Enzymes

Dr Hadiyanto
 What are
enzymes?

Enzymes are
proteins
(tertiary and
quaternary
structures).

From the Virtual Cell Biology Classroom on ScienceProfOnline.com Image: Levels of protein structure, M Ruiz
 What do enzymes do?

• Enzymes act as
catalyst in cellular
reactions.

• Q: What does a
catalyst do?

From the Virtual Cell Biology Classroom on ScienceProfOnline.com Images: Activation energy graph, Wiki
 How do enzymes work?

Enzymes catalyze
reactions by
weakening chemical
bonds, which
________ activation
energy.

From the Virtual Cell Biology Classroom on ScienceProfOnline.com Image: Activation energy graph, Wiki
 How do enzymes work?
• Each enzyme has a unique 3-D shape, including a surface groove called
an active site

• The enzyme works by binding a specific chemical reactant (substrate)

to its active site, causing the substrate to become unstable and react.

• The resulting product (s) is then released from the active site.

From the Virtual Cell Biology Classroom on ScienceProfOnline.com Image: Enzymatic reaction, Jerry Crimson Manni
Enzymes…

• are specific for what

they will catalyze.

• fit with substrate

like a lock and key.

From the Virtual Cell Biology Classroom on ScienceProfOnline.com

 When an enzyme is interacting with
it’s substrate, during the chemical
reaction, together they are referred
to as the …

From the Virtual Cell Biology Classroom on ScienceProfOnline.com Image: Enzyme –substrate complex, UC Davis
 Enzymes…

…are reusable.

They are not

consumed (used up)
in the reactions
they catalyze.

From the Virtual Cell Biology Classroom on ScienceProfOnline.com

Enzymes are like tiny machines within living things.

$
$
$
$
$
$
$
The more cans (substrate), the more $ (product).
The more recycling machines (enzymes), the faster the cans turn into $.

From the Virtual Cell Biology Classroom on ScienceProfOnline.com

 Enzymes…

• Have names that

usually end in -_____.
-Sucrase
-Lactase
-Maltase
 Why Are Enzymes So Important?

Why are we devoting

one whole lecture topic
to a protein molecule?

Nearly all chemical

reactions in
biological cells need
enzymes to make
the reaction occur
fast enough to
support life.
 Formats for writing a enzymatic
reaction.

Enzyme

Substrate + Substrate -----------> Product

( Enzyme )

Substrate -----------> Product+ Product

 Meet the Enzyme: ___ _______
Important metabolic enzyme that
harnesses energy for biological cells
to use.

Involved in synthesis of adenosine

triphosphate (ATP), from:
- adenosine diphosphate (ADP)
- a phosphate group and
- energy from H+ ion gradient

ATP is the most commonly used

"energy currency" of cells.

Reaction:
(ATP synthase)

ADP + Pi -------ATP
substrate substrate product
 How do you stop
an enzyme?

Denaturate it

• Alteration of a protein shape through

Irreversible egg
some form of external stress protein
denaturation
• Example, by applying heat or changing pH. caused by high
temperature
• Denatured protein can’t carry out its (while cooking it).
cellular function .

From the Virtual Cell Biology Classroom on ScienceProfOnline.com

 Factors That Influence Enzyme Activity

• Temperature

• pH

• Cofactors & Coenzymes

• Inhibitors

From the Virtual Cell Biology Classroom on ScienceProfOnline.com Image: Animation of Enzyme, Wiki
 Temperature & pH

• Think about what kind of cell or

organism an enzyme may work in…

• Temperatures far above the normal

range denaturate enzymes. (This is why
very high fevers are so dangerous. They can cook the
body’s proteins.)

• Most enzymes work best near

neutral pH (6 to 8).

