AC(Enzyme Kinetic) Rev
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Transcript of AC(Enzyme Kinetic) Rev
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ENZYME KINETIC
By: Luh Lian Pertiwi (0913031030)
Chemistry Education Department, Faculty of Mathematics and Natural Sciences
Ganesha Education University
Abstract
The enzyme is a biocatalyst that can increase the speed of reactions in biological systems and
enzymes affect reaction rates when equilibrium is reached, but did not affect the total
equilibrium of the reaction. Enzymes assist the reaction by providing a reaction pathway that
has a low activation energy for the substrate into a product transition. Reaction rate of the
reaction catalyzed by the enzyme increases with the addition of substrate concentration, until
reaching a state with the addition of the substrate concentration does not affect the initial
reaction rate. This situation is referred to as the maximum reaction rate (Vmax). The aim of this
experiment is to determine Vmax and KM for enzymetic rate reaction and substrat concentration.
According to graph of relationship 1/Vo to 1/[S], it can be determined Vmax and KM value.According to the experiment, KM value and VMax value of enzymetic rate recation of tripsin with
kasein is 8,84 mg/mL and 1,938 mol/menit.
Keyword : enzyme, Vmax and KM.
Introduction
In the reaction, enzyme can react with the reactants and also can be recovered at the time
of formation of the product. The phenomenon is what causes the enzyme can be regarded as a
catalyst. Enzymes are the catalysts of biochemical reactions that play a role in the specific
enzyme is often referred to as a biocatalyst. As a biocatalyst, the enzyme can increase the speed
of reactions in biological systems and the enzyme itself does not change during the reaction. Inaddition, the enzyme also affects the reaction rate when equilibrium is reached, but did not affect
the total equilibrium of the reaction. Enzymes assist the reaction by providing a reaction pathway
that has low activation energy for the transition into a product compared to the substrate without
the catalysis process. The catalytic activity of the enzyme is very sensitive to conditions such as
temperature, pH, and the strength of the product. Enzyme reaction kinetics assumes that a
complex between the enzyme and the substrate is formed immediately and reversibly. This
complex then breaks down slowly and produces enzymes.
The speed of biochemical reactions that are catalyzed by an enzyme commonly used as
the value at time zero (Vo symbol, within minutes mol-1), because the highest velocity occurs in a
state where the reaction product has not been established. In these circumstances, the greatest
concentration of substrate and enzyme is not inhibited by its product feedback. Plot of the
reaction products formed with time on the enzymatic reaction showed that the initial stage occurs
a linear graph, and then followed by a decrease in reaction rate because most of the substrate has
been used in the reaction or the enzyme activity decreased. Initial reaction velocity (Vo) is
obtained by drawing a straight line through the linear curve at zero time point and the slope of a
straight line equal to Vo.
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Reaction rate depends on the concentration of enzyme that acts as a catalyst in a reaction.
At low substrate concentration, if the substrate concentration is increased two times, then the
initial reaction velocity (Vo) has doubled. This means that at low substrate concentration of
enzymatic reaction rates of order one. However, at high concentrations, increasing the substrate
concentration will lead to changes in Vo is very small and at very high substrate concentrations,
increasing the substrate concentration will not affect the price of V o (Vo constant prices). This
situation is caused because the enzyme was saturated by the substrate, or in other words the
entire active enzyme has a substrate binding.
The reaction rate increases as substrate concentration increased up to a point where said
enzyme is saturated with substrate. The measured initial velocity reaches a maximum speed and
is not affected by the subsequent increase in substrate concentration, because the substrate is in
molar excess. Overall reaction rate depends on the speed of product dissociation from the
enzyme, and the addition of the substrate will not affect Vo. Plot Vo against [S]-shaped
hyperbolic.
(a) (b)
(b)Figure 1 (a). The relation concentration of substrate and Vo plot
(b). The relation of 1/Vo and 1/[S] plot Lineweaver Burk
Michaelis and his friends claimed that the enzymatic reaction at various concentrations of
substrates having two phases, namely: (1) when a low substrate concentration ([S]) is low, then
not all of the active enzyme bound to the substrate and (2) if molecule of the substrate increases,
the active side was entirely bound to the substrate. At this time the enzyme has been working
complete. From the capacity Michaelis and friends statement can also be proven by metematics.
