Beruflich Dokumente
Kultur Dokumente
Kinetics in
Biology
Important resources at:
http://glutxi.umassmed.edu/index.html
http://glutxi.umassmed.edu/grad.html
Goals
Chemical kinetics
1.! Understanding reaction order and rate constants
2.! Analysis of reaction rates
Steady-state Enzyme kinetics
3.! How to model steady-state kinetics
a.! The King-Altman method
b.! The method of Cha
How do we analyze time course data and then what do we do with it?
dc
v=
dt
but it 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.
AND
WILLIAM S. AGNEW
Proc. Natl. Acad. Sci. USA Vol. 81, pp. 5594-5598, 1984
2)
These concentrations do not change during a single run but may be changed from
one experiment to the next. The concentrations of these components frequently
do influence the rates of reactions.
Characteristics of a Reaction
Lets consider 3 aspects of a reaction:
the stoichiometry
the mechanism
the order
The stoichiometry of the reaction tells us how many moles of each reactant are needed
to form each mole of product:
H2 + 0.5 O2 = H2O
or
The order of the reaction describes how the velocity of the reaction depends upon
the concentration of reactants.
Consider the following reaction:
A+BP
dP
v=
= kC AmC Bn
dt
where the concentrations CA, CB are raised to powers m and n that are usually
integers or zero (C0A = constant). The order of the reaction with respect to a
particular component (A or B) is just the exponent of the concentration term.
For example, if the reaction is 3A + 2B P
v=
dP
= kC A3 C B2
dt
Because the velocity of the reaction may depend on the concentrations of several
species, we must distinguish between order with respect to a particular component
and the overall order which is the sum of exponents of all components.
10
Zero-order kinetics
First order kinetics
True Second order kinetics
Second order kinetics characterized by pseudo-first order behavior.
In order to analyze time course data, you need a good software tool.
11
Step 1
GraphPad Prism version 6 download link(s) and license information for
2014-2015 are:
Prism 6 Windows
http://cdn.graphpad.com/downloads/prism/6/InstallPrism6.exe
Prism 6 Windows serial number: GPW6-200512-LEM5-16772
Prism 6 Mac OSX
http://cdn.graphpad.com/downloads/prism/6/InstallPrism6.dmg
Prism 6 Mac serial number: GPM6-200513-LEM5-F3EF2
12
Step 2
When you launch Prism you will asked for your email address and will subsequently receive
an email stating:
Thank you for registering GraphPad Prism. To activate Prism on your computer,
copy the code below and paste it into the Prism registration wizard:
XXXXX-XXXXXXXX-XXXXXXXX-XXXXXXXX-XXXXXXXX (the email will contain the
actual code)
This code will activate serial number (the email will insert the Windows or Mac
serial # here) to run on the computer identified by this machine ID:
YYYYYYYYYYY (the email will identify your computers true ID).
If you have any problems registering Prism, please contact GraphPad technical
support at support@graphpad.com
13
Support
GraphPad Prism is very easy to learn and to use but extensive
support is available through:
http://www.graphpad.com/scientific-software/prism/
14
A zero-order reaction.
Zero-order reaction
Substrate
Product
zero-order kinetics
100
80
v (d[P]/dt)
[Substrate] or [Product]
10
60
40
20
0
2
0
0
100
200
[S] M
10
TIME
Note that [substrate] decreases linearly with time and [product] increases linearly with time. This
observation suggests that we should perform Linear Regression analysis of the data to obtain
constants (slopes) for substrate loss and product formation.
16
dC
dt = k 0
The units of k0 are molarity per sec. This is a zero-order 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
or
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 (yintercept) and k0 (the slope).
16
Zero-order reaction
[Substrate] or [Product]
10
Substrate
Product
8
6
4
2
0
0
10
TIME
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
17
18
18
19
A first-order reaction.
1stOrder
5
4
100
[Substrate]
[Product]
v (d[P]/dt)
[A] or [B]
80
60
40
20
first-order kinetics
0
1
0
0
100
200
[S] M
10
TIME
Note here that [substrate] decreases in a curvilinear fashion with time and [product]
increases in a curvilinear manner with time. This observation indicates that the
reaction is NOT zero-order. How can we analyze this further?
