Enzymes

Review Chapter 1
Read Chapter 6

5e Chapter 1: p20-27
6e Chapter 1: p21-25
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tion 5: Enzymes (Chapter 6 Lehning

1) Physiological
significance of
enzymes

Examples of enzymes
Why enzymes?

2) Thermodynamics a
review

Gibbs free energy and Reaction

equilibria
Activation energy and reaction
rates
Coupling of exergonic and endergonic
reactions
Enzyme substrate complex
Enzymatic reduction of activation energy
Types of enzymatic reactions

) Mechanisms of catalysis

4) Enzyme kinetics

Steady of kinetics to inform about

enzymes
V,
VO, VMAX, KM, S
Michaelis-Menten equation
Enzymatic inhibitors

5) Chymotrypsin

Assigned reading: pg 205- 209

2

Enzymes and substrates

Recall from chapter 5:
Proteins bind ligand molecules ex: myoblobin binds O2
No change to O2 or the protein simple binding reaction
Compare with enzymes:
Enzymes also bind ligands but these are now called
substrates.
Substrates are chemically modified by enzymes substrate
is changed into the product.
The basic equation for an enzyme reaction is:

E(Enzyme) + S(Substrate)
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Chymotrypsin active site

Chymotrypsin

Substrate

Activesite

Enzymatic Substrate Selectivity

Phenylalanine
hydroxylase

H
-

+
NH3

OOC

+
NH3

No binding

+
NH3

OOC

H
OOC

H
-

OH

OH
HO
H
H

OH

H
NH
CH3

All (almost all) natural amino acids are of

stereochemistry.

Binding but no
reaction

Why biocatalysis?
In other words, why enzymes?

What do enzymes do for a cell?

Co-Enzymes

Co-enzymes are organic molecules that carry specific functional

groups needed for enzymatic function

Enzyme structure
A complete enzyme
(functional) =

holoenzyme
Alcohol
dehydrogenase:
Needs Zn (prosthetic
group, also a cofactor): a holoenzyme
with Zn2+ atom

The protein part of the

enzyme without co-enzyme
or co-factor = apoenzyme
or apoprotein
Alcohol
dehydrogenase:
Without Zn2+ atom, it
is in the apoenzyme
form

Enzyme classification
and nomenclature (1)
Usually enzyme names end with a ase suffix
Usually ase is attached to a name that designates the
substrate or the activity
Example 1: urease catalyses hydrolysis of urea
Example 2: DNA polymerase catalyses
polymerization of DNA.
But it is not always the case: example pepsin
and trypsin there is a systematic classification

Enzyme classification and nomenclature

(2)

See. Pg. 185 5e, Pg 191 6e for details

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How do enzymes work?

Before we get into
that we need to
review
thermodynamics.
5e Chapter 1: 20-27
6e Chapter 1: 21-25

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Entropy and reactions

Entropy, S: randomness and disorder of the universe
+ S: increased entropy = increased disorder = spontaneous
- S: decreased entropy = decreased disorder = nonspontaneous but

S
+S

12

Enthalpy and reactions

Enthalpy, H: energy content (heat) derived from volume,
pressure
and different types and number of bonds.
Intuitively: the heat content of chemical bonds
+ H: positive enthalpy= increased heat/energy content
in a bond =
energy must be transferred into system
- H: negative enthalpy = decreased heat/energy
content in bond =
energy is liberated into the
universe
C-H
C=O

Less energy content

More energy content
~ 416kJ/mol
~ 736kJ/mol
Bond dissociation energy ("energy to break a
bond")
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Gibbs Free Energy

Gibbs free energy (G) is total energy balance
derived from enthalpy and entropy
G = H TS, where T is temperature Kelvin

Whats important is change in G between conditions!!

G = H - TS Change in parameter between conditions

G positive
reaction
favours
SUBSTRATE
S

G negative
reaction
favours
PRODUCTS
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Change in Gibbs Free Energy

determines direction of a process
Favours SUBSTRATES
Positive G, which is (H [TS])
+H
S
+S
H
Negative G, which is (H [TS])
Favours Products
15

Spontaneous reactions (negative G)

do not necessarily happen that well
Glucose does not react with O2 to form
CO2 and water, despite being highly
exergonic (at room temperature)!!!
WHY NOT?

16

Properties of enzymatic
reactions

In other words, how much product versus substrate?

HOW MUCH?

G
+G

HOW FAST?
17

Reaction rate depends on

activation energy

For any reaction, including exergonic reactions,

there is a barrier the activation energy, an energy
quantity of the transition state.
transition state represents a high-energy distortion
of bonds that reactants must go through to become
a product! A transient distortion that often has very
unfavorable positions
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The likelihood of a reaction depends on

the activation energy
G = must define
conditions
G = standard; 298
K, 101.3 kPa,
concentration 1 M.
G = biological
conditions
G = activation energy

S
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G
+G

Metabolism

Photosynthesis

How do cells drive +ve G reactions?