The curve in Figure 1 (a) Michaelis-Menten equation satisfies is follows:
[S]K
[S]VV
M
maks
o+
=
Where : Vo = the velocity of enzyme by the concentration of substrate [S]
KM = Michaelis Menten constant (mol perliter)
Vmaks = Maximum velocity
From the equation above its can be:
Kinetic Zero-order (Fase II)
Mixture kinetic zero and
first-order
KineticFirst-order(Fase I)
Maximum Velocity (V)
V0
Vmaks
Vmaks
KM
[S]
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E + Sk1
k2
k3k4
ES E + P
The specificity of an enzyme catalyzed by the enzyme appears to the formation of the
enzyme substrate complex (ES), which then decomposes into enzyme and products (E and P). In
this case k1, k2, k3, and K4 is the reaction rate constants. Initially be achieved disintegration of thestate of the enzyme rate equal to the speed of formation of ES.
k1[S][E] + k4 [E][P] = k2 [ES] + k3 [ES]
32
4
32
1
kk
[P]k
kk
[S]k
[E]
[ES]
+
+
+
=
In the condition of concentration is P (product) is very small so the constant of k1, k2, k3
and k4 can be written is as follows:
KM =1
32
k
kk +.
The equation is:
[S]
K
[ES]
[E] M=
If the amount of total concentration of enzyme [E]t and [ES] so the concentration [E] =
[E]t [ES], as follows:
[S]
K1
[ES]
[E]
[ES]
[ES][E]
[ES]
[E] Mtt==
=
1[S]
K
[ES]
[E] Mt+=
Maximum celocity (V if maks) if the enzyme the shape is complete if all of enzyme in
complex form with the substrate. But Vo directly proportional with [ES]. Its can be written is as
follows:
1[S]
K
V
VsO
[ES]
[E]
V
V Mmakstmaks+==
In the figure 1 (a) can be seen that KM as mole perliter. And KM is higher so can be written
is as follows:
M
maks
K
[S]VV=
In this case V depends on the substrate concentration (filtrate reaction order) and in
Figure 1 (a) can also be seen kinetic and first-order zero. Thus, the Michaelis-Menten equation iseligible for a simple reaction that is catalyzed by the enzyme. KM is often defined as the
dissociation constant of the reaction catalyzed by the enzyme. In the simple reaction can be seen:
E + Sk1
k2ES maka K s=
[E][S]
[ES]=
k2k1
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Because KM is1
32
k
kk +so Ks =
1
32
k
kk +
So, KM will always same or bigger than Ks. can be seen from the equation:
maksmaks
M
M
maks
V
1
[S]
1
V
K
V
1So
[S]K
[S]VV +
=
+
=
The equation above is identical with line equation is y = ax + b, where:
y =maksmaks
M
V
1bdan
V
Ka;
[S]
1x;
V
1===
The equation can be formulated by Lineweaver-Burk and like figure 1 (b). Based in the
figure 1 (b), line equation with the gradient inoV
1is
maksV
1, in
[S]
1is
MK
1 . The plot of
line ismaks
M
V
K. Plot Lineweaver-Burk can be used to determine inhibition that occurs in the
enzymatic reaction.
Spectrophotometer
Spectrophotometer is the method that used to qualitative and quantitative analysis in
solution. Spectroscopy imply the measurements how far the light energy absorption as a function
of the wavelength of radiation. Instruments that used in this method its called by
spectrophotometer. In analyzing the uses of UV-Vis spectrophotometer should note the
following:
a. Formation of colored compounds
This step is conducted if the analyzed compounds not absorbing visible region. In thiscase the compound must be converted into other compounds that can do the absorption or
reacted with a reagent that form color so can absorb visible light. Reagents that cause
color, must meet several requirements such as: reaction with a substance that is analyzed
to be selective and sensitive, do not form colored compounds with other substances that
exist in solution, they react with other substances which must be analyzed quickly and
quantitatively (perfect), color or compounds that are formed must be sufficiently stable
for a specified period, and not too fast changes with changes in pH.
b. Selection of wavelength
Wavelengths required in a quantitative analysis by spectrophotometry is the wavelengthcorresponding to maximum absorbance (peak absorption). This is caused by changes in
absorbance for each unit of concentration is greatest at the maximum wavelength, it will
obtain the maximum sensitivity as well. Wavelengths required in a quantitative analysis
by spectrophotometry.
