CHM 8304

Physical Organic Chemistry

Catalysis

Outline: General principles of catalysis

•  see section 9.1 of A&D

–  principles of catalysis
–  differential bonding

Catalysis
1

CHM 8304

General principles

•  a catalyst accelerates a reaction without being consumed

•  the rate of catalysis is given by the turnover number

•  a reaction may alternatively be “promoted” (accelerated, rather than

catalysed) by an additive that is consumed

•  a heterogeneous catalyst is not dissolved in solution; catalysis typically

takes place on its surface

•  a homogeneous catalyst is dissolved in solution, where catalysis takes

place

•  all catalysis is due to a decrease in the activation barrier, ΔG‡

Catalysts

•  efficient at low concentrations

–  e.g. [Enz]cell << 10-5 M; [Substrates]cell < 10-4 - 10-5 M

•  not consumed during the reaction

–  e.g. each enzyme molecule can catalyse the transformation of 20 - 36 x 106
molecules of substrate per minute

•  do not affect the equilibrium of reversible chemical reactions

–  only accelerate the rate of approach to equilibrium end point

•  most chemical catalysts operate in extreme reaction conditions while

enzymes generally operate under mild conditions (10° - 50 °C, neutral
pH)

•  enzymes are specific to a reaction and to substrates; chemical catalysts

are far less selective

Catalysis
2

CHM 8304

Catalysis and free energy

•  catalysis accelerates a reaction by stabilising a TS relative to the ground

state
–  free energy of activation, ΔG‡, decreases
–  rate constant, k, increases

•  catalysis does not affect the end point of an equilibrium, but only
accelerates how quickly equilibrium is attained
–  free energy of the reaction, ΔG°, remains unchanged
–  equilibrium constant, Keq, remains unchanged

Energy profile of catalysis

uncatalysed
Free energy

ΔG‡uncat

catalysed ΔG‡cat

A P • cat ΔGºrxn
A • cat
P

Reaction coordinate

unchanged

Catalysis
3

CHM 8304

Transition state binding

•  interaction between a catalyst and reactant or activated complex can

stabilise one or the other

•  if the activated complex is bound more strongly than the substrate, the
activation barrier will be decreased

•  HOWEVER, the activated complex is not a molecule – so the catalysts

must first of all interact with the substrate, and then release the product
at the end of the reaction :

A + cat A•cat P•cat P + cat

Differential binding
•  consider 4 scenarios :

ΔG‡non-cat ΔG‡non-cat
Energy

Energy

ΔG‡cat ΔG‡cat

binding
binding

Rxn. coord. Rxn. coord.

binding
ΔG‡non-cat ΔG‡non-cat
binding
Energy

Energy

ΔG‡cat
ΔG‡cat

binding binding

8
Rxn. coord. Rxn. coord.

Catalysis
4

CHM 8304

Differential binding
•  to accelerate a reaction, a catalyst must stabilise the TS more than it
stabilises the substrate
–  even if this stabilisation takes place over less time than that of a bond
vibration, by definition

+ +
transition
state
substrate
product

Outline: Types of catalysis

•  see section 9.2 of A&D

–  approximation
–  electrostatic
–  covalent
–  strain and distortion

10

Catalysis
5

CHM 8304

Catalysis by approximation

•  the catalyst brings together the reactants, increasing their effective

concentrations, and orients them with respect to the reactive groups

Jencks :
•  the loss of entropy associated with the restriction of rotation and
translation of substrate must be compensated by the intrinsic energy of
binding (favourable non-bonding interactions)

Bruice / Kirby :
•  the magnitude of this effect is given by the effective concentration,
determined by comparison if the rate constants of the bimolecular and
intramolecular reactions

11

Intramolecular approximation
•  an intramolecular reaction implies a smaller decrease in entropy (and
therefore a decrease in the free energy of activation)

intramolecular
ΔG ΔG = ΔH - TΔS
Free A B
Énergie
energy
libre note trade-off
between enthalpy
intermolecular (in bond formation)
A +B
and entropy
réactifs
reactants

Coordonnée
Reaction de réaction
co-ordinate
12

Catalysis
6

CHM 8304

Example of catalysis by approximation

•  the catalyst brings together the reactants, increasing their effective
concentrations, and orients them with respect to the reactive groups
O
CH3
obs -1 -1 O
O2N O k 2 = 4,3 M s
+ O2N O- + CH3
(H3C)3+N
(H3C)2NCH3