19
20
dC
dt = k 1 C
The units of k1 are time-1 (e.g. s-1). There are no concentration units in k1 so we do not
need to know absolute concentrations - only relative concentrations are needed.
The reaction:
k1
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).
20
-d[A] = k1 [A]0 dt
21
[A] = [A] 0 e -k t
1
ln [A] =- k 1 t + ln [A] 0
slope x + intercept
ln2 0.693
t 1/2 = k = k
1
1
and because = 1/k1,
t1/2 = 0.693
21
22
[B] = [B] {1 - e -k t}
1
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
22
23
[A] or [B]
4
3
[Substrate]
[Product]
2
1
0
0
10
TIME
The data suggest that [substrate] falls from 5 mM to an equilibrium value of 0 mM.
If we plot the log [substrate] vs time (or show the y-axis data on a log scale),
we obtain
1stOrder
10
[Substrate]
[A]
0.1
0.01
0
10
TIME
This produces a linear plot which is consistent with 1st order kinetics!
23
24
1st Order
5
[Substrate]
4
3
2
1.4 sec
1
0
0
1.4 sec
1.4 sec
10
TIME
A second clue comes from the measurement of half-times. 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.
This is characteristic of first-order decay as observed with radioactive decay.
Constant decay times and the linear relationship between log {[S]t - [S]} vs time indicate a first
order process. Let us check this by applying a first-order analysis to the data.
24
25
[A] = [A] 0 e -k t
1
Nonlinear regression finds the values of those parameters of the equation (k1
and [A]0) that generate a curve that comes closest to the data. The result is the
best possible estimate of the values of those parameters.
To use nonlinear regression, therefore, you must choose a model or enter one.
GraphPad Prism oers a model for first-order reactions called One-Phase
Decay
The equation is:
2.
3.
4.
25
26
27
1stOrder
[Substrate] or [Product]
5
4
[Substrate]
[Product]
3
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
27
28
General rules for 1st order reactions
1. First-order enzyme catalyzed kinetics are typically observed at subsaturating [S]
k1
k2
k1
AB
k1 = 0.5 s-1
[A] or [B]
1.0
0.5
0.0
0
A
B
One-phase association
Best-fit values
Y0
Plateau
K
Tau
Half-time
Span
1.000
4.699e-010
0.5000
2.000
1.386
-1.000
-5.029e-009
1.000
0.5000
2.000
1.386
1.000
10
time
29
k1
A?B
k-1
[B] eq
k
K eq = k 1 =
-1
[A] neq
k obs = k 1 + k -1
[A] or [B]
1.0
One-phase association
Best-fit values
Y0
Plateau
K
Tau
Half-time
Span
Perfect fit
1.000
0.5000
1.000
1.000
0.6931
-0.5000
-1.618e-009
0.5000
1.000
1.000
0.6931
0.5000
0.5
A
B
0.0
10
Time sec
30
http://www.berkeleymadonna.com/jmadonna/
jmadrelease.html#!
Berkeley Madonna is an extremely fast, general purpose differential equation solver. It
runs on both Windows and Mac OS. Developed on the Berkeley campus under the
sponsorship of NSF and NIH, it is currently used by academic and commercial
institutions for constructing mathematical models for research and teaching
http://www.berkeleymadonna.com/features.html
31
Conclusion
1. Irreversible first-order reactions have explicit
solutions
2. Reversible first-order reactions have explicit
solutions but may also be solved
numerically.
32
Class 1 (A+A P)
v=k2[A]2
Although one or more reactants may be involved, the rate law for many reactions depends only
on the second power of a single component. e.g.
[Proflavin]2
2 proflavin
]2
AAGCUU
2 AAGCUU
UUCGAA
2[
2
]
2
33
[A] or [B]
[Substrate]
[Product]
3
2
1
0
0
10
TIME
[substrate] decreases in a curvilinear fashion with time and [product] increases in a curvilinear
manner with time. This observation indicates that the reaction is NOT zero-order. How can
we analyze this further?
The curves drawn through the points were made by nonlinear regression assuming first order
kinetics (one-phase decay equation). Note the systematic deviations from the fit. This strongly
suggests that this reaction does not follow first order kinetics.