Description:
Violet : 400 - 420 nm
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Indigo : 420 - 440 nm
Blue : 440 - 490 nm
Green : 490 - 570 nm
Yellow : 570 - 585 nm
Orange : 585 - 620 nm
Red : 680 780 nm
c. Preparation of calibration curve
For calibration curves, created a standard solution with various concentrations of the
unknown. Absorbance of standard solution is measured, and then plots the absorbance
(A) against concentration (C), the curve formed is called the calibration curve.
In the analysis using UV-Vis spectroscopy is used the basic law of Lambert-Beer.
Lambert-Beer law states the relationship between the intensity of light absorbed by the
concentration and the thick solution through which the beam. If a beam of light with a
certain length is passed in a solution containing absorbent material, some light will be
absorbed and some light transmitted. Simply, Lambert-Beer law can be shown in thefollowing scheme.
Light or radiation with the intensity Po of that passes through a medium-thick b
containing a solution with concentration C, will result in reduced intensity of P so that P
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Information obtained from ultraviolet and visible spectra:
1. The maximum wave length ( maks )
Quantitative analyse, for all the measure based on analisis kuatitatif, maks , but forthe compound that already know frequently used approach Woodward-Fieser.
Qualitative analyses : less informatif.
2. Absorptivity maximum of molar( maks ); qualitative analyses is the information transisi
electronic.
Lambert-Beer Laws
If a beam of light with specific wavelength is passed in a solution containing absorbent
material, then some light will be absorbed and some will be forwarded. The relationship between
the intensity of light absorbed by the concentration and the thick solution through which thebeam expressed by Lambert-Beer equation as follows.
A = -log I / Io = bC
(Muderawan,2009)
Where, A = absorbance
I = Intensity of light emitted by the solution in the cell
Io = intensity of light emitted by the solvent in the same cell at I
= coefficient of extinct species absorbent or constant comparison.
b = Length of solution through which the light (typically 1 cm)
C = Concentration of absorbing species in units of mol L-1 (M)
Slope = b
The relation between concentration
and absorbance
The relation between
concentrations with transmittance
C
(mg/
L)
T
Lambert-Beer diagram
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(Ismono,1978)
The greater the intensity of light is absorbed then the value of A will become bigger and
the intensity of the transmitted beam will be smaller. Often times in real terms in the
measurement result, polt. Beer law is not linear throughout the entire concentration observed.
Curvature states that is not a constant, which depends on the concentration. value is expected
to depend on the nature absorbance agent species in solution and at a wavelength of radiation. In
practice the deviation is usually caused by the deviation on the basis of chemical and diversion of
the instruments used.
Materials and Method
In this experiment about the determine the protein content of the egg whites in the
sample solution through the use of methods of Lowry at Thursday 13 April, 2012. This
experiment conducted in Organic Laboratory Undiksha.
Materials and equipment
The materials that used in this experiment are casein solution, Folin-Ciocalteu reagent,
aquades, trypsine solution, NaOH solution, and TCA solution and the equipment that used are
test tube, volumetric pipette, volumetric flask, drop pipette, graduated cylinder glass and beaker
glass.
Procedure
Prepare 10 test tubes. In each of test tube added by casein, buffer and trypsin in different
concentration like is as follows:
No Test tube Casein (mL) Buffer phosphate
(mL)
Trypsin (mL)
It= 0 minute 0,1 5,9 1
t = 20 minute 0,1 5,9 1
IIt= 0 minute 0,5 5,5 1
t = 20 minute 0,5 5,5 1
IIIt= 0 minute 1,0 5,0 1
t = 20 minute 1,0 5,0 1
IVt= 0 minute 3,0 3,0 1
t = 20 minute 3,0 3,0 1
V t= 0 minute 5,0 1,0 1
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t = 20 minute 5,0 1,0 1
At the incubation 20 minutes
1. In each test tube that contain casein incube 5 minute in water incubator 35oC. stirrer and
add buffer phosphate solution pH 8,0 and trypsin solution (based on the table).
2. The solution 20 minutes in water incubator35oC after added trypsin.
3. After incubation 20 minutes add 3 mL of TCA 20% solution into each of test tube. Than
cooled in 30 minute in ice batch to get the precipitate perfect.