Potential Energy

20

Energy beltway in life (1):

Catabolism supports anabolism

21

Energy beltways in life (2) corrected

Photosynthesis: light energy drives an electron chain to reduce
carbon into sugars

G = +H (T(-S)) = +ve
+H
- S
+S
-H
G = -H (T(+S))= -ve
Chemical energy of glucose oxidation is coupled in the
respiratory chain to drive +G reactions like making
ATP
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Coupling of exergonic reactions to drive

endergonic reactions
(endergonic, G1= +ve)

Glucose + Pi

Glucose 6-phosphate

(exergonic, G2= very -ve)

ATP

ATP + Pi

Glucose + ATP

Glucose 6-phosphate + ADP

(exergonic, G3= G1+G2 = -ve)

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But what about bypassing the

activation energy problem?
Back to enzymes

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The likelihood of a reaction depends on

the activation energy
G = must define
conditions
G = standard; 298
K, 101.3 kPa,
concentration 1 M.
G = biological
conditions
G = activation energy

S
25

G
+G

The enzyme-substrate complex

reduces activation energy

26

The enzyme-substrate
complex
-G , k1

No enzyme S
P
+G , k-1

-G = -G
+G = +G

cat

-G cat , kcat1

E+S

ES
+G

EP

cat

, kcat-1

cat

E + P k >>>k
cat1
1
kcat-1 >>>k-1

E = enzyme; S = substrate; P = product

ES and EP are intermediate complexes
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Enzymes accelerate
reaction
rates
Enzymes (and catalysts in general) lower the
activation barrier compared to the uncatalyzed reaction

Permits reactions to reach equilibrium

faster!!
Enzymes are not unidirectional!
They can convert P to S depends
on [P] and [S]
Enzymes
dont
affectequilibrium
equilibrium or G
Time
to reach
S

-Gk1 No enzyme: years

+Gk-1 Enzyme: seconds

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How to lower the

activation energy?
enzyme active sites are most
complementary to the transition state of
the reaction
enzymes bind transition states better
than substrates
stronger interactions with the transition
state as compared to the ground state
lower the activation barrier

Enzymes bind transition states best

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Activation energy G (uncatalyzed)

is offset by GB to produce a lower
catalyzed Gcat

30

Illustration of Transition State

Stabilization Idea: Imaginary
Stickase

The enzyme helps to

_________ the substrate.

How to lower the

activation energy?
Covalent Mechanism: Formation of transient
covalent intermediates between substrate functional
groups and enzyme functional groups in active site
alternate states of lower energy!
Non-covalent Mechanism: weak, favorable
non-covalent bonds between enzyme and
substrate (and transient state). Each interaction
releases a small amount of free energy called
binding energy GB.
Many interactions add to a large ve GB. This
lowers the activation energy of reactions!
GB = weak interactions during binding
specificity + weak interactions that stabilize the
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transient states!!

How to lower the activation

energy? (2)
Through binding substrate, transition state,
product:
increases local concentration
orients and restricts movement of
functional groups (entropy reduction)
specificity of reaction
microenvironment desolvation for
example

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How to lower the activation

energy? (3)

Example: Some enzymes organize reactive groups into

proximity/space

Chemical example:
The rate of anhydride formation
from esters and carboxylates
shows a strong dependence on
proximity of two reactive groups

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How is catalysis actually driven

by enzymes?

37

Catalysis is driven by specific

functional groups
metal ion catalysis: use redox cofactors, pKa
shifters
acid-base catalysis: give and take protons
covalent catalysis: change reaction paths

38

Metal Ion Catalysis

Ionic interaction with substrate

Ionic stabilization of transition
states
Mediate redox reactions by
reversible changes in metal
oxidation state (ex. Fe2+ to Fe3+
- electron transfer)

39

General acidbase catalysis

is common in
enzymes

40

The role of Amino Acids found in the

active site of enzymes in General
Acid-Base catalysis
Amino acid Rgroups are
precisely
positioned in
active site with
respect to
intermediate
Rates
accelerated
species
2
10
to 105
Very common
mechanism!!!
41

Covalent catalysis: A chemical

example
O

CH3

H3C

H2O
slow

O
O

H3C

2 H

CH3

H3C

fast

+
N

CH3

..
N
..