We can investigate this further by plotting the residuals of the fit (how each point deviates
from the calculated fit) vs time.
34
[Substrate]
[Product]
0.2
0.1
0.0
-0.1
-0.2
5
TIME
10
This confirms the poor fit and that we should consider either an error in data sampling or
another model for the data.
35
1
1
= k2t
[ A] [ A]0
1
1
= k2t +
[ A]
[ A]0
[ A]0
= [ A]0 k2t + 1
[ A]
Thus one expects a linear relation between the reciprocal of the reactant concentration and time.
[A]0/[A]
6
slope==[A]
[A]00kk22
slope
0
0
10
TIME
[A]0/[A] versus time for normalized 1st and 2nd order kinetics with identical t
36
half-time vs [A]
1
2
3
4
5
6
7
8
9
10
[A]0/[A]
Increasing
[A]0
5
2
0
0
0
0
10
1.5
1.0
10
TIME
[A]
t1/2 sec
10
0.5
0.0
0
10
[A]o
37
38
A+A
k1
k -1
1.0
A
0.5
1/A
[A] or [B]
15
time
1.000 1.028e-008
1.000 5.950e-008
-1.000
1.000
A
B
0.0
0
10
Best-fit values
Slope
Y-intercept when X=0.0
X-intercept when Y=0.0
1/slope
10
0
0
10
time
39
A+A
k1
k -1
One-phase association
Best-fit values
Y0
Plateau
K
Tau
Half-time
Span
0.9896
0.5007
1.740
0.5747
0.3984
-0.4888
0.005212
0.2496
1.740
0.5747
0.3984
0.2444
200
150
1/(At-0.5)
[A] or [B]
1.0
0.5
A
B
100
50
0
10
time
0.0
10
time
[B] eq
k
K eq = k 1 =
-1
[A] neq
40
Conclusion
1. Irreversible second-order reactions have
explicit solutions
2. Reversible second-order reactions do not
have useful explicit solutions and must be
solved numerically.
41
k1
E+S
ES
k2
If the enzyme E were maintained at a constant low [] (e.g. [E] < [S]/100) and the substrate
were varied, the reaction could be written as:
v= [E]k1[S]
R+ L
kf
kr
LR
Upon rapid mixing of R and L, the receptor may undergo a fluorescence change allowing
measurement of ligand binding. Alternatively, it may be possible to measure ligand binding by
use of radiolabeled Ligand and filter-bound receptor. Either way, the time course of ligand binding
may be examined to determine whether it displays first or second order kinetics.
42
At zero-time, various concentrations of L (M) were mixed with 1 nM R. The time course of
LR formation was monitored at each [L].
[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
10
TIME
The data were fitted with the one-phase decay equation and the fit is excellent in each
case (the residuals < [LR]/100)
You can also see that the reaction becomes faster at higher [L] - k increases and t1/2
falls with increasing [L].
43
1.
2.
25
20
15
Best-fit values
Slope
Y-intercept when X=0.0
X-intercept when Y=0.0
1/slope
1.999 0.0001861
0.5012 0.0007352
-0.2507
0.5002
10
5
0
0
10
[L] M
The slope is kf
The y-intercept is kr
The x-intercept is -kr/kf
44
1.
2.
3.
In a plot of kobs versus [L], kobs increases linearly with [L] (slope = kf) and
the y-intercept = kr.
4.
5.
6.
This is ONLY true when [L] >> [R]. Under these conditions first-order
kinetics are observed ([L] does not change significantly). If [L] [R] the
system will behave like a class 1 second order reaction.
45
kobs for a class 1 2nd order reaction is k[S]0 and when [S]0 is 0, kobs =
0
kobs for a class 2 2nd order reaction is kf[S]0 + kr and when [S]0 is 0,
kobs = kr.
