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Chemical

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?

Chemical Kinetics in Biology


What is Chemical Kinetics?
The study of reaction rates.
Why do we study Chemical Kinetics?
This method, in combination with steady-state
kinetic analysis reveals fundamental reaction
pathways.
What is Relationship between Chemical Kinetics and
thermodynamics?
Thermodynamics tells us whether a reaction can
proceed spontaneously but does not inform us about
the rate at which the reaction will proceed. This
information has to be obtained experimentally.

Chemical Kinetics in Biology - studying rates of Biological reactions


The methods of reaction rate analysis were developed for studying relatively
simple systems encountered by chemists. These approaches are also valuable
in analyzing more complex biological processes because, oftentimes, one or a
few steps control the rate of an extensive chain of reactions.

All steps involved in metabolism, replication, cell division, muscle contraction


etc. are subject to the same basic principles as the elementary reactions of
the chemist.
The rate or velocity, v, of a reaction or process describes how fast it occurs.
Usually, the velocity is expressed as a change in concentration per unit time,

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.

Primary reactions of sensory rhodopsins


I. Lutz, A. Sieg, A. A. Wegener, M. Engelhard, I. Boche, M. Otsuka, D.
Oesterhelt, J. Wachtveitl, and W. Zinthi

A Continuous-Flow Capillary Mixing Method to Monitor


Reactions on the
Microsecond Time Scale
M. C. Ramachandra Shastry,* Stanley D. Luck,* and Heinrich Roder*

Single-channel properties of the reconstituted voltage-regulated Na


channel isolated from the electroplax of Electrophorus electricus
ROBERT L. ROSENBERG, SALLY A. TOMIKO,

AND

WILLIAM S. AGNEW

962967 PNAS January 30, 2001 vol. 98 no. 3

Biophysical Journal Volume 74 May 1998 27142721

Proc. Natl. Acad. Sci. USA Vol. 81, pp. 5594-5598, 1984

ATP Regulation of the Human Red Cell Sugar Transporter


Anthony Carruthers
The Journal of Biological Chemistry
vol. 261, pp. 11028-11037,1986

Kinetics of removal and reappearance of non-transferrin-bound


plasma iron with deferoxamine therapy
JB Porter,, RD Abeysinghe, L Marshall, RC Hider and S Singh

Blood, Vol 88, No 2 (July 15), 1996: pp 705-713

Kinetics of Corneal Epithelium Turnover In Vivo


Richard J. Cenedella and Charles R. Fleschner
Investigative Ophthalmology & Visual Science, Vol. 31 p 1957, (1990)

Chemical kinetics is the study of the rates of reactions.


Some reactions (e.g. 2H2 + O2 2H2O) proceed so slowly as to be
unmeasurable.
Radioisotopes of some nuclei have very long lifetimes (e.g. for
238
U = 2.3 x 1017 s (4.47 billion years)).
Other reactions, such as the growth of bacterial cells, are slow (
= 1 x 104 s) but measurable.
The biological reactions depicted on the last 2 slides range from
picoseconds (10-12 s) to days (105 s).
Clearly, the methods of observation must be very different to include
processes over such an enormous range of time.

Quick review of Rate Law


The rate of a process depends in some way on the concentrations or amounts involved.
The reaction rate is a function of concentration:
v= (concentrations)

Substances that influence v can be grouped into 2 categories:


1)

Those whose [] changes with time:


e.g.
A. reactants decrease with time
B. products increase with time
C. intermediates increase then decrease during a reaction
e.g. consider C in the following reaction
ACB

2)

Those whose [] do not change with time:


e.g.
A. catalysts (promoters/inhibitors) including enzymes and active surfaces
B. Intermediates in a steady-state process including reactions under flow
C. Components buffered by means of equilibrium with large reservoirs
D. Solvents and the environment in general

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

2H2 + O2= 2H2O

(both are correct stoichiometric expressions).


The mechanism of the reaction tells us how the molecules react to form products. For
the above reaction, the mechanism is thought to involve H, O and OH radicals:
H2 2H
H + O2 OH + O
OH + H2 H2O + H
O + H2 OH + H
each reaction is an elementary reaction; the 4 reactions describe the proposed
mechanism.
9

The order of the reaction describes how the velocity of the reaction depends upon
the concentration of reactants.
Consider the following reaction:

A+BP

For this reaction, the rate law is of the form:

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

The reaction rate may be given by:

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

Examples of reaction orders encountered in nature


The next several slides illustrate 4 types of reactions you may observe in the
research setting.

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

Download and install GraphPad Prism

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

Using GraphPad Prism

Support
GraphPad Prism is very easy to learn and to use but extensive
support is available through:

The built in help system


http://www.graphpad.com/scientific-software/prism/#learn
https://twitter.com/graphpad

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.

You can download this file at:


http://inside.umassmed.edu/Global/Kinetics.pzf.zip
15

Theory of Zero-order Reactions

16

A zero-order reaction corresponds to the dierential rate law

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

General rules for zero-order reactions


1. Plot of St or Pt vs time produces a straight line with slope = -k (for
St) or k (for Pt)
2. k has units of mols produced or consumed per unit time
3. Zero-order, enzyme catalyzed kinetics are typically observed at
saturating [S]

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

Theory of First-order Reactions


A first-order reaction corresponds to the dierential rate-law:

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

has the rate law:

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

First Order reactions - loss of substrate


Theory

-d[A] = k1 [A]0 dt

21

Integrated rate law

[A] = [A] 0 e -k t
1

Defining [A]o as [A] at [A] at zero-time and


integrating between A at time 0 and time t gives

ln [A] =- k 1 t + ln [A] 0

slope x + intercept

Defining [A] at t1/2 as [A]0/2

ln2 0.693
t 1/2 = k = k
1
1
and because = 1/k1,
t1/2 = 0.693

21

First Order reactions - product formation


Integrated rate law

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

Returning to our example of a first order reaction,


1stOrder
5

[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

Non-linear regression analysis


To do this we subject the data to nonlinear regression (the plot is nonlinear)
using an appropriate equation for first-order reactions.
The integrated rate law for first-order substrate loss is

[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:

Y=(Y0 - Plateau)*exp(-k*X) + Plateau

In which the parameters are defined as:


1.

Y0 is the Y value when X (time) is zero or [A]0 in this case.

2.

Plateau is the Y value at infinite time (0 for our data set).

3.

k is the rate constant k1 (per unit time).

4.

Span is the dierence between Y0 and Plateau

25

Every nonlinear regression method follows these steps:

26

1. Start with initial estimated values for each parameter in the


equation.
2. Generate the curve defined by the initial values. Calculate the
sum-of-squares - the sum of the squares of the vertical distances
of the points from the curve.
3. Adjust the parameters to make the curve come closer to the data
points - to reduce the sum-of-squares. There are several
algorithms for adjusting the parameters - Prism uses the
Marquardt algorithm.
4. Adjust the parameters again so that the curve comes even closer
to the points. Repeat.
5. Stop the calculations when the adjustments make virtually no
dierence in the sum-of-squares.
6. Report the best-fit results. The precise values you obtain will
depend in part on the initial values chosen in step 1 and the
stopping criteria of step 5. This means that repeat analyses of the
same data will not always give exactly the same results.
URL: http://www.graphpad.com/help/Prism5/Prism5Help.html?how_regression_works2.htm
26

27

1stOrder

[Substrate] or [Product]

5
4

[Substrate]
[Product]

3
2
1
0
0

10

TIME

Y=(Y0 - Plateau)*exp(-k*t) + Plateau


One phase decay!Perfect t!
Best-t values! !
Y0!!
!
!
!
Plateau! !
!
!
k ! !
!
!
!
Half Life!!
!
!
Tau = 1/k!
!
!
Goodness of Fit!
!
Degrees of Freedom!
R square!!
!
!

[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]

2. Plot of log (St-S) vs time produces a straight line with slope = -k


3. The half-time (t1/2) and k are invariant of the starting value of St chosen.
4. Plot of log (P-Pt) vs time produces a straight line with slope = -k
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

The equations are unchanged but now


k = k1 + k2
28

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

k1 = k-1 = 0.5 s-1

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

Check out its features at:

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

Second Order Reactions


Fall into 2 main categories depending on whether the rate law depends:
1) upon the second power of a single reactant species, or
2) the product of the concentrations of two different reagents.

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

2nd Order class 1

[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

Nonlin fit of 2ndOrderIrrev:Residuals


0.4
0.3

[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

Theory of Class 1 Second-order Reactions


Defining [A] at zero-time = [A]0, it can be shown that

1
1

= k2t
[ A] [ A]0

1
1
= k2t +
[ A]
[ A]0

multiply both sides


by [A]0

[ A]0
= [ A]0 k2t + 1
[ A]

Thus one expects a linear relation between the reciprocal of the reactant concentration and time.

Class 1, 2nd order Transform


of data

[A]0/[A]

6
slope==[A]
[A]00kk22
slope

1st order data


2nd order data

0
0

Linear regression analysis


Best-t values!
Slope! 0.66 0
Y-intercept when X=0.0! 1.0 0
X-intercept when Y=0.0! -1.515

Goodness of Fit!
R square!1.000

2nd order data

10

TIME

[A]0/[A] versus time for normalized 1st and 2nd order kinetics with identical t
36

half-time vs [A]

How starting [A] affects rate of 2nd order reaction


15

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]

[A]0 k per sec

t1/2 sec

10

A0k (slope) vs A0 second order


Best-fit values
Slope
Y-intercept when X=0.0
X-intercept when Y=0.0
1/slope

[A]0 k per sec


0.1320 2.842e-009
-3.974e-009 1.763e-008
3.010e-008
7.576

0.5

0.0
0

10

[A]o

37

General rules for 2nd order reactions (Class 1)

1. Standard 1st order analysis does not work


2. Plotting [A]0/[A] vs time produces a straight line with
slope [A]0 k
3. Plotting slope vs [A]0 produces a straight line with
slope k and y-intercept 0.
4. The half-time (t1/2) falls with increasing [A].
5. The units of k are concentration-1.time-1.
6. This analysis breaks down when the reaction is
reversible (i.e. when kr kf/10)

38

A+A

k1
k -1

k1 = 1 M-1. s-1; k-1 = 0

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

k1 = k-1 = 0.5 M-1. s-1

k1= 0.5 M-1.s-1; k-1 = 0.5 s-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

2nd order reactions Class 2 (pseudo-first order; A+BP)


A reaction that is 2nd order overall may be first order with respect to each of the two reactants.

For example, in the reaction

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]

Let us review this by examining ligand (L) binding to a receptor (R).

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].

Pseudo 1st Order

[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.

We obtain k or kobs from the one-phase decay equation fits.


If we then plot kobs versus [L], you can see that the plot
is linear with 2 constants - slope and y-intercept.
kobs vs L

25

kobs per sec

kobs per sec

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

We will show below that:


1.
2.
3.

The slope is kf
The y-intercept is kr
The x-intercept is -kr/kf
44

Theory for pseudo first-order reactions


For our reaction

The rate of LR formation is given by:

Defining [R]0 as the amount of receptor at t= 0, it can be shown that:

1.

The time dependent component of this expression is e-t(kr+kf[L]).

2.

Thus kobs = (kr+kf[L])

3.

In a plot of kobs versus [L], kobs increases linearly with [L] (slope = kf) and
the y-intercept = kr.

4.

The x-intercept (when kobs = 0) = -kr/kf

5.

Hence, analysis of the time course of L binding to R at varying [L] permits


computation of kf, kr and kf/kr = Keq for the reaction.

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

What is the difference between a first order reaction and a


second-order reaction that behaves like a first order reaction?

A true first-order reaction is characterized by a rate-constant, k, that


is independent of [substrate] or [product].

A second-order reaction that behaves like a first order reaction is called a


pseudo-first-order reaction. Its rate constant, kobs, increases linearly with
[S] (i.e. kobs = kr+kf[S]).

What is the difference between a class 1 second order reaction


and a class 2 second-order (pseudo-first-order) reaction?

A class 1 2nd order reaction is not described accurately by first


order equations but when 1/[S] is plotted vs time, the plot is linear.

kobs for a class 1 2nd order reaction is k[S]0 and when [S]0 is 0, kobs =
0

A class 2 2nd order reaction is described accurately by first order


equations.

kobs for a class 2 2nd order reaction is kf[S]0 + kr and when [S]0 is 0,
kobs = kr.
46

Parallel Reactions

Parallel reactions are of the type:


k1

A
k2

Two separate routes of A breakdown exist. The rate expressions are:

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

[A], [B] or [C]

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

All reactions are


limited by the
fastest rate constant

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

Now B peaks then


declines. Changes
in all 3 species
governed by
separate rate
constants

Now C peaks then


declines.

