten equation by
direct non-linear regression analysis is the
77
1. Km = -x-intercept
2. Vm = 1/slope
Vm [S]
v = K + [S]
m
78
[I]
Vm
V m (app) - 1
79
80