From the Virtual Cell Biology Classroom on ScienceProfOnline.com Images: pH scale, Edward Stevens, Wiki
 Factors That Influence Enzyme Activity

• Temperature

• pH

• Cofactors & Coenzymes

• Inhibitors

From the Virtual Cell Biology Classroom on ScienceProfOnline.com Image: Animation of Enzyme, Wiki
 Cofactors & Coenzymes

• Non-protein substances (zinc,

iron, copper, vitamins) are sometimes
need for proper enzymatic
activity.

• Coenzyme vs Cofactor: What’s

the difference?

Cofactor more general term.

Includes inorganic and organic
molecules.

Coenyzyme type of cofactor,

But specifically organic
molecules.

Image: Enzyme with Cofactor, Wiki. Ribbon-diagram showing carbonic

From the Virtual Cell Biology Classroom on ScienceProfOnline.com anhydrase II. The grey sphere is the zinc cofactor in the active site.
Coenzyme: Vitamin B12

• Most vitmin are coenzymes

essential in helping move
atoms between molecules in
the formation of
carbohydrates, fats, and
proteins.

• Exclusively synthesized by
bacteria.

• Dietary sources include

meat, eggs, dairy products
and supplements.
Factors That Influence Enzyme Activity

• Temperature

• pH

• Cofactors & Coenzymes

• Inhibitors
 Two Types of Enzyme Inhibitors

1. Competitive
inhibitor
Chemicals that resemble
an enzyme’s normal
substrate and
compete with it for
the active site.

Reversible depending on
concentration of
inhibitor and
substrate.
EXAMPLE: The drug Antabuse is used to help alcoholics
quit drinking. Antabuse inhibits aldehyde oxidase, resulting
in the accumulation of acetaldehyde (say a-si-’tell-de-hide)
during the metabolism of alcohol. Elevated acetaldehyde
levels cause symptoms of nausea and vomiting.

From the Virtual Cell Biology Classroom on ScienceProfOnline.com Image: Competitive inhibition of enzyme, Jerry Crimson Mann
 Two Types of Enzyme Inhibitors

2. no-competitive
inhibitor
____________

Do not enter active

site, but bind to
another part of the
enzyme, causing the
enzyme & active site to
change shape.

Usually reversible,
depending on
concentration of
inhibitor & substrate.

EXAMPLE: You may know that compounds containing

heavy metals such as lead, mercury, copper or silver
are poisonous. This is because ions of these metals
are non-competitive inhibitors for several enzymes.

From the Virtual Cell Biology Classroom on ScienceProfOnline.com Image: Pouring liquid mercury, Bionerd
 Enzyme Inhibitors

Blocking an enzyme's activity can kill a

pathogen or correct a metabolic
imbalance. EXAMPLE:

•Another example of
competitive inhibition is
protease inhibitors.
Many medications are enzyme
inhibitors.
•They are a class of anti-
retroviral drugs used to
treat HIV.

Enzyme inhibitors are

also used as herbicides and pesticides. •The structure of the drug
ritonavir (say ri-TAHN-a-veer)
resembles the substrate of
HIV protease, an enzyme
required for HIV to be made.

From the Virtual Cell Biology Classroom on ScienceProfOnline.com Images: Prescription bottle, T. Port; Dead cockroach, Wiki
 Meet the Enzyme: Catecholase
• Catecholase is present in most fruits and vegetables.

• It is the enzyme that facilitates the browing of cut or bruised fruits

and vegetables by catalyzing the following reaction:

(______________)

Catechol + Oxygen ----------------- polyphenol

colorless substrate brown product
 Meet the Enzyme:
Catecholase
Lemon juice and other acids are used to preserve color
in fruit, particularly apples, by lowering the pH and
removing the copper (cofactor) necessary for the enzyme
to function.

