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(Chem 43) Expt. 5 ENZYME KINETICS OF ALPHA-AMYLASE PDF
(Chem 43) Expt. 5 ENZYME KINETICS OF ALPHA-AMYLASE PDF
5
ENZYME KINETICS OF α -AMYLASE
Cecilia, Jasmin S. (1), Chan, Janelle Mae A. (2), So, Shannen Louise T. (3)
CHEM 43 LB1A
Ms. Fatsy Cruz
ABSTRACT
Enzymes, most of which are proteins, play an important role as they increase the otherwise slow reaction
rates of biochemical reactions. This is possible when the substrate binds to the active site of an enzyme and
turns into its respective product, as is the case of the enzyme α -amylase used in this activity, which catalyzes
the hydrolysis of starch into glucose. In order to determine the effects of incubation time, temperature, pH,
and substrate concentration on the activity of α -amylase acquired from human saliva, a starch standard curve
was first prepared. This was used in determining starch concentrations from the absorbances of iodine-starch
complexes obtained at 620 nm using a UV-Vis spectrophotometer, as the time was varied from 0, 3, 5, 7, to
10 minutes; the temperature from 10, 40, 60, to 80 ° C; the pH from 2.0, 4.0, 6.7, 7.4, to 9.0; and finally, the
substrate concentration from 0.10, 0.08, 0.05, 0.02, to 0.01%. Plots of starch concentration versus each of the
variables were created, except for the variable of substrate concentration, for which a 1/V versus 1/[S] plot
was made and the KM and Vmax values were calculated through the Lineweaver-Burk equation. The standard
curve obtained, with an R2 value of 0.9307, had an equation of y = 95.432x + 1.2697. The following trends
regarding the four factors were observed: the longer the incubation time, the less the substrate concentration;
the plot for incubation temperature had a graph with an upward concavity; the plot for pH had a relative
minimum at around 7.05; and the greater the initial substrate concentration, the faster the reaction velocity.
Based from the Lineweaver-Burk plot obtained, a KM value of 0.011 and a Vmax value of 5.27*10-4. These were
calculated from the linear equation y = 21.212x + 1897.6, which had an R2 value of 0.9456. In conclusion,
optimal enzyme activity occurs at 10 minutes of incubation time, 40 ° C incubation temperature, a pH value of
around 7.05, and a relatively greater initial substrate concentration.
KEYWORDS: enzyme kinetics, α -amylase, iodine-starch complex, factors on enzyme activity, Lineweaver-Burk
equation, UV-Vis spectrophotometry
Enzymes have active sites that the target substrates Figure 1. Formation and Breakdown of the ES Complex
would bind to. These active sites are composed of amino Source: http://2016.igem.org/Team:Freiburg/Delivery
acids that would form a cleft or a pocket where the
substrate stays during the reaction. The cleft would provide This relationship can also be represented
a better environment for the reaction to proceed – by mathematically using Equation 1.
excluding water, for example. [3]
In this experiment, the researchers aimed to determine Effect of pH on Enzyme Activity. In 5 separate test
the effects of various factors that affect enzyme kinetics, to tubes, 2.50 mL of 0.2% starch solution, 0.5 mL of 0.9%
construct a standard curve for the determination of product NaCl solution, and 2.0 mL of 0.1 M phosphate buffer with
concentration, and to determine the values of kinetic varying pH were mixed, making the solutions have the
parameters, KM and Vmax. following pH: 2.0, 4.0, 6.7, 7.4, 9.0. To each test tube, 100
µL of the saliva solution was added. They were then
EXPERIMENTAL incubated for 5 minutes. From each test tube, 1 mL aliquot
Preparation of Starch Standard Curve. From 0.2% of each solution was put into separate test tubes, and 100
starch solution, 2 mL of the following starch concentrations µL of concentrated HCl was added to each. Before reading
were prepared in 0.1 M phosphate buffer (pH 6.7): 0% their absorbance at 620 nm, 20 µL of I2 in
KI solution and 5
(blank solution), 0.10%, 0.08%, 0.05%, 0.02%, 0.01%, and mL of distilled water was added to all test tubes. The starch
0.005%. An mL of each starch concentration was concentration vs pH was then graphed.
transferred to separate test tubes, and 20 µL of KI, along
with 5 mