ENZYME

Hadiyanto
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
reactants
products
H
E
A
T.S.
catalyst
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

pH
Enzyme pH Optimum
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


cheese
soy products
yoghourt
wine, beer
bread


Enzymes

adjust food flavour
adjust food texture
improve nutritional
quality
high fructose corn syrup



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
substrate
products
Lock and Key
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
n
S m
n
S m
C K
C V
V

"
Enzyme with cooperative binding that has more than one active site. Mostly is
regulatory enzyme.
n: cooperative coefficient
Langmuir plot

Lineweaver-Burk plot

Exercise
-2,00
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
-0,60 -0,40 -0,20 0,00 0,20 0,40 0,60 0,80
1
/
V
o

1/[S]
conditio
n A
Eadie-Hofstee plot

Inhibitor
Competitive
Non-competitive
Substrate inhibitor
Inhibited Enzyme Kinetics
Competitive inhibitors (I)
Assume rapid equilibrium and with the
definitions of
] [ ] [ ] [ ]
0
[ EI ES E E
] [
] [
2
ES k
dt
P d
v
] [
] ][ [
1
1 '
ES
S E
k
k
m
K

] [
] ][ [
EI
I E
I
K
Inhibited Enzyme Kinetics
Competitive inhibitors (I),
we can obtain,
] [ ) / ] [ 1 (
'
] [
S
I
K I
m
K
S
m
V
v

] [
'
,
] [
S
app m
K
S
m
V
v

) / ] [ 1 (
' '
,
I
K I
m
K
app m
K When [I] =0,
' '
, m
K
app m
K
Inhibited Enzyme Kinetics
Noncompetitive inhibitors (I)
Assume:
- rapid equilibrium

- same equilibrium constants of inhibitor binding
to E and ES K
I

- same equilibrium constants of substrate binding
to E and EI
Km
Inhibited Enzyme Kinetics
Noncompetitive inhibitors (I)
] [ ] [ ] [ ] [ ]
0
[ ESI EI ES E E
] [
] [
2
ES k
dt
P d
v
] [
] ][ [
] [
] ][ [
1
1 '
ESI
S EI
ES
S E
k
k
m
K

] [
] ][ [
] [
] ][ [
ESI
I ES
EI
I E
I
K
Inhibited Enzyme Kinetics
Noncompetitive inhibitors (I)
we can obtain,
]) [
'
)( / ] [ 1 (
] [
S
m
K
I
K I
S
m
V
v

] [
'
] [
,
S
m
K
S
app m
V
v

) / ] [ 1 /(
,
I
K I
m
V
app m
V
When [I] =0,
m
V
app m
V
,
Uncompetitive inhibition
Inhibited Enzyme Kinetics
Uncompetitive inhibitors (I)
] [ ] [ ] [ ]
0
[ ESI ES E E
] [
] [
2
ES k
dt
P d
v
] [
] ][ [
1
1 '
ES
S E
k
k
m
K

] [
] ][ [
ESI
I ES
I
K
Inhibited Enzyme Kinetics
Uncompetitive inhibitors (I)
we can obtain,
] [ )) / ] [ 1 /(
'
(
] ))[ / ] [ 1 /( (
S
I
K I
m
K
S
I
K I
m
V
v

] [ ,
'
] [
,
S app
m
K
S
app m
V
v

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

- in Lineweaver-Burk
Plot
Inhibited Enzyme Kinetics
Uncompetitive substrate inhibitors
- can cause inhibition at substrate
concentration
- bind only to the .
Assume rapid equilibrium,
E P
k
ES
2
E+S
+
S
ES2
K
SI
Km
Substrate Inhibition
Inhibited Enzyme Kinetics
Uncompetitive substrate inhibitors (I)
] [ ] [ ] [ ]
0
[ 2 ES ES E E
] [
] [
2
ES k
dt
P d
v
] [
] ][ [
1
1 '
ES
S E
k
k
m
K

] [
] ][ [
2 ES
S ES
SI
K
Inhibited Enzyme Kinetics
Uncompetitive substrate inhibitors (I)
we can obtain,
SI
K S S
m
K
S
m
V
v
/
2
] [ ] [
'
] [

] [
'
] [
S
m
K
S
m
V
v

At low substrate concentration

SI
K S /
2
] [
<<1
SI
K S
m
V
v
/ ] [ 1

At high substrate concentration 1 ] /[

'
S
m
K
Michaelis-Menten Equation
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
0 ] [ / )
/
2
] [ ] [
'
] [
( ] [ /

S d
SI
K S S
m
K
S
m
V
d S d dv
2 / 1
)
'
(
max
] [
SI
K
m
K S
2 / 1
)
'
(
max
] [
0 /
2
] [
'
0 )
2
) /
2
] [ ] [
'
(
/
2
] [
'
0 )
2
) /
2
] [ ] [
'
(
) / ] [ 2 1 ]( [ ) /
2
] [ ] [
'
(
(
0 )
2
) /
2
] [ ] [
'
(
) / ] [ 2 1 ]( [
(
/
2
] [ ] [
'
0 ] [ / )
/
2
] [ ] [
'
] [
( ] [ /
SI
K
m
K S
SI
K S
m
V
m
K
m
V
SI
K S S
m
K
SI
K S
m
V
m
K
m
V
SI
K S S
m
K
SI
K S S
m
V
SI
K S S
m
K
m
V
SI
K S S
m
K
SI
K S S
m
V
SI
K S S
m
K
m
V
S d
SI
K S S
m
K
S
m
V
d S d dv

Uncompetitive substrate inhibitors (I)

Determine [S]max:
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



If no, then examine the data with and without inhibitors in 1/v
~ 1/[S] plot (Lineweaver-Burk Plot).
2 / 1
)
'
(
max
] [
SI
K
m
K S
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
OD
540
(V
i
) 0.081 0.048 0.035 0.020
1/V
i
12.3 20.8 31.7 50.0

V
max
= 0.10
K
m
= 1.25 mM
[S] = 1.25 mM V
i
= 0.05
or 1/2x V
max
.)

Dengan 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
OD
540

(V
i
)
0.040 0.024 0.016 0.010
1/V
i
25 41 62 102


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
OD
540
V
i
) 0.060 0.032 0.019 0.011
1/V
i
16.7 31.3 52.6 90.9
Vmax = 0.10.
Km = 2.50 mM
[S] of 2.50 mM1/2 Vmax.)


V
max
= 0.05.
K
m
= 1.25 mM
[S] = 1.25 1/2 V
max

para-hydroxybenzoic acid (PHBA)
phenylthiourea
Tanpa Comp Non-Comp
vmax 0.1 0.1 0.05
Km 1.25 2.5 1.25
0
0,01
0,02
0,03
0,04
0,05
0,06
0,07
0,08
0,09
0,1
0 2 4 6 8 10
[
v
p
]

[S]
Tanpa inhibition
Competitive
Non-competitive