Reaction:
catecholase

catechol + O2 ---------- polyphenol

colorless substrate brown product

Images: Apples, T. Port; Lemons, André Karwath; Enzyme

with Cofactor, Wiki; pH scale, Edward Stevens, Wiki From the Virtual Cell Biology Classroom on ScienceProfOnline.com
 Meet the Enzyme: Bromelain

• Pineapple contains enzyme bromelain,

which can digest protein

• Collagen = big, fibrous molecule makes

skin, bones, and tendons both strong
and elastic.
 Enzymatic Reactions
• Enzyme combines with a specific
substrate to a form an enzyme-
substrate complex in a lock and key
concept before forming new
products.
 Enzyme action
“Lock and Key”

products
substrate

enzyme
E + S E-S E + P
Enzyme Kinetics
Enzyme Kinetics
 Reaction rate approach
• Michaeles-Menten (MM)

slower
 Reaction rate approach
• Briggs-Haldane approach

Assume: dCEs/dt=0,
compare to Cp or Cs
Exercise
 Allosteric enzyme

Enzyme with cooperative binding that has more than one active
site. Mostly is regulatory enzyme.
VmCSn n: cooperative coefficient
V "
K m  CSn
Langmuir plot
Lineweaver-Burk plot
Exercise
Eadie-Hofstee plot
 Inhibitor
• Competitive
• Non-competitive
• Substrate inhibitor
Inhibited Enzyme Kinetics
• Competitive inhibitors (I)
Assume rapid equilibrium and with the
definitions of
d[P]
v  k 2 [ ES]
dt
' k1 [ E][S ]
Km  
k1 [ ES]
[ E ][I ] [E0 ]  [E]  [ES]  [EI ]
KI 
[ EI ]
Inhibited Enzyme Kinetics
• Competitive inhibitors (I),
we can obtain,
Vm [S ]
v
' (1  [ I ] / K )  [S ]
Km I
Vm[S ]
v
'
Km , app  [S ]
'  ' (1  [I ] / K ) When [I] =0, ' '
Km ,app K m I Km , app  K m
Inhibited Enzyme Kinetics
• Noncompetitive inhibitors (I)
Assume:
- rapid equilibrium
- same equilibrium constants of inhibitor
binding to E and ES KI
- same equilibrium constants of substrate
binding to E and EI Km’
Inhibited Enzyme Kinetics
• Noncompetitive inhibitors (I)
d[P]
v  k 2 [ ES]
dt
' k1 [ E][S ] [ EI ][S ]
Km   
k1 [ ES] [ ESI]
[ E ][I ] [ ES ][I ]
KI  
[ EI ] [ ESI]

[E0 ]  [E]  [ES]  [EI ]  [ESI]

Inhibited Enzyme Kinetics
• Noncompetitive inhibitors (I)
we can obtain,

Vm[S ]
v
'  [S ])
(1  [ I ] / K I )(K m

Vm, app[S ]
v
'  [S ]
Km

Vm,app  Vm /(1  [I ] / K I ) When [I] =0, Vm,app  Vm

Uncompetitive inhibition
Inhibited Enzyme Kinetics
• Uncompetitive inhibitors (I)
d[P]
v  k 2 [ ES]
dt
' k1 [ E][S ]
Km  
k1 [ ES]
[ ES ][I ]
KI 
[ ESI]
[E0 ]  [ E]  [ ES]  [ESI]
 Inhibited Enzyme Kinetics
• Uncompetitive inhibitors (I)
we can obtain,
(Vm /(1  [ I ] / K I ))[S ]
v
' /(1  [ I ] / K ))  [S ]
(Km I
Vm, app[ S ]
v
' , app  [ S ]
Km

Vm,app  Vm /(1  [I ] / K I ) ' '

Km,app  Km /(1  [I ] / K I )
' '
Km,app / Vm,app  Km / Vm
Substrate Inhibition
Inhibited Enzyme Kinetics
• Uncompetitive substrate inhibitors (I)
d[P]
v  k 2 [ ES]
dt
' k1 [ E][S ]
Km  
k1 [ ES]
[ ES ][S ]
K SI 
[ ES 2]

[E0 ]  [ E]  [ ES]  [ES 2]

