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
 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
its 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

Have names that

usually end in -ase.
-Sucrase
-Lactase
-Maltase
 Formats for writing a enzymatic
reaction.
Enzyme

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

( Enzyme )

Substrate -----------> Product+ 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 cant 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
bodys 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: Whats

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
 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, Andr Karwath; Enzyme

with Cofactor, Wiki; pH scale, Edward Stevens, Wiki From the Virtual Cell Biology Classroom on ScienceProfOnline.com
 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 ) ' '
Km , app K m I When [I] =0, K m,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 ]
(K m I
Vm, app[ S ]
v
Km ' , app [ S ]

Vm, app Vm /(1 [ I ] / K I ) K m ' K ' /(1 [ I ] / K )

, app m I
' '
K m, app / Vm, app K m / 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 <<1
SI
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 (Km 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 its 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 (Km ' 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
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
mM1/2 Vmax.)
OD540Vi) 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
OD540 [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]