46
Parallel Reactions
A
k2
d[A]
= k1 [A]+ k2 [A] = (k1 + k2 )[A]
dt
d[B]
= k1 [A]
dt
d[C]
= k2 [A]
dt
The solution to the first eqn has the form of a first-order rate law
[A]
= (k1 + k2 )t
ln
[A]0
[A] = [A]0 e(k1 +k 2 )t
47
To find out how [B] and [C] change with time, we substitute for [A] from the last eqn.
d[B]
= k1 [A] = k1[A]0 e( k1 +k2 )t
dt
d[C]
= k2 [A] = k2 [A]0 e( k1 +k2 )t
dt
Separating variables and integrating and assuming [B] = [C] = 0
o
o
k1[A]0
(1 e(k1 +k 2 )t )
k1
+ k2
k [A]
[C] = 2 0 (1 e(k1 +k 2 )t )
k1 + k2
[B] =
Thus in parallel reactions, if one step is much faster than the others, it dominates the
reactions.
Time
0
1
2
3
4.62
5
7
9
11
13
15
20
12
0.1
0.05
10
A
10.00
8.61
7.41
6.38
5.00
4.72
3.50
2.59
1.92
1.42
1.05
0.50
10
B
0.00
0.93
1.73
2.42
3.33
3.52
4.33
4.94
5.39
5.72
5.96
6.33
C
0.00
0.46
0.86
1.21
1.67
1.76
2.17
2.47
2.69
2.86
2.98
3.17
k1
k2
[A]o
A
B
C
t1/2
=
4.62
s
thus
k=0.693/
4.62
=
0.15
s-1
6
4
2
0
0
10
time
15
20
25
48
k1=0.1
k-1=0
k2=0.05
k-2=0
Introduce reversibility
k1=0.1
k-1=0.1
k2=0.05
k-2=0
k1=0.1
k-1=0
k2=0.05
k-2=0.05
49
Conclusion
1. Irreversible parallel reactions have explicit
solutions
2. Reversible parallel reactions do not have
useful explicit solutions and must be solved
numerically
50
A
These are difficult to solve for:
k1
k2
d[ A]
v1 =
= k1[ A]
dt
k t
[ A] = [ A]0 e 1
d[B]
= k1[ A] k2 [B]
dt
= k1[ A]0 e
k1t
k2 [B]
[C] = [ A]0 [1
Ao
k1
k2
10
5
0.05
t
0
0.1
0.25
0.5
1
2
3
4
5
7
9
13
15
20
25
A
10
6.07
2.87
0.82
0.07
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
B
0
3.92
7.08
9.02
9.54
9.14
8.69
8.27
7.87
7.12
6.44
5.27
4.77
3.72
2.89
C
0
0.01
0.05
0.16
0.39
0.86
1.31
1.73
2.13
2.88
3.56
4.73
5.23
6.28
7.11
A
B
C
k2
12
10
k1
6
4
2
0
0
10
15
20
25
-2
time min
52
k1=5
k-1=0
k2=0.05
k-2=0
Introduce reversibility
k1=5
k-1=2
k2=0.05
k-2=0
k1=5
k-1=0
k2=0.05
k-2=0.05
53
Ao
k1
k2
10
0.05
0.2
t
0
1
2
3
4
5
6.93
9
13
15
20
25
A
10
9.51
9.05
8.61
8.19
7.79
7.07
6.38
5.22
4.72
3.68
2.87
B
0
0.44
0.78
1.04
1.23
1.37
1.52
1.57
1.49
1.41
1.17
0.93
C
0
0.05
0.17
0.35
0.58
0.84
1.40
2.05
3.29
3.87
5.16
6.20
[A],
[B]
or
[C]
k2
12
A
B
C
10
k1
8
6
4
2
0
0
10
15
20
25
time
min
54
k1=0.05
k-1=0
k2=0.2
k-2=0
Introduce reversibility
k1=.05
k-1=.05
k2=0.2
k-2=0
k1=.05
k-1=0
k2=0.2
k-2=0.2
55
Conclusion
1. Irreversible sequential reactions have explicit
solutions
2. Reversible sequential reactions do not have
useful explicit solutions and must be solved
numerically
56
All reactions approach equilibrium. For every forward step there is a reverse reaction. In
practice we sometimes ignore the reverse step because the concentrations of products are kept
very small. However, you have seen how the reverse reaction influences time courses.
Furthermore, it is important to know the relationship between kinetic rate constants (k) and the
thermodynamic equilibrium constant (K).