O
CH2
obs -1 O
O2N O k 1 = 21 500 s
CH2 O2N O- + CH2
(H3C)2+N
(H3C)2NCH2

obs
k1
obs
= 5000 M = la concentration
effective effective
concentration,

k2 or effective molarity (EM)

13

Example: Ester hydrolysis

Constante de Effective
Concentration
concentration
Reaction
Réaction
kvitesse relative
rel(k )
rel
effective
(M) (M)
O

parent OAr
1 M-1s-1 -
reaction
+

CH3CO2-
O

OAr
220 s-1 220 M
O-

O
O
decreasing
entropy of
OAr
rotation and 5.1 × 104 s-1 5.1 × 104 M
O-
translation
O
O

OAr
2.3 × 106 s-1 2.3 × 106 M
O-

O O

O-
OAr 1.2 × 107 s-1 1.2 × 107 M

O
14

Catalysis
7

CHM 8304

More notions of catalysis by approximation

•  many notions have been advanced by many different researchers, to

describe the subtleties of catalysis by approximation:
–  orbital steering: the alignment of orbitals is proposed to accelerate the
reaction
–  stereopopulation control: one reactive conformer among several is favoured
–  near attack conformations: conformations are favoured whose spatial
orientation lead to the desired reaction

CAUTION: one must not forget the

Curtin-Hammett principle!!

15

Electrostatic catalysis

•  stabilisation of charge developed at TS

•  for example, serine and cysteine proteases favour the formation of a
tetrahedral intermediate by stabilising the negative charge developed
on oxygen, in an oxyanion hole
–  e.g.: consider papain, a Cys protease
oxyanion
hole Cys25
O S

N
H
R

16

Catalysis
8

CHM 8304

Electrostatic catalysis
•  e.g.: oxyanion hole of subtilisin:

J. Mol. Biol. 1991, 221, 309-325. 17

Electrostatic catalysis

•  can be very important :

–  consider the triple mutant of subtilisin where each residue of its
catalytic triad is replaced (S221A-H64A-D32A)
•  catalyses proteolysis 106-fold less than the native enzyme
•  BUT the reaction with the mutant is still 103-fold faster than the
uncatalysed reaction!!
•  an important part of catalysis is due to the electrostatic
environment

18

Catalysis
9

CHM 8304

Metal catalysis

•  electrostatic charges developed at the TS can also be stabilised by metal

ions

•  coordination of a ligand by a metal (as a Lewis acid) can also lead to

polarisation of a ligand
–  e.g. pKa of metal-bound H2O is 7.2, making it easier to deprotonate, therby
generating –OH as a nucleophile
•  for example, zinc-bound water in carbonic anhydrase, a highly efficient
metalloenzyme as well as certain enzyme models

19

Covalent catalysis

•  catalyst forms a covalent intermediate that reacts faster than uncatalysed

reaction :

A + B P

vs

A + B + C AC + B P + C

intermédiare
intermediate

more reactive

20

Catalysis
10

CHM 8304

Covalent catalysis

•  in order for catalysis to be efficient, the activation energy for formation of

the intermediate and for its subsequent reaction must both be lower than
that of the uncatalysed reaction :

ΔG‡uncat
Energy

ΔG‡cat

Rxn. coord.

21

Covalent catalysis
•  an example of non-enzymatic covalent catalysis and anchimeric
assistance :
–  mustard gas
H 2O

S S S
Cl Cl Cl Cl OH2

Cl

S + H+
Cl OH

•  enzymes use nucleophilic groups (e.g. Asp, Glu, Ser, Cys, Lys,
His, Arg) and cofactors to form covalent bonds (nucleophilic
catalysis)

22

Catalysis
11

CHM 8304

Nucleophilic catalysis

•  catalyst attacks substrate to form intermediate that is even more

susceptible to nucleophilic attack, by a second reactant
–  e.g. reaction of acid chlorides with alcohols, catalysed by addition of a
tertiary amine:

O R'OH O
R'
R Cl (lente) R O
R'OH ΔG‡uncat
(rapide)

Energy
NEt3 NEt3
O ΔG‡cat
Et
R N Et
Et

Rxn. coord.