Inhibited Enzyme Kinetics
• Uncompetitive substrate inhibitors (I)
we can obtain,
Vm[S ]
v
'  [S ]  [S ]2 / K
Km SI
At low substrate concentration [S ]2 / K SI <<1
Vm[S ]
v Michaelis-Menten Equation
Km'  [S ]
'
At high substrate concentration K m /[S ]  1
Vm
v
1  [S ] / K SI
Inhibited Enzyme Kinetics
• Uncompetitive substrate inhibitors (I)

The substrate concentration resulting in the

maximum reaction rate can be determined by
setting dv/d[S]=0, [S]max is given by

Vm[S ]
dv / d[S ]  d ( ) / d[S ]  0
'  [S ]  [S ]2 / K
Km SI

' K )1/ 2
[S ]max  (K m SI
• Uncompetitive substrate inhibitors (I)
Determine [S]max:
Vm [S ]
dv / d[S ]  d ( ) / d[S ]  0
'  [S ]  [S ]2 / K
Km SI

Vm [S ](1  2[S ] / K SI )
Vm
( )0
'  [S ]  [S ]2 / K
Km ( K '  [S ]  [S ]2 / K ) 2
SI m SI
'  [S ]  [S ]2 / K )  V [S ](1  2[S ] / K )
Vm ( K m SI m SI
( )0
(K m'  [S ]  [S ]2 / K ) 2
SI
'  V [S ]2 / K
Vm K m m SI
)0
'  [S ]  [S ]2 / K ) 2
(K m SI
'  V [S ]2 / K  0
Vm K m m SI
' K )1/ 2
[S ]max  ( K m SI
 Inhibition Estimation
• Product formation rate v ~ [S]: v has a peak?
If yes, then it’s substrate inhibition.
- get [S]max from the plot of v~[s].
- at low substrate concentration, obtain Vm and Km’
graphicallyor through direct calculation.
- calculate KI through
[S ]max  (K m' K )1/ 2
SI

If no, then examine the data with and without inhibitors in

1/v ~ 1/[S] plot (Lineweaver-Burk Plot).
Estimation of Inhibited Enzyme
Kinetics
Inhibitor
 Example
 Apple turns into brown due to enzyme o-diphenol oxidase
 Tanpa inhibitor
Tube A Tube B Tube C Tube D
[S] 4.8 mM 1.2 mM 0.6 mM 0.3 mM
1/[S] 0.21 0.83 1.67 3.33
Δ OD540 (Vi) 0.081 0.048 0.035 0.020
1/Vi 12.3 20.8 31.7 50.0

•Vmax = 0.10
•Km = 1.25 mM
• [S] = 1.25 mM  Vi = 0.05
or 1/2x Vmax.)
 Dengan inhibitor
para-hydroxybenzoic acid (PHBA)

Tube A Tube B Tube C Tube D Vmax = 0.10.

Km = 2.50 mM
[S] 4.8 mM 1.2 mM 0.6 mM 0.3 mM
[S] of 2.50
1/[S] 0.21 0.83 1.67 3.33
mM1/2 Vmax.)
ΔOD540Vi) 0.060 0.032 0.019 0.011
1/Vi 16.7 31.3 52.6 90.9
phenylthiourea
Tube A Tube B Tube C Tube D
[S] 4.8 mM 1.2 mM 0.6 mM 0.3 mM •Vmax = 0.05.
1/[S] 0.21 0.83 1.67 3.33 •Km = 1.25 mM
ΔOD540 [S] = 1.25 1/2 Vmax
0.040 0.024 0.016 0.010
(Vi)
1/Vi 25 41 62 102
 Tanpa Comp Non-Comp
vmax 0.1 0.1 0.05
Km 1.25 2.5 1.25
0,1

0,09

0,08

0,07

0,06
[vp]

0,05 Tanpa inhibition

0,04 Competitive
0,03 Non-competitive
0,02

0,01

0
0 2 4 6 8 10
[S]