Enzymes and Enzyme Kinetics:

Enzyme Catalysis

With Dr. Kevin Ahern

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Enzymes and Enzyme Kinetics
Outline
• Background

• Flexibility

• Activation energy

• Mechanism of catalysis

• Kinetic considerations

• Michaelis Menten

Hexokinase and its

2 substrates (upper right)

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Types of Enzymatic Reactions
Four Reaction Possibilities

A B A B C

A B C A B C D

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Types of Enzymatic Reactions
Background
• Single substrate Single product : A <=> B

• Single substrate Multiple products : A <=> B + C

• Multiple substrates Single products : A + B <=> C

• Multiple substrates Multiple products : A + B <=> C + D

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 Enzymes Bind Substrates
Background

Enzyme

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Substrates Affect Enzymes on Binding
Background

Enzyme

ES complex

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Substrates Affect Enzymes on Binding
Background

Enzyme changes result in reaction

between substrates A and B

ES* complex

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Substrates Affect Enzymes on Binding
Background

has moved

EP complex

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Substrates Affect Enzymes on Binding
Background

Product D released
Product C released

Enzyme freed to bind more

substrates

E+P

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 Steps In Catalysis
Background

E+S Free enzyme & substrates

<=>
ES Substrate binding

<=>
Reversible
ES* Reaction

<=>
EP
<=> Product formation

E+P Release of products

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 Enzymes
Energy

1,2
∆G* uncatalyzed transition state

1 Activation energy necessary

for uncatalyzed reaction
Free energy of
0,8
substrate(s) Free energy

0,6

∆GTotal
Reaction
0,4

0,2

Free energy of
0
0 0,2 0,4 0,6 0,8 1 1,2
product(s)
Reaction progress

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 Enzymes
Energy

1,2
∆G* uncatalyzed transition state

1 Activation energy necessary

for uncatalyzed reaction
Free energy of
0,8
substrate(s) Free energy

0,6

∆GTotal
Overall free
Reaction energy change
0,4

0,2

Free energy of
0
0 0,2 0,4 0,6 0,8 1 1,2
product(s)
Reaction progress

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Enzymes
Energy

1,2
∆G* uncatalyzed transition state

1 Activation energy necessary

for uncatalyzed reaction
Free energy of
0,8
substrate(s) Free energy

0,6
Reaction Reaction
reverses goes
∆GTotal
Overall free
forward energy change
Reaction
0,4

0,2

Free energy of
0
0 0,2 0,4 0,6 0,8 1 1,2
product(s)
Reaction progress

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Enzymes
Energy

1,2

1 Activation energy necessary

for catalyzed reaction
Free energy of
0,8
substrate(s) Free energy
∆G* catalyzed transition state

0,6
Reaction Reaction
reverses goes
∆GTotal
forward
Reaction
0,4

0,2

Free energy of
0
0 0,2 0,4 0,6 0,8 1 1,2
product(s)
Reaction progress

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Enzymes
Energy

1,2

1 Activation energy necessary

for catalyzed reaction
Free energy of
0,8
substrate(s) Free energy
∆G* catalyzed transition state

0,6
Reaction Reaction
reverses goes
∆GTotal
No overall free
forward energy change
Reaction
0,4

0,2

Free energy of
0
0 0,2 0,4 0,6 0,8 1 1,2
product(s)
Reaction progress

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Enzymes
Mechanistics
• Binding specificity

• Flexibility

• Electronic environment

• Coenzymes

A serine protease

http://www.ebi.ac.uk
Derek Bryan Guerrero, derekbguerrero@gmail.com
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 Serine Proteases
Background
• Cleave peptide bonds

• Specificity of cutting

• Common active site composition/structure

• Mechanistically well studied

A serine protease

http://www.ebi.ac.uk
Derek Bryan Guerrero, derekbguerrero@gmail.com
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 Serine Proteases
Catalytic Mechanism

Folded polypeptide chain of enzyme

Enzyme backbone

Enzyme backbone
Enzyme backbone
Ser His Asp
O
C NH
R R´
OH H
N -O O
Substrate for enzyme

N
Catalytic triad of active site

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Serine Proteases
Catalytic Mechanism

Enzyme backbone

Enzyme backbone
Enzyme backbone
Ser His Asp

OH H
N -O O

O
Region of enzyme that N
determines what substrate C NH S1 Pocket
the enzyme binds R R´ of enzyme

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Serine Proteases
Catalytic Mechanism

Enzyme backbone

Enzyme backbone
Enzyme backbone
Ser His Asp

OH H
N -O O

O
N
1. Binding of substrate stimulates C NH S1 Pocket
slight structural changes R R´ of enzyme

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Serine Proteases
Catalytic Mechanism

2. Structural changes
induced by binding Enzyme backbone

Enzyme backbone
Enzyme backbone
change electronic
environment of Ser His Asp
catalytic triad

OH H
N -O O

O
N
1. Binding of substrate stimulates C NH S1 Pocket
slight structural changes R R´ of enzyme

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Serine Proteases
Catalytic Mechanism

2. Structural changes
induced by binding Enzyme backbone

Enzyme backbone
Enzyme backbone
change electronic
environment of Ser His Asp
catalytic triad

3. N abstracts a proton
O- H
chain, creating an
N -O O
alkoxide ion
+HN
O

1. Binding of substrate stimulates C NH S1 Pocket

slight structural changes R R´ of enzyme

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Serine Proteases
Catalytic Mechanism
Enzyme backbone

Enzyme backbone
Enzyme backbone
Ser His Asp

O- H
Alkoxide ion makes N -O O
nucleophilic attack on
carbonyl carbon of +HN

peptide bond O
C NH S1
R R´ Pocket

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Serine Proteases
Catalytic Mechanism
Enzyme backbone