4. After that filter the solution by Anson method. 2 mL of filtrate add by 4 mL NaOH 0,5
M, then add the reagent Folin-Ciocalteu wait 10 minute. Measure the absorbance by
spektronik 20+.
At the incubation 0 minute
1. In each test tube add buffer fosfat solution and trypsin. Than add the TCA solution 20%
incube 5 minute in water incubator 35oC.
2. In each test tube added by casein solution ditambahkan larutan kasein. Than cooled in 30
minute in ice batch to get the precipitate perfect.
3. After that filter the solution by Anson method. 2 mL of filtrate add by 4 mL NaOH 0,5
M, then add the reagent folin ciocalteu wait 10 minute. Measure the absorbance by
spektronik 20+.
Result and Discussion
In this experiment tests performed is the enzyme kinetics reaction, where the value of
these tests can be determined Vmax and KM (Michaelis-Menten constant) over the curve of therelationship between the reaction rate and substrate concentration.
In this experiment used as the substrate is a casein solution, while being used as the
enzyme is trypsin solution. Trypsin is a proteolytic enzyme, this enzyme catalyzes the hydrolysis
of the peptides bond, where in the peptide bonds were broken in the substrate casein is located on
the carboxyl side of lysine or arginine residues are charged.
N C
H
H
C
R1
O
N
H
C
H
R2
C
O
+ H2O N
H
C C
R1
O
O-
H
++H3N C C
H
R2
O
Peptides Carboxcyl Amine
There are two treatments (time) is done in this experiment the incubation time of 0
minutes and 20 minutes. In this experiment provided 10 tubes, for an incubation time of 0
minutes, while the tubes for 20 min incubation. In this case also do a variation of the
concentration of substrate in each tube. For the incubation of 20 minute, casein solution is
incubation first
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Incubation of casein in 5 minute
This treatment of substrate conditions at maximum temperature is 35oC. Into the casein
solution that incube add the buffer fosfat solution (pH 8,0) so the volume become 6 mL in each
test tube. The objective of dilution is to make the substrate condition in each test tube are
different so the plot of reaction rate is different. Then the solution is added by trypsine solution
1 mL in each test tube. The addition of trypsin solution aims to catalyze the hydrolysis reaction
under on the peptide bonds in casein. Peptide bonds were broken by trypsin is the bond that lies
at the carboxyl side of lysine and arginine residues.
Incubation at 20 minute
After the incubation the solution is added by TCA ( tri chloro acetat). The aim of this
solution is to stop the trypsine enzyme so can be analysis the effect of substrate concentration
in enzyme of reaction rate. The addition of TCA 20% solution that caused the precipitate form
in the test tube after cooled 30 minute.
Brown precipitate which formed a precipitate of casein protein and the enzyme trypsin. The
addition of a solution of TCA was able to stop the activity of the enzyme and substrate solution
of casein given TCA solution is acidic, so the addition of either TCA can cause denaturation of
the enzyme and the casein. Structures that cause clotting TCA are as follows.
C CCl
Cl
Cl
O
OH
Structure of TCA
With the addition of acid to the solution, causing denaturation protein both the substrate and the
enzyme trypsin. Also, the addition of TCA solution causes the pH of the acidic solution to be
down (acid) so that the substrate complex formed is unstable and the activity of the enzyme to
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catalyze the hydrolysis of casein in a standstill due to changes in pH. This is because the enzyme
trypsin to work at optimum pH is pH 8.0.
After filtering the precipitate, the resulting filtrate treated with the method of Anson,
where the filtrate to each tube to 2 mL after adding 4 ml of 0.5 M NaOH was added Folin-
Ciocalteu reagent. Folin-Ciocalteu reagent is a mixture between fosfotungstat and fosfomolibat
in yellow. These reagents can be reduced by the phenolic group on the amino acid tyrosine in
the filtrate contained tungstate and molybdenum producing blue-green. Based on the results of
the experiment, when the filtrate was added Folin-Ciocalteu reagent green in solution. The
resulting solution is then measured by using UV vis 20+.
For the 0-minute incubation time, the order is different from the incubation of 20
minutes, which at time 0 minute incubation in a row was added phosphate buffer, trypsin
solution, a solution of TCA to each tube. After incubation for 30 minutes then added a solution
of casein at varying volume for each test tube. Calculation of substrate concentration for each
tube as follows.