23

Covalent catalysis

•  another important example is activation of a carbonyl by formation of an

imine
–  very common in organocatalysis
–  used by many enzymes
•  e.g.: acetoacetate decarboxylase :

Chemical reaction:
CO2 H+
O O O O
H 3C O H 3C

Enzymatic reaction:
RNH 2 RNH 2

-OH -OH

CO2
R H+
NH O R R
NH NH
H 3C O H 3C
24

Catalysis
12

CHM 8304

Strain and distortion

•  destabilisation of the ground state induced in the substrate or in the
catalyst (such as an enzyme)

Koshland :
–  induced complementarity hypothesis: the approach of substrate serves to
provoke a conformational change in the enzyme, to adopt a form that better
binds the substrate, but in a higher energy (strained) form and/or to better
orient reactive groups ("orbital steering")
–  the substrate can also be deformed to adopt a strained form

Jencks :
–  strain and distortion in the substrate are essential for the catalysis
–  TSs are stabilised, rather than E•S and E•P complexes (so as not to form
overly stable intermediates)
–  binding energy must therefore be used to destabilise the E•S and E•P
complexes

25

Strain and distortion

•  binding energy is used to destabilise the E•S and E•P
complexes

Without enzyme
With enzyme

‡

S
P
E+S
E+P

E•S
E•P

26

Catalysis
13

CHM 8304

Strain and distortion

•  binding energy is used to destabilise the E•S and E•P
complexes

Without enzyme
With enzyme

‡

S
P
E+S
E•S
E•P
E+P

27

Productive strain

•  in order for a reaction to be facilitated by strain, two conditions must be

met:

1.  the strain must be along the reaction pathway

–  strain “pushes” the reactants towards the TS

2.  the strain must be at least partly alleviated at the TS

–  if the strain were still present at the TS, it would not contribute
to catalysis

28

Catalysis
14

CHM 8304

Outline: Acid-base catalysis

•  see section 9.3 of A&D

–  specific acid/base catalysis
–  general acid/base catalysis
–  kinetic equivalence
–  Brønsted catalysis law
–  prediction of acid-base catalysis
–  energy surfaces of acid-base catalysis

29

Acid-base catalysis

•  the catalyst (namely an acid or a base) accelerates the reaction through

protonation or deprotonation

30

Catalysis
15

CHM 8304

Specific acid-base catalysis

•  catalysis by H+ or –OH, controlled only by pH, where a fast equilibrium

precedes the rls :
–  e.g.:
fast H H H
O O slow O O
H+
O O O O
O O
H H H H

H 2O

H
O O
+ H+ +
OH OH HO

31

Rate laws of specific acid-base catalysis

•  when a substrate must be protonated before its reaction in the rls, this
appears as a pH dependence in the rate law:
–  e.g.: v = k[R]×[H+]/Ka,RH

•  when a substrate must be deprotonated before its reaction in the rls, this
appears as a pH dependence in the rate law:
–  e.g.: v = k[RH]×Ka,RH /[H+]

32

Catalysis
16

CHM 8304

Specific acid-base catalysis plots

•  pH dependence :
log kobs

log kobs
slope = -1; slope = +1;
specific acid specific base
catalysis catalysis

pH pH

•  at constant pH, independence of [HA] or [B] :

log kobs

log kobs
slope = 0; pente = 0;
specific acid specific base
catalysis catalysis

[HA] [B]
33

General acid catalysis

•  catalysis by an acid or a base (not H+ nor –OH) where a proton is
transferred during the rls
–  rate is proportional to the concentration of acid or base, at
constant pH
NO2 NO2
uncatalysed:
O O- -
O NO2
C C
CH3 O CH3 O
OH +
+ lente rapide CH3CO2H
O +
H H
H H H
O O+
H H

catalysed:
O
NO2
O-
NO2
-
O NO2
C C
CH3 O lente CH3 O rapide
OH +
+ CH3CO2H
O +
H H

Base H +
Base 34

Catalysis
17

CHM 8304

Rate laws

•  if a substrate is protonated during the rls, this appears as a dependence

on [HA] in the rate law :
–  e.g.: v = k[R]×[HA] à = kobs[R] where kobs = k[HA]

•  if a substrate is deprotonated during the rls, this appears as a

dependence on [B] in the rate law:
–  e.g.: v = k[R]× [B] à = kobs[R] where kobs = k[B]

35

General acid-base catalysis plots

•  pH dependence :
log kobs

log kobs

slope = 0 à -1;
general acid slope = 1 à 0;
catalysis general acid
catalysis

pH pH

•  at constant pH, dependence on [HA] or [B]:

log kobs

log kobs

slope = 1; slope = -1;

general acid general base
catalysis catalysis

[HA] [B]
36

Catalysis
18

CHM 8304

General acid-base catalysis

•  can increase the nucleophilicity or the electrophilicity of a functional
group

ΔG‡uncat
Free energy

intermediate
ΔG‡cat
products
reactants

Reaction coordinate

37

General acid-base catalysis

•  very common in enzymes
–  e.g. chymotrypsin uses a catalytic triad whose His plays the roles of a
general base :