..
N

H3C

H3C

+
N

OH
H

CH3

H3C

The anhydride hydrolysis

reaction is catalyzed by
pyridine, a better
nucleophile than water
(pKa=5.5).
Hydrolysis is accelerated
because of charge loss in
the transition state makes
pyridine a good leaving
group.
42

Covalent Catalysis: Enzyme Example

A-B
A-B + X:

H2O

H2O
A
A-X+ B

+ B
+ X: + B

Amino acid functional (R-) group can react to

form transient covalent species with a
reactant
Sometimes this is a coenzyme (e.g.
prosthetic) functional group
At the end of reaction, functional group is
reformed
43

Covalent Catalysis: In
Enzymes
Proteases and peptidases
chymotrypsin, elastase, subtilisin
reactive serine nucleophile
Some aldehyde dehydrogenase
glyceraldehyde-3phosphate dehydrogenase
reactive thiolate nucleophile
Aldolases and decarboxylases
amine nucleophile
Dehalogenases and some glycosidases
carboxylate nucleophile
NH2

HO

O
S

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45

What is Enzyme Kinetics?

Kinetics is the study of the rate at which
compounds react
Rate of enzymatic reaction is affected by
Enzyme concentration
Substrate concentration
Effectors
Temperature
pH

46

Why Study Enzyme

Kinetics?
It is a quantitative description of biocatalysis
Determine the order of binding of substrates
Elucidate acid-base catalysis
Understand catalytic mechanism
Find effective inhibitors like potential drugs
Understand regulation of activity

47

EnzymeSubstrate EnzymeSubstrate
complex

ES
V

EnzymeProduct
complex

Enzyme Product

EP

= reaction rate
= has units of (moles of Product)/s

48

Rate Equations and

Constants
S

k
k-1

S1 + S2

k-1

Equilibrium constant
Keq = [P]
[S]
Reaction equation
V = k[S]

Second-order reaction
[P]
Keq =
[S1][S2]
V = k[S1][S2]

k has units of s-1

k has units of M-1s-1

First-order reaction

49

Reaction rate depends

on [S]
V depends on concentration of S
BUT [S] changes with time to become P!!
Thus reaction rate changes over time as [S] decreases and [P]
increases
To avoid this complication, one studies the initial rate, V0, at
the beginning of the reaction
At this stage, [S] can be assumed to be nearly constant
Enzyme-Substrate complex is nearly constant
This is steady-state kinetics

50

V0 (initial) rates are assumed to be

linear

Initial time
of
reaction;
rates are
nearlinear

51

V0 can be saturated at high [S]

52

Effect on V0 by varying [S]

Saturation of E to ES

Why this shape?

Key assumption: [E] << [S]

53

ES complex formation and

Vmax
E+S
ES

k1

k-1

ES

Fast and reversible

k2 = (kcat)
E + PSlower, rate-limiting
[ES] is going to determine overall rate
k-2

At low [S], there is little ES most enzyme is

unoccupied
At low [S], rate is proportional to [S] more S, forces
ES formation!
At high [S], enzyme is mostly in ES thus [ES]
becomes main parameter further increase in [S]
has little effect cant make more ES! Thus Vmax is
54
achieved.

Steady-state Kinetics
Studies the initial rate, V0
At this stage, [S] can be assumed to be nearly constant
[P] is very small
ES breakdown to E + P is rate limiting step;
Enzyme-Substrate complex is nearly constant
Overall rate proportional to [ES]

E+S

k1
k-1

ES

k2

E+P

k-2 is negligible in
early reaction
because there is
little P! 55

The Michaelis-Menten Equation

E S

ES

EP

56

What happens when [S] is very

small or very large?

Vmax [ S ]
Vvo
Km S

57

When V0=1/2 Vmax, then [S]=Km

58

Double-Reciprocal Plot
(Lineweaver-Burke plot)

59

Double-Reciprocal Plot

60

Competitive inhibition

ES

EI

(Inhibitor)
61

Uncompetitive
inhibition

ES
+

(inhibitor)

ESI
62

Types of enzyme
inhibitors
Competitive

Non-competitive

ASSIGNED: See BOX 6-2 (page 202):

Kinetic tests for determining inhibition

63

Chapter 6: Summary
Assigned Reading:

Section 6.4 Examples

of enzymatic reactions: Chymotrypsin (pages
205-209)

You Should Know

hysiological functions of enzymes

Changes in Gibbs free energy determines direction of reaction but not
How unfavourable reactions occur in a cell
he impact of enzymes on activation energy
Mechanisms by which enzymes catalyze reactions
How to use Michaelis-Menten enzyme kinetics to study enzymes
he mechanism of catalysis of chymotrypsin

Suggested problems
Worked example 6-1 and 6-2 (page 199-200 5e, page 206 6e)
Problems (page 229-233): #1, 3, 6, 7-13
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