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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 (pancreas) 8.0
Lipase (stomach) 4.0 - 5.0
Lipase (castor oil) 4.7
Pepsin 1.5 - 1.6
Trypsin 7.8 - 8.7
Urease 7.0
Invertase 4.5
Maltase 6.1 - 6.8
Amylase (pancreas) 6.7 - 7.0
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
wine, beer improve nutritional
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" CSn
Langmuir plot
Lineweaver-Burk plot
14.00
condition
A 12.00

10.00

8.00

Exercise

1/Vo
6.00

4.00

2.00

0.00
-0.60 -0.40 -0.20 0.00 0.20 0.40 0.60 0.80
-2.00
1/[S]
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 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 - 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 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 ]
' /[S ] 1
At high substrate concentration K m
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
graphically or 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 Vmax = 0.10
1/[S] 0.21 0.83 1.67 3.33 Km = 1.25 mM
OD540 (Vi) 0.081 0.048 0.035 0.020 [S] = 1.25 mM Vi = 0.05
or 1/2x Vmax.)
1/Vi 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
mM1/2 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
1/[S] 0.21 0.83 1.67 3.33 Vmax = 0.05.
OD540 Km = 1.25 mM
(Vi)
0.040 0.024 0.016 0.010 [S] = 1.25 1/2 Vmax
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]

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