δ−
O
Asp R
O
102 N
C
H R'
O H
N
orients and N
δ+
H O
activates
the His
nucleophile
His 57
Ser 195
general base

38

Catalysis
19

CHM 8304

Kinetic equivalence
•  one cannot distinguish, kinetically, between :
1.  general acid catalysis;
v = kobs[R][HA] :
H A
A
O lente OH OH
+ HA
O O OH
H H H H

2.  specific acid catalysis followed by general base catalysis;

v = kobs[R][H+][A-] :
H
H OH
O rapide O lente
+ HA
OH
O
H H A

39

Kinetic equivalence
•  by analogy, one cannot distinguish between :
1.  general base catalysis and
2.  specific base catalysis followed by general acid catalysis

v = kobs[R][B] = kobs[R][OH-][HB+]

40

Catalysis
20

CHM 8304

Brønsted
•  Johannes Brønsted (1879-1947)
–  Danish physical chemist (Copenhagen)
–  studied protonic theory of acid-base reactions (as
did Lowry)
–  acid-base catalysis

41

Brønsted catalysis law

•  Brønsted noted that the rate constants for reactions catalysed by a
general acid (having a proton in flight in the rate limiting transition
state) are proportional to the acidity constants of the general acids :
k obs ∝ K aα log k obs = α ⋅ logK a = −α ⋅ pK a

and for general bases : k obs ∝ K bβ log k obs = β ⋅ logK b = −β ⋅ pK b

•  Brønsted plots (log k vs pK) have slopes between 0 and 1:

•  slope of 0 : no proton transfer in rds
•  slope of 1 : proton already transferred before rds
•  intermediate slope: proportional with charge developed at TS of rds

42

Catalysis
21

CHM 8304

Brønsted plot (acids)

Graphique de Brønsted
General acid
Catalyse par catalysis
acides généraux

-3.5

-4.0

-4.5
pente
slope = -α
log k2

-5.0

-5.5

-6.0

-6.5

5 6 7 8 9
pKpK
a de l'acide général
a of general acid

43

Brønsted plot (bases)

Graphique de Brønsted
Catalyse par bases générales
General base catalysis

-3.5

-4.0

-4.5
log k2

-5.0
pente = β
slope

-5.5

-6.0

-6.5

5 6 7 8 9
pKapK
dea l'acide conjugué
of conjugate aciddeoflageneral
base générale
base

44

Catalysis
22

CHM 8304

Example: gac ketal hydrolysis

•  Brønsted coefficient α = 0.78
–  significant (partial) proton transfer at TS of rds

H A

OEt O
HA
Ph
+ 2 EtOH
EtO Ph Ph Ph

slow
‡ significant
δ−
H A charge
OEt development
Ph OH
Ph Ph
EtO Ph
δ+ EtO
H

EtOH + A- H 2O
+

Ph Ph f ast

OEt
45

Example: gbc gem-diol dehydration

•  Brønsted coefficient β = 0.4
–  slight (partial) proton transfer at TS of rds

B
H O
O B
H
+ H 2O
O H H H
H slight
charge
δ+ ‡
slow B
H
development
O OH- + BH +
H +

Oδ− H H H
H
O

46

Catalysis
23

CHM 8304

Step-wise vs concerted mechanism

•  in general, a reaction will take place by a step-wise mechanism unless it
is forced to take place by a concerted mechanism

–  if the intermediates of a

Energy
reaction pathway are all
fairly stable, this pathway
will have the lowest
energy….
Rxn. coord.

–  but when an intermediate

becomes too unstable to
exist, it becomes a TS: Energy

Rxn. coord. 47

Prediction of general acid-base catalysis

•  catalysis by a general acid or base is a concerted mechanistic step
–  proton transfer AND heavy atom bond formation/cleavage, in one step
–  cf specific acid/base catalysis, which is step-wise
•  concerted catalysis becomes necessary when proton transfer to or from
the reactant is only possible at the TS, owing to changes in heavy atom
bonding
–  predictable, according to relative acidities
–  e.g.:
δ−
H A
O
H A
H H A H
O O relative H > >
O
δ+
O O
H H acidities:
O H
O H O H
H H H
Energy

Rxn. co-ord.
48

Catalysis
24

CHM 8304

Energy surfaces and catalysis

•  the order of the reaction steps of a mechanism can vary according to the
stability of the corresponding intermediates
–  e.g.: addition/elimination on carbonyl
–  Example #1: strong nucleophile, no acid catalysis:

rate-limiting
attack R nuc. attack. T.I.
O
fast
Nuc protonation
‡

Nuc + HA

protonation on O
+ OH
Energy

O
Nuc
+
good Nuc; +
HA
stable T.I.
A

>C=O+H P
Rxn. coord.

49

Energy surfaces and catalysis

•  the order of the reaction steps of a mechanism can vary according to the
stability of the corresponding intermediates
–  e.g.: addition/elimination on carbonyl
–  Example #2: weak nucleophile, “required” catalysis:
slow
difficult O protonation
R nuc. attack T.I.
attack Nuc
+ HA
Nuc
protonation on O

+ weak Nuc; OH ‡

unstable T.I.
Energy

O
Nuc
+
+
HA
A

>C=O+H P
Rxn. coord..

50

Catalysis
25

CHM 8304

Energy surfaces and catalysis

•  the order of the reaction steps of a mechanism can vary according to the
stability of the corresponding intermediates
–  e.g.: addition/elimination on carbonyl
–  Example #3: both nuc. attack AND protonation are difficult, and concerted

protonation
difficult at T.S.; gac R nuc. attack T.I.

attack δ−
δ−
H A
Nuc O

protonation on O
+ δ− OH
Nuc
Energy

O
Nuc
+ ‡

intermediate +
HA
so unstable A
it becomes T.S.
>C=O+H P
Rxn. coord.

51

Energy surfaces and catalysis

•  the order of the reaction steps of a mechanism can vary according to the
stability of the corresponding intermediates
–  e.g.: addition/elimination on carbonyl
–  Example #4: weak electrophile, protonation required, spec. acid catalysis:

rate-limiting
attack T.I.
R nuc. attack

Nuc
protonation on O

Nuc +A
+ OH
+
Energy

O H Nuc
+ O activated +
HA substrate
A ‡

fast
protonation
>C=O+H P
Rxn. coord.

52

Catalysis
26

CHM 8304

Outline: Enzyme catalysis

•  see section 9.4 of A&D

–  enzymes and non-bonding interactions (review)
–  Michaelis-Menten kinetics
–  significance of kinetic parameters
–  energy diagrams
–  enzyme catalysis and “supramolecular”
•  example: chymotrypsin and an enzyme model

53

Enzymes

•  proteins that play functional biological roles

•  responsible for the catalysis of nearly all chemical reactions that take
place in living organisms
–  acceleration of reactions by factors of 106 to 1017
•  biological catalysts that bind and catalyse the transformation of substrates
•  the three-dimensional structures of many enzymes have been solved
(through X-ray crystallography)

54

Catalysis
27

CHM 8304

Rappel: Structures des acides aminés

•  as proteins, enzymes are polymers of amino acids whose side chains
interact with bound ligands (substrates)

CO2H
H 2N H
R

55

Coenzymes and cofactors

•  indispensable for the activity of some enzymes

•  can regulate enzymatic activity

•  the active enzyme-cofactor complex is called a haloenzyme

•  an enzyme without its cofactor is called an apoenzyme

56

Catalysis
28

CHM 8304

Cofactors
•  metal ions (Mg2+, Mn2+, Fe3+, Cu2+, Zn2+, etc.)

•  three possible modes of action:

1.  primary catalytic centre
2.  facilitate substrate binding (through coordination bonding)
3.  stabilise the three-dimensional conformation of an enzyme

57

Coenzymes
•  organic molecules, very often vitamins
–  e.g.: nicotinic acid gives NAD; pantothenic acid gives CoA

•  intermediates in the transport of functional groups

–  e.g. H (NAD), acyl (CoA), CO2 (biotin), etc

•  also known as prosthetic groups

58

Catalysis
29

CHM 8304

Enzymes as catalysts

Jencks :
•  enzymes use binding energy to effect catalysis

Wolfenden :
•  reaction acceleration is proportional to the affinity of an enzyme for the
transition state of the catalysed reaction
•  the reaction rate is proportional to the concentration of substrate in the
activated complex at the TS
•  substrate affinity is therefore also important and enzymes use protein
conformational changes during the reaction to better stabilise the TS

Knowles :
•  often the various steps of an enzymatic reaction are stabilised so as to
level the energies of the various ground states and TSs

59

Protein-ligand interactions

•  covalent bonds
•  ionic bonds
•  ion-dipole and dipole-dipole interactions
•  hydrogen bonds
•  charge transfer complexes
•  hydrophobic interactions
•  van der Waals interactions

60

Catalysis
30

CHM 8304

Covalent bond

•  the formation of a covalent bond can represent a stabilisation of

40 to 110 kcal/mol

e.g.:

activé

O R
N
R H

H2N

61

Ionic bonds

•  Coulombic attraction between full positive and negative charges

–  ~5 kcal/mol of stabilisation

NH2
H O2 C
O δ−
δ+ O N

OH O2 C

62

Catalysis
31

CHM 8304

Ion-dipole and dipole-dipole interactions

•  electrostatic interactions that involve partial charges

–  ~1 kcal/mol of stabilisation

NH2
H O2 C
O δ−
δ+ O N

OH O2 C

63

Hydrogen bonds
•  special type of dipole-dipole interaction
–  donors / acceptors : N, O, F
–  stabilisation of around 3-10 kcal/mol

NH2
H O2 C
O δ−
δ+ O N

OH O2 C

64

Catalysis
32

CHM 8304

Charge transfer complex

•  special type of dipole-dipole interaction
•  involves π electrons, often in aromatic rings (Phe, Tyr, Trp, His)
–  stabilisation : < 3 kcal/mol

65

Hydrophobic interactions

•  stabilisation largely due to desolvatation (entropy increase)

–  stabilisation : ~0.5 kcal/mol

H H
O H
O H
H H H CH O
H 2 H
surface
hydrophobic
O + O H
CH2 H O

hydrophobe
surface
CH2 H O
H
H O
H CH2 H O
O H
H H
H
H 6 O
H

(desolvation)

(désolvatation)




66

Catalysis
33

CHM 8304

van der Waals interactions

•  special type of dipole-dipole interaction
–  movement of electrons in electron cloud of alkyl chains induces
the formation of temporary dipoles
–  very important over short distances
–  stabilisation : ~0.5 kcal/mol (per interaction)

67

Enzyme kinetics
•  same rules, laws and methods as analysis of non-enzymatic
(“chemical”) kinetics

•  treated separately simply to emphasise the kinetic equations of enzyme

activity and inhibition that are so pertinent in bioorganic and medicinal
chemistry

68

Catalysis
34

CHM 8304

Steady state

k1 k2
E + S E•S E•P E + P
k-1
•  at the beginning of an enzymatic reaction, there is an induction
period (see the treatment of consecutive reactions) where the
concentrations of intermediates build to a certain level

•  when the rate of formation of these intermediates equals their rate

of disappearance, they are said to be in a steady state

•  enzymatic reaction rates are typically measured during this time

period

69

Initial rates
•  normally, [E]0 << [S]

•  at the beginning of the reaction (<10%), [S] ≈ [S]0

•  under these conditions, [E•S] doesn’t change appreciably and the

rate is therefore considered to be constant

70

Catalysis
35

CHM 8304

Saturation kinetics
•  the rate of an enzymatic reaction is linearly proportional to the
concentration of enzyme

•  however, these rates show saturation kinetic (hyperbolic) behaviour

with respect to the concentration of substrate
–  at low concentrations of S, the rate increases linearly with [S]
–  at higher concentrations of S, the rate increases less and less with
increasing [S]
–  at saturating concentrations of S, the rate approaches a limiting
value called the maximum rate, Vmax

71

Michaelis
•  Leonor Michaelis (1875 – 1949)
–  German biochemist and physician (Berlin,
Johns Hopkins, Rockefeller)
–  develop enzyme kinetic equations with Menten
–  studied urinary tract infections
–  developed chemical denaturation of keratin
(‘perm’!) and depilation

72

Catalysis
36

CHM 8304

Menten
•  Maude Menten (1879-1960)
–  Canadian medical scientist
–  MD/PhD with Michaelis
–  developed enzyme kinetics equations
–  later became pathologist (Pittsburgh)
–  developed enzyme assays and
electrophoretic separation of proteins

73

Michaelis-Menten equation
•  in 1913, Michaelis and Menten proposed the following simplified
kinetic scheme:
–  NB: rapid equilibrium to form the Michaelis complex, followed by
its reaction in the slow step
KS kcat
E + S E•S E + P

[E][S]
v = kcat[E•S] and K S = and [E]0 = [E] + [E•S]
[E • S]
K S [E • S] [E]0 [S]
[E] = = [E]0 − [E • S] KS[E•S] = [E]0[S] - [E•S][S] [E • S] =
[S] K S + [S]

hyperbolic [E]0 [S]k cat

v= Vmax = kcat[E]0
equation K S + [S]