For the elementary first order reaction
k1
A
The rate of disappearance of A is
k-1
d[ A]
= k1[ A] k1[B]
dt
k1
[B]eq
=
=K
eq
k1
[ A]
Often, there is more than one path for the reaction of A to form B. To be consistent with the
principles of equilibrium thermodynamics, we MUST apply the principles of microscopic
reversibility. This states that if A can react to form B by 2 or more paths, we cannot have a
mechanism by which AB only by one path and BA by another.
is not possible.
C
57
Each step in the reaction must be reversible. Thus the mechanism is:
k1
A
k-3
k-1
k3
B
k2
k-2
k1
k 2 k3
[B]eq
K=
=
=
eq
k1 k2 k3
[ A]
thus
k1k2 k3 = k1k2 k3
(note the product of rate constants in one direction = the product of all rate constants in
the opposite direction). Thus the 6 rate constants are not independent.
58
Complex reactions
k1
A+B
k-1
k2
k-2
P+Q
v2 =X k2
The exact solution to the rate equations in this case is very complex. Because the
elementary reactions are bimolecular and 5 molecular species are involved, it is useful to
learn some approximations that may be applied.
Prior- or Rapid-equilibrium approximation
Here, we assume steps k1 and k-1 are rapid relative to k2. A, B and X rapidly attain a
state of quasi-equilibrium, such that v1 = v-1
k1[A][B] = k-1[X]
Thus we obtain the equilibrium expression
[X]
k
K = k 1 = [A] [B]
-1
59
[X]
K = k1 =
k-1
[A] [B]
thus
[X] =
k1 [A] [B]
k-1
Step 2 is the rate-limiting step and the rate of product formation is given by
d [Q]
d [P]
=
= k2 [X]
v=
dt
dt
substituting from above we see that:
v = k2 k1 [A] [B]
k-1
Thus v can be expressed in terms of reactant concentrations only. The criterion for the prior
equilibration approximation is
v v <<v
v
2
1
-1
which may be read as:
The overall velocity of the reaction is limited by the slow step 2 and the velocity is much slower
than the forward and reverse reactions of step 1 which are essentially in equilibrium
60
Steady-state approximation
Sometimes an intermediate is formed that is highly reactive. Thus it never truly builds up to
an appreciable level.
k1
A +B
(slow)
(fast)
k2
X+D
d[P]
v=
= k1[A][B]
dt
Another approach is to consider all steps involving the formation and disappearance of the
reactive intermediate and to set the sum of their rates to zero. e.g.
d[ X]
k2 [X][D] 0
= k1 [A][B]
dt
The significance of this is not that [X] is constant throughout the reaction. Such is never the
case. However, it is true that the slope d[X]/dt of the curve of [X] versus time is much
smaller than that of the other reactants or products.
61
k1
E+S
k3
ES
E+P
k2
1)
Formation of ES
2)
62
E+S
k1
k2
ES
k3
k4
E+P
S
ES
k1
k2
INIT E
INIT ES
INIT S
k3
k4
INIT P
10
1
0.01
0
1
1
2
0
E
P
63
! !
v = k3 [ES]!
(1
64
i.e.
k1 [E] [S] = k2[ES] (5
hence
[E][S]k1
[ES] =
k2
[E][S]
[ES] =
k2
k1
(6
(7
65
v
k 3 [ES]
=
[E t ] [E] + [ES]
(9
66
v
[ES]
=
[E t ]k 3 [E] + [ES]
(10
k1
k1
[E][S]
[S]
v
k2
k2
=
=
k
[Et ]k3 [E] + k1 [E][S]
1+ 1 [S]
k2
k2
[S]
(11
k2
+ [S]
k1
67
Define ! Vm as:! !
or
[Et]k3 = Vm!
(12
kk2 + k
KKm == 2 = K3S
m
k1 k 1
(13
[S]
v
Km
=
Vm 1+ [S]
Km
(14
v=
Vm [S]
K m + [S]
(15
68
Steady-state assumption
When the concentration of intermediates in a reaction are in a steadystate, this means that the rate of their formation and breakdown are
identical.
69