The filtrate obtained is done by Anson, where the filtrate to each tube to 2 mL after
adding 4 ml of 0.5 M NaOH was added Folin-Ciocalteu reagent. Folin-Ciocalteu reagent is a
mixture between fosfotungstat and fosfomolibat in yellow. These reagents can be reduced by the
phenolic group on the amino acid tyrosine in the filtrate contained tungstate and molybdenum
producing blue-green. Based on the results of the experiment, when the filtrate was added Folin-
Ciocalteu reagent the color is green. The resulting solution is then measured using a
spectrophotometer at a wavelength of 700 nm. Based on the experiments conducted, the
following is a comparison of observations absorbance for tube t = 0 and t = 20.
Table. Observation result of absorbance
No Test tube Absorbance Transmitant (%)
I t = 0 minute 0.096 81t = 20 minute 0.139 85
IIt = 0 minute 0.184 92
t = 20 minute 0.187 83
IIIt = 0 minute 0.211 72
t = 20 minute 0.229 89
IVt = 0 minute 0.259 75
t = 20 minute 0.387 88
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To calculate and determine the value of KM and VMax, so be must determine the value of
[S] and [Vo]. The value [S] concentration of casein in the test tube is different value. The
calculation is as follows:
Massa of casein that added is 5 gram in 250 mL aquades.
[casein] = mL500
gram5
= 10 mg/mL
Test tube 1
V1 = 0,1 mL; V2 = 6 mL; M1 = 10
mg/mL
M2=
mg/mL6
1
mL6
mg/mL10xmL0,1=
[S] = mg/mL6
1
6.mL/mg[S]
1=
Test tube 4
V1 = 3,0 mL; V2 = 6 mL; M1 = 10
mg/mL
M2 =
mg/mL5mL6
mg/mL10xmL3,0=
[S] = mg/mL5
mL/mg2.0[S]
1=
Test tube 2
V1 = 0,5 mL; V2 = 6 mL; M1 = 10
mg/mL
M2 =
mg/mL6
5
mL6
mg/mL10xmL0,5=
[S] = mg/mL6
5
mL/mg1.2[S]1 =
Test tube 3
V1 = 1,0 mL; V2 = 6 mL; M1 = 10
mg/mL
M2 =
mg/mL3
5
mL6
mg/mL10xmL1,0=
[S] = mg/mL3
5
mL/mg0.6[S]1 =
For the calculation of Vo can be determine the value of absorbance by spectrophotometer
dapat ditentukan dengan besaran absorbansi ketika diukur dengan spektronik 20+. The absorbance
is as follows:
A = .b.C
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Because .b is constant, so can assume that .b is constants. So, A = k x C, where A =
absorbance C = concentration k = constants. Based on the equation so AC
So the equation of velocity iso
ot
[C]V =
ot
AV =
oV
1
A
1=
The calculation of absorbance can be calculating the absorbance in t=20 and t=0 so theabsorbance:
Incubation time o minute Incubation time 20 minute
Test tube I
%T = 81; T = 0.81
A = -log T = -log 0.81 = 0.096
1/A = 1/0.096 = 10.41
1/V0 = 10.41
Test tube II
%T = 92; T = 0.92
A = -log T = -log 0.92 = 0.184
1/A = 1/0.184= 5.43
1/V0 = 5.43
Test tube III
%T = 72; T = 0.72
A = -log T = -log 0.72= 0.211
1/A = 1/0.211= 4.73
1/V0 = 4.73
Test tube IV
%T = 75; T = 0.75
A = -log T = -log 0.75=0.259
1/A = 1/0.259= 3.86
1/V0 = 3.86
Test tube I
%T = 85; T = 0.85
A = -log T = -log 0.85 = 0.139
1/A = 1/0.139 = 7.19
1/V0 = 7.19
Test tube II
%T = 83; T = 0.83
A = -log T = -log 0.83 = 0.187
1/A = 1/0.187= 5.34
1/V0 = 5.34
Test tube III
%T = 89; T = 0.89
A = -log T = -log 0.89= 0.229
1/A = 1/0.229= 4.36
1/V0 = 4.36
Test tube IV
%T = 88; T = 0.88
A = -log T = -log 0.88= 0.387
1/A = 1/0.387 = 2.58
1/V0 = = 2.58
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Based the calculation the table is as follows:
Tabel 2. Calculation table of 1/A
No Test tube %T T A 1/A
It = 20 minute 81 0.81 0.096 10.41
t = 0 minute 85 0.85 0.139 7.19
IIt = 20 minute 92 0.92 0.184 5.43
t = 0 minute 83 0.83 0.187 5.34
IIIt = 20 minute 72 0.72 0.211 4.73
t = 0 minute 89 0.89 0.229 4.36
IVt = 20 minute 75 0.75 0.259 3.86
t = 0 minute 88 0.88 0.387 2.58
Based on the table can make curve of the relation between 1/V 0 versus 1/[S] at the time of
incubation 0 minute and 20 minute.