74

Catalysis
37

CHM 8304

Michaelis-Menten plot

v = Vm ax = k cat [E]0

v = Vm ax [S] / KM
Rate
v
Vitesse,
Vmax / 2

KM is the concentration
of substrate necessary
to reach half the
maximal rate

KM

[S]
75

M-M equation cf M-M mechanism

•  the Michaelis-Menten equation (hyperbolic) accurately describes the

kinetics observed for most enzymatic reactions

•  however, the mechanism suggested by the simplistic Michaelis-Menten

scheme (fast equilibrium binding, followed by one slow step) is rarely
appropriate

•  a more rigorous kinetic treatment of the same scheme invokes the steady
state approximation

76

Catalysis
38

CHM 8304

Steady state equation

k1 k2
E + S E•S E + P
k-1

•  at the steady state, the rate of formation of E•S equals that of its
disappearance:

d[E • S] k 2 + k −1 [E][S]
= k1[E][S] − (k 2 + k -1 )[E • S] = 0 =
dt k1 [E • S]

•  this is the only difference compared to the previous treatment;

KS is simply replaced by KM, the Michaelis constant :

k 2 + k −1 [E]0 [S]k cat Vmax [S]

= KM v= v= Vmax = kcat[E]0
k1 K M + [S] K M + [S]

77

Mechanistic implication

•  saturation kinetics refers to the the hyperbolic relation between the

reaction rate and the concentration of reactant (substrate)

•  in general, this is consistent with (and often due to) the rapid pre-
formation of a complex before its reaction to give product

A+C A•C P+C

78

Catalysis
39

CHM 8304

Constant catalytic : kcat

•  in general, kcat represents the rate constant of the rds, the slowest step of
the enzymatic reaction
–  more strictly speaking, it is affected by first order rate constants of all
steps in the mechanism

•  also called the turnover number because it represents the number of

substrate molecules converted into product, per enzyme active site, per
unit of time

79

Apparent equilibrium constant : KM

•  related to KS, the dissociation constant

•  can be considered as an apparent dissociation constant

–  more strictly, it is the dissociation constant for the sum of all
enzyme species bound by substrate
–  related to the affinity of the enzyme for the substrate
•  lower KM corresponds to higher affinity
–  less substrate necessary to saturate enzyme

•  always represents the concentration of substrate necessary to give

half of the maximal rate
–  (derives mathematically from the hyperbolic equation)

80

Catalysis
40

CHM 8304

Specificity constant : kcat / KM

•  second order rate constant for the reaction of enzyme and

substrate

•  since the value of (kcat / KM) varies for each substrate and its
affinity for the enzyme, this ratio is also called the specificity
constant

•  can be considered as an indicator of the efficiency of the

reaction of free enzyme with a given substrate

81

Energy diagrams
•  consider energy profiles for enzymatic reactions, at the native
concentration of substrates:
•  for [S] >> KM
–  the uniform binding of substrate and activated complex would not
lead to catalysis :

uniform
binding no additional
catalysis
ΔG‡cat
ΔG*

ΔG*

ΔG‡cat
E+S E•P E+S E•P
E•S E+P E+P
E•S
Rxn co-ord. Rxn co-ord.

82

Catalysis
41

CHM 8304

Energy diagrams
•  consider energy profiles for enzymatic reactions, at the native
concentration of substrates:
•  for [S] >> KM
–  the differential binding of substrate and activated complex can lead
to catalysis :

differential
binding more
catalysis
ΔG‡cat
ΔG*

ΔG*
ΔG‡cat
E+S E•P E+S E•P
E•S E+P E•S E+P

Rxn. co-ord. Rxn. co-ord.

83

Energy diagrams
•  consider energy profiles for enzymatic reactions, at the native
concentration of substrates:
•  for [S] < KM
–  the uniform binding of substrate and activated complex can lead to
catalysis :

uniform
binding more
catalysis
ΔG‡cat
ΔG*

ΔG*

E•S ΔG‡cat
E+S E•P E+S E•S E•P
E+P E+P

Rxn. co-ord. Rxn. co-ord.

84

Catalysis
42

CHM 8304

Energy diagrams
•  consider energy profiles for enzymatic reactions, at the native
concentration of substrates:
•  for [S] < KM
–  the differential binding of substrate and activated complex can lead
to catalysis :

differential
binding more
catalysis
ΔG‡cat
ΔG*

ΔG*
ΔG‡cat
E•S
E+S E•P E+S E•S E•P
E+P E+P

Rxn. co-ord. Rxn. co-ord.