Enzyme backbone
Enzyme backbone
Ser His Asp

H
N -O O

+HN
O
Tetrahedral intermediate
stabilized by oxyanion hole -O C NH
S1 Pocket
R
Oxyanion hole R

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 Serine Proteases
Catalytic Mechanism
Enzyme backbone

Enzyme backbone
Enzyme backbone
Ser His Asp

Peptide bond broken as H

N binds to H on histidine N -O O

+HN
O
Tetrahedral intermediate
stabilized by oxyanion hole -O C NH
S1 Pocket
R
Oxyanion hole R

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 Serine Proteases
Catalytic Mechanism
Enzyme backbone

Enzyme backbone
Enzyme backbone
Ser His Asp

O C H
N -O O
Half of polypeptide R
released from enzyme N
H
HN
Released R`

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Serine Proteases
Catalytic Mechanism
Enzyme backbone

Enzyme backbone
Enzyme backbone
Ser His Asp

Other half of polypeptide O

covalently linked to serine
O C H
N -O O
Half of polypeptide R
released from enzyme N
H
HN
Released R`

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Serine Proteases
Catalytic Mechanism
Enzyme backbone

Enzyme backbone
Enzyme backbone
Ser His Asp

Other half of polypeptide O

covalently linked to serine
O C H
N -O O
Half of polypeptide R
released from enzyme N
H
HN
Released R`
Fast phase of catalysis completed

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 Serine Proteases
Catalytic Mechanism
Enzyme backbone

Enzyme backbone
Enzyme backbone
Ser His Asp

H
N -O O

O
N
O C
O H
R
1. Water enters active site
H

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 Serine Proteases
Catalytic Mechanism
Enzyme backbone

Enzyme backbone
Enzyme backbone
Ser His Asp

H
N -O O

O
N
O C 2. Nitrogen attacks
O H water proton
R
1. Water enters active site
H

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 Serine Proteases
Catalytic Mechanism
Enzyme backbone

Enzyme backbone
Enzyme backbone
Ser His Asp

H
N -O O
3. Hydroxide attacks O
carbonyl carbon N
O C 2. Nitrogen attacks
O H water proton
R
1. Water enters active site
H

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 Serine Proteases
Catalytic Mechanism
Enzyme backbone

Enzyme backbone
Enzyme backbone
Ser His Asp

H
N -O O
Oxyanion hole stabilizes O +HN
tetrahedral intermediate
-O C OH

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 Serine Proteases
Catalytic Mechanism
Enzyme backbone

Enzyme backbone
Enzyme backbone
Ser His Asp

Oxygen abstracts H on
N-group, breaking bond
with serine H
N -O O
Oxyanion hole stabilizes O +HN
tetrahedral intermediate
-O C OH

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 Serine Proteases
Catalytic Mechanism
Enzyme backbone

Enzyme backbone
Enzyme backbone
Ser His Asp

Oxygen abstracts H on
N-group, breaking bond
with serine H
N -O O
Oxyanion hole stabilizes O +HN
tetrahedral intermediate
-O C OH

R
Slow phase of catalysis complete

Derek Bryan Guerrero, derekbguerrero@gmail.com

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 Serine Proteases
Catalytic Mechanism
Enzyme backbone

Enzyme backbone
Enzyme backbone
Ser His Asp

Other polypeptide
H
fragment released OH
N -O O

O N

C OH
Released
R

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 Serine Proteases
Catalytic Mechanism
Enzyme backbone

Enzyme backbone
Enzyme backbone
Enzyme returned
to original state Ser His Asp

Other polypeptide
H
fragment released OH
N -O O

O N

C OH
Released
R
Cycle complete. Enzyme ready to start anew

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 Enzymes
Kinetic Considerations

Rate of formation of product

of primary interest

E + S <=> ES <=> ES* <=> EP <=> E + P

If we assume in the simple case that ES proceeds directly to E + P,

kf kcat
E+S kr
ES E+P
Where kf, kr, and kcat refer to the rate constants for formation of ES,
reversible breakdown of ES, and conversion of ES to E + P, respectively

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 Enzymes
Kinetic Considerations

Substrate Enzyme

Low substrate High substrate

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 Enzymes
Kinetic Considerations

1,2

High [S], high V0

(enzymes almost always busy)
0,8

Velocity
measured as
V0

0,6

[product]/time
0,4

Low [S], low V0 (enzymes often idle)

0,2

0
0 0,2 0,4 0,6 0,8 1 1,2

[S] Substrate concentration (molarity)

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 Enzymes
Kinetic Considerations

1,2

The longer an enzymatic

1 reaction goes, the more the reverse
reaction becomes a factor
0,8

Enzyme kinetic reactions should be

studied early, before products accumulate
[P]

0,6

0,4
Linear initial accumulation
of product
0,2

0
0 0,2 0,4 0,6 0,8 1 1,2

Time

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 Michaelis-Menten Kinetics
Considerations

1,2

At the beginning [S] As the reaction

of the reaction: [P] proceeds:
1

• [P] is low • [S] is low

0,8

• [ES] is low
Concentration
• [ES] is higher
• [E] is high 0,6

• [E] is lower
• [S] is high [E]
0,4 • [P] is high

0,2

[ES]

0
0 0,2 0,4 0,6 0,8 1 1,2

Time

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 Michaelis-Menten Kinetics
Considerations

1,2

[S]
1
[P]

Pre-steady
Steady state conditions
state conditions 0,8
Concentration

0,6
[E] and [ES] not varying rapidly

[E]
0,4

[E] and [ES]

varying rapidly 0,2

[ES]

0
0 0,2 0,4 0,6 0,8 1 1,2

Time

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 This document is a property of: Derek Bryan Guerrero

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distributed in any way without the written authorization of Lecturio GmbH.

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