The curve of the relation between 1/V0 versus 1/[S] at the incubation time o minute is as
follows:
Test Tube 1/V0 1/[S] (mL/mg )
1 10.41 6
2 5.43 1.2
3 4.73 0.6
4 3.86 0.2
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From the curve we y get the line equation is 1.086x + 3.934The relation with the Lineweaver-Burk can be calculate by 1/Vmax, 1/[S] and -1/KM. Lineweaver-
Burk equation:
maksmaks
M
oV
1
[S]
1
V
K
V
1+
=
From the equation, the value ofmaksV
1when the x = 0, so
y = 1.086x + 3.934
y = 1.086 0 + 3.934
y = 3.934so that,
maksV
1= 3.934
Vmaks = utemol min/25.03.934
1=
So when the[S]
1is -
MK
1, its same with y = 0, so
y = 1.086x + 3.934
0 = 1.086x + 3.934
1.086x = -3.934
x = -3.62
its means the -MK
1= -3.62
KM =3.62
1= 0.276 mg/mL
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The curve of the relation between 1/V0 versus 1/[S] at the incubation time 20 minute is as
follows:
Test Tube 1/V0 1/[S] (mL/mg )
1 7.19 6
2 5.34 1.2
3 4.36 0.6
4 2.58 0.2
From the curve we get the line equation is 0.629x+ 3.609
The relation with the Lineweaver-Burk can be calculate by 1/Vmax, 1/[S] and -1/KM. Lineweaver-
Burk equation:
maksmaks
M
oV
1
[S]
1
V
K
V
1+
=
From the equation, the value ofmaksV
1when the x = 0, so
y = 0.629x+ 3.609y = 0.629 0+ 3.609
y = 3.609
so that,
maksV
1= 3.609
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Vmaks = utemol min/277.03.609
1=
So when the[S]
1is -
MK
1, its same with y = 0, so
y = 0.629x+ 3.609
0 = 0.629x+ 3.609
0.629x = -3.609
x = -5.73
its means the -MK
1= -5.73
KM =5.73
1= 0.174mg/mL
The value of KM at the incubation 20 minutes is 0.174 mg/mL and the incubation time at 0
minute is 0.276 mg/mL. From the experiment data the value of KM at the incubation 20 minutelower than the incubation in 0 minute. The value of KM show the stability complex of enzyme
and the substrate where the value of KM is higer its mean the bond of enzyme and substrate is
less. From the value of KM can be concluded that if the time is increase so the complex enzyme
and substrate will stable because the bond of the enzyme and substrate is strong.
Conclusion
Based on the data result and data analyze that we have been done, so can be concluded
the value of KM from the experiment that have been done is 0.174 mg/mL in 20 minute and at 0
minute the value of KM is 0.276 mg/mL.
Acknowledgment
Thanks to the god almighty who has bless me to finishing this article with the title Enzyme
Kinetic. This article is made for me assignment in Biochemistry Practice lesson. In process to
create this paper, the author found some problem. It because of my knowledge still need to
improvement. But because of the guidance and big support from many peoples, so I can finish it.
Therefore, the authors want to say thanks to the:
1. Mr. Dr. I Nyoman Tika, M.Si. as my lecturer who has guide me,
2. Mrs. Dra. Siti Maryam, M.Kes. as my lecturer who has guide me,
3. My best friend in RKBI Class who has supported me, and
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4. Many other peoples who has supported me.
This article is still need improvement and still contains many of mistakes, because of that
authors need advice and critics to make this article will better and can useful for many people.
Thank you.
References
Ismono. 1978. Cara-cara Optik Dalam Analisis Kimia. Diktat. Bandung: Jurusan Kimia ITB.
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