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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?

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.

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.

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.

I. Lutz, A. Sieg, A. A. Wegener, M. Engelhard, I. Boche, M. Otsuka, D.

Oesterhelt, J. Wachtveitl, and W. Zinthi

Reactions on the

Microsecond Time Scale

M. C. Ramachandra Shastry,* Stanley D. Luck,* and Heinrich Roder*

channel isolated from the electroplax of Electrophorus electricus

ROBERT L. ROSENBERG, SALLY A. TOMIKO,

AND

WILLIAM S. AGNEW

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

Anthony Carruthers

The Journal of Biological Chemistry

vol. 261, pp. 11028-11037,1986

plasma iron with deferoxamine therapy

JB Porter,, RD Abeysinghe, L Marshall, RC Hider and S Singh

Richard J. Cenedella and Charles R. Fleschner

Investigative Ophthalmology & Visual Science, Vol. 31 p 1957, (1990)

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.

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)

1)

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)

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

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

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

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

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/#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.

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

15

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

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

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

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

Theory

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

21

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

1

integrating between A at time 0 and time t gives

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

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

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

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:

1.

2.

3.

4.

25

26

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

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]

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

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

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

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

[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

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

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

1

1

= k2t

[ A] [ A]0

1

1

= k2t +

[ A]

[ A]0

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.

of data

[A]0/[A]

6

slope==[A]

[A]00kk22

slope

2nd order data

0

0

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

10

TIME

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

36

half-time vs [A]

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]

t1/2 sec

10

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

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

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

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

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

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.

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

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

1.

2.

3.

The slope is kf

The y-intercept is kr

The x-intercept is -kr/kf

44

For our reaction

1.

2.

3.

In a plot of kobs versus [L], kobs increases linearly with [L] (slope = kf) and

the y-intercept = kr.

4.

5.

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

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

is independent of [substrate] or [product].

pseudo-first-order reaction. Its rate constant, kobs, increases linearly with

[S] (i.e. kobs = kr+kf[S]).

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

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

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

k1

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

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

declines. Changes

in all 3 species

governed by

separate rate

constants

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

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]

k t

[B] =

{e e 2 }

k2 k1

d[C]

v2 =

= k2 [B]

dt

Integrating - assuming that [C]0 = 0

[C] = [ A]0 [1

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

k2

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

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

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.

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

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

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

reversible, it remains the ratedetermining step and the

differences between this condition

and irreversibility in the 1st step

are subtle.

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

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

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

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

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

assumption)

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

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

64

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

[Et] = [E] + [ES] (8

where Et is total enzyme

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

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.

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.

rate constants permits definition of the intermediates in terms of

[reactant], [product] and rate constants.

69

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