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

Series Reactions (first order)


Some reactions are of the type:

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]

Integrating - assuming that [B]0 = 0

k1[ A]0 k1t


k t
[B] =
{e e 2 }
k2 k1
d[C]
v2 =
= k2 [B]
dt
Integrating - assuming that [C]0 = 0

[C] = [ A]0 [1

k2 k1[ A]0 k1t


k t
=
{e e 2 }
k2 k1
1
k t
k t
{k2 e 1 k1e 2 }]
k2 k1
51

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

[A], [B] or [C]

k2

When k1 >> k2, the second


reaction is the rate-determining
step. A will be rapidly converted
to B and, during most of the
reaction, B undergoes a firstorder conversion to C. If the
appearance of C is our measure
of velocity, when k1 >> k2 its
appearance follows simple first
order kinetics.

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

When k1 >> k2, the second


reaction is the rate-determining
step. A is rapidly converted to B
and B undergoes a first-order
conversion to C. If the appearance
of C is our measure of velocity,
when k1 >> k2 its appearance
follows simple first order kinetics.

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

When the first reaction is made


reversible, the second reaction
remains the rate-determining step
but now the peak of intermediate
B is lower and A declines as a 2phase decay. The appearance of C
follows simple first order kinetics.

When the second reaction is made


reversible, the second reaction
remains the rate-determining step,
the peak of intermediate B is
unchanged. The appearance of C
follows simple first order kinetics,
is faster (kobs =k2+k-2) and B and
C achieve equilibrium (Keq=k2/k-2)

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

When k2 >> k1, the reaction


begins with the very slow
conversion of A to B followed by
a very rapid conversion of B to C.
In this case, [B] remains low
throughout and C appears as A
disappears. The time course of C
formation indicates a lag phase.

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

When k2 >> k1, the reaction


begins with the very slow
conversion of A to B followed by a
very rapid conversion of B to C. In
this case, [B] remains low
throughout and C appears as A
disappears. The time course of C
formation indicates a lag phase.

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

When the first reaction is made


reversible, it remains the ratedetermining step and the
differences between this condition
and irreversibility in the 1st step
are subtle.

When the second reaction is made


reversible, the first reaction
remains the rate-determining step,
B does not peak over this time
course, A declines as in the
original condition but C increases
more slowly.

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

Equilibrium and Kinetics

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

At equilibrium -d[A]/dt=0, therefore

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.

Thus the reaction

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

The relation to thermodynamics requires further that:

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

Enzyme mediated reactions involve a series of reversible steps. For example:

k1

A+B

k-1

k2

k-2

P+Q

v1 =A B k1; v-1 =X k-1

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

To a first approximation, X reacts as rapidly as it is formed.


v1 = v2
k1 [A][B] = k2 [X][D]
hence

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

Steady state approximation in chemical kinetics


This approximation consists of assuming that the concentration of a reactive
intermediate in a reaction mechanism is constant, i. e. its derivative is zero.
Its use helps us to solve the differential equations that arise from rate equations, which
lack an analytical solution for most mechanisms beyond the most simple ones. The
steady state approximation is applied, for example in Michaelis-Menten kinetics.

k1
E+S

k3
ES

E+P

k2

There are two parts to this reaction:

1)

Formation of ES

2)

ES breakdown to product P and free enzyme E

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

Defining the rate of product formation, v as


!

! !

v = k3 [ES]!

(1

If k3[ES] << k1 [E][S] k2[ES] (i.e. we make the rapid equilibrium


assumption)
+d[ES]/dt = k1 [E][S] (2

Rate of breakdown of [ES]


-d[ES]/dt = k2[ES] (3

64

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 (4
!

i.e.
k1 [E] [S] = k2[ES] (5

hence

[E][S]k1
[ES] =
k2

[E][S]
[ES] =
k2
k1

(6

(7

65

The following steps are algebraic tricks


[Et] = [E] + [ES] (8
where Et is total enzyme

if we divide the velocity equation (1 by Et we obtain

v
k 3 [ES]
=
[E t ] [E] + [ES]

(9

66

We can rearrange this to

v
[ES]
=
[E t ]k 3 [E] + [ES]

(10

then substitute for [ES] from equation (6 to give

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

The rate constants describing a reversible reaction are not independent.

The law of Microscopic Reversibility states that when the reaction is


drawn as a King Altman diagram, the product of rate constants for the
forward reaction is identical to the product of rate constants for the
reverse reaction.

When d[S]/dt or d[P]/dt are constant, the concentration of intermediates


in a reaction are said to be in a steady-state or an internal equilibrium.

When the concentration of intermediates in a reaction are in a steadystate, this means that the rate of their formation and breakdown are
identical.

Knowing this and the inter-relationship between forward and reverse


rate constants permits definition of the intermediates in terms of
[reactant], [product] and rate constants.

The next class will build on this.

69