85

“Perfect” enzymes

•  some enzymes are so efficient, their kcat/KM values approach the

diffusion-controlled limit, at [S]<KM
–  e.g.: triosephosphate isomerase
O O O O
P kcat/KM = 2.4x108 M-1s -1 P
O O O O
KM = 25 µM

O OH

OH O

–  e.g. carbonic anhydrase

k cat/KM = 8.3x10 7 M-1 s -1

KM = 12 mM
H 2 CO 3 CO2 + H 2O

86

Catalysis
43

CHM 8304

Serine proteases

•  catalyses the hydrolysis of proteins and peptides

–  esters, too

•  studied for over 40 years, their mechanism is very well known

–  especially true for trypsin, chymotrypsin, elastase and subtilisin

•  contain a catalytic triad, composed of Ser, His and Asp

δ−
O
Asp R
O
102 N
C
H R'
O H
N δ+
N H O

His 57
Ser 195
87

Chymotrypsin
•  e.g. catalytic triad of chymotrypsin :

J. Mol. Biol. 1985, 184, 703-711. 88

Catalysis
44

CHM 8304

Chymotrypsin

•  secreted by the pancreas, it aids in the digestion proteins in the intestine

•  catalyses the hydrolysis of the peptide bond on the C-terminal side of an
amino acid having a side chain containing an aromatic group :
–  P1 = Phe, Trp, Tyr

P2 P1 P'1 P'2

S2 S1 S'1 S'2

89

pH-rate profile of chymotrypsin

•  plateau in pH-rate profile implies dependence on ionisation state of
one residue (His57)

pKa = 6.8

O
- H N
O N
log kcat

HO

basic form
necessary for
catalysis

O H
- H N
O N HO

pH
90

Catalysis
45

CHM 8304

Chymotrypsin mechanism
O R' O R' O R'
N N HN
R H
R H R H
O H O O
N NH H N NH N NH
O O O
Ser Ser Ser
O Asp O Asp O Asp
His His His
Michaelis complex tetrahedral acyl-enzyme
RCO2 H intermediate H 2O

O RCONHR' O O R'NH 2
OH OH HO
R R R
H
O H O O
N NH H N NH N NH
O O O
Ser Ser Ser
O Asp O Asp O Asp
His His His

tetrahedral
intermediate

91

Bender’s model (Acc. Chem. Res. 1987, 20, 146)

•  Bender coupled a binding site model (a cyclodextrin) with a model of the

catalytic triad of the serine proteases

•  one of the first enzyme models to incorporate all the components of an

enzymatic system in the same molecule

•  particpates in an intermolecular reaction with a model substrate, p-t-

butylphenyl acetate
–  caution! a phenyl ester is not an amide!

92

Catalysis
46

CHM 8304

Cyclodextrin
•  cyclic oligomer of 1,4-α-D-glucose units
–  6 glucoses = α-CD, 7 glucoses = β-CD, 8 glucoses = γ-CD

•  hydrophobic cavity, with OH groups around the entry, often used as

sites for attachment of other functional groups

93

Bender’s model (Acc. Chem. Res. 1987, 20, 146)

O

H3C O

good +
affinity CO2- O
for CD
N CO2-
N
H N
H H3C O H N
H
O
O
S
S

CO2-

H O HO H N CO2-
N N
N
H
H H3C O
O S
S

CH3CO2- 94

Catalysis
47

CHM 8304

Bender’s model (Acc. Chem. Res. 1987, 20, 146)

•  saturation kinetics observed

–  consistent with formation of a bound complex
–  allows the measurement of kcat and KM values

•  1 mmol of catalyst effected the hydrolysis of > 10 mmol of substrate

–  regeneration of catalyst; true catalysis

95

Bender’s model (Acc. Chem. Res. 1987, 20, 146)

•  reaction is three-fold slower in D2O than in water

–  proton in flight at TS
–  consistent with general base catalysis
–  not consistent with nucleophilic catalysis (which is also possible with
imidazoles)

•  pH-kcat profile plateaus above pH 10

–  basic form is active
–  supports a role for a general base

96

Catalysis
48

CHM 8304

Bender’s model (Acc. Chem. Res. 1987, 20, 146)

•  comparisons (made by the author) between chymotrypsin and his

“artificial chymotrypsin”:
–  model is more stable with respect to pH and temperature
–  model is more efficient than the enzyme, with respect to their molecular
weights
–  starting point for the synthesis of “artificial enzymes”
–  “the ultimate proof of the mechanism of chymotrypsin catalysis”

•  HOWEVER, chymotrypsin catalyses the hydrolysis of amides at pH 7...

•  note that other authors have used more realistic substrate models,
namely amides
–  see Brown et al., JACS 1989, 111, 1445 :

N
O
97

Catalysis
49