Enzyme

Hadiyanto
What is an enzyme?
A biological catalyst that promotes and
speeds up a chemical reaction without
itself being altered in the process.
Lowers the activation energies of a
substance
Energy Profile
T.S.
catalyst

EA

products
reactants

H
Structure of an
enzyme
Contains both a protein and
a nonprotein.
Nonprotein is either a
coenzyme (usually a vitamin)
or a cofactor (usually a
mineral).
Enzyme Nomenclature
Names usually end in ase.
Usually named after substrates they
act upon e.g. urea --- urease
lactose --- lactase
or the resulting type of chemical
reaction e.g. hydrolysis --- hydrolases
oxidation --- oxidases
This rule does not always apply. E.g.
ficin found in figs and papain in
papayas.
Factors influencing
enzyme activity
Operate under optimum
conditions of pH and
temperature.
Easily inactivated
(denatured) in presence
of inhibitors.
T dan pH
 Enzyme pH Optimum
pH Lipase 8.0
(pancreas)
Lipase (stomach) 4.0 - 5.0
Lipase (castor 4.7
oil)
Pepsin 1.5 - 1.6
Trypsin 7.8 - 8.7
Urease 7.0
Invertase 4.5
Maltase 6.1 - 6.8
Amylase 6.7 - 7.0
(pancreas)
Amylase (malt) 4.6 - 5.2
Catalase 7.0
Effect of substrate
Properties of enzymes
Control ripening.
Cause food spoilage (rotting).
Responsible for changes in flavor, color, texture
and nutritional properties.
Can be inactivated by heat to extend storage
stability of foods.
Used for fermentation purposes in foods.
Can be immobilized to a surface of a membrane
or other inert object in contact with the food
being processed.
Can be extracted and purified to a high degree.
Properties
Used for fermentation purposes in foods.
Can be immobilized to a surface of a membrane
or other inert object in contact with the food
being processed.
Can be extracted and purified to a high
degree.
Applications in food industry
Carbohydrases: production of corn syrups
from starch (glucoamylase); conversion of
cereal starches into fermentable sugars in
malting, brewing, distillery, baking industry
(amylase).
Proteases: meat tenderizers (bromelin,
papain, ficin)
Lipases: Flavor production in chocolate and
cheese
Applications
Glucose oxidase: desugaring of eggs, flour and
potatoes; preparation of salad dressings.
Pectinases: clarification of fruit juices;
increase of yield of juice from grapes and
other products; removal of excess pectin from
juices before concentration.
Applications contd
Lipoxygenase: bleaching of flours.
Phosphatase: quality testing of food
products
Phenol oxidase: imparts the
characteristic dark hue to tea, cocoa,
coffee and raisins.
Renin (chymosin): cheese production
 Applications
Flavorases: restoration and enrichment of
flavor by addition of enzyme preparations to
food products e.g. fresh corn enzyme
extracts to improve flavor of cannned goods
or addition of alliinase to convert alliin of
garlic into garlic oil.
Industry Focus: Food and
beverage
Fermentation Products Enzymes

cheese adjust food flavour

soy products adjust food texture
yoghourt improve nutritional
wine, beer
bread quality
high fructose corn
syrup
Industry Focus: Food and
beverage
Food Enzymology
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
Reaksi pembentukan
produk
Pada konsentrasi rendah: reaksi order 1
Pada konsentrasi tinggi: perubahan dari order reaksi 1 ke 0
Maximum kecepatan reaksi proporsional terhadap konsentrasi
enzim
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.
n: cooperative coefficient
n
VmC
V S

K m" C Sn
Langmuir plot
Lineweaver-Burk plot
 14.00

12.00

10.00

8.00

Exercise
1/Vo
6.00

4.00

2.00

0.00
0.00 0.10 0.20 0.30 0.40
1/[S] 0.50 0.60 0.70 0.80
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 [ ES ]
dt 2

' 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 ]
'
Km K ' (1 [ I ] / K ) ' '
, app 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
KmEI

Inhibited Enzyme Kinetics
Noncompetitive inhibitors (I)
d [ P]
v k [ ES ]
dt 2

' 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 [ ES ]
dt 2

' 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
' , app [ S ]
Km

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

Km K ' /(1 [ I ] / K )
, app m I
' '
K m, app / Vm, app K m / Vm - in Lineweaver-
Burk Plot
Inhibited Enzyme Kinetics
Uncompetitive substrate inhibitors
- can cause inhibition at substrate
concentration
- bind only to the .
Assume rapid equilibrium,
Km
k2
E+S ES P E
+
S
K SI
ES2
Substrate Inhibition
Inhibited Enzyme Kinetics
Uncompetitive substrate inhibitors (I)
d [ P]
v k [ ES ]
dt 2

' 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
[ S ]2 / K SI <<1
At low substrate concentration
Vm [ S ]
v Michaelis-Menten Equation
'
K m [S ]
Km' /[ S ] 1
At high substrate concentration
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 its substrate inhibition.
- get [S]max from the plot of v~[s].
- at low substrate concentration, obtain Vm and
Km graphically or through direct calculation.
- calculate KI through
' K )1 / 2
[ S ]max ( K m 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

4.8 Vmax = 0.10
[S] 1.2 mM 0.6 mM 0.3 mM
mM
Km = 1.25 mM
1/[S] 0.21 0.83 1.67 3.33
OD540 (Vi) 0.081 0.048
[S] = 1.25 mM Vi
0.035 0.020
1/Vi
= 0.05 or 1/2x Vmax.)
12.3 20.8 31.7 50.0
 Dengan inhibitor
para-hydroxybenzoic acid (PHBA)

Tube A Tube B Tube C Tube D

Vmax= 0.10.
[S] 4.8 mM 1.2 mM 0.6 mM 0.3 mM Km= 2.50 mM
[S]of 2.50
1/[S] 0.21 0.83 1.67 3.33
mM1/2Vmax.)
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
1/[S] 0.21 0.83 1.67 3.33 Vmax = 0.05.
OD540 Km = 1.25 mM
0.040 0.024 0.016 0.010
(Vi) [S] = 1.25 1/2 Vmax
1/Vi 25 41 62 102
 Non-
Tanpa Comp Comp
vmax 0.1 0.1 0.05
0.1
Km 1.25 2.5 1.25
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 1 2 3 4 5 6 7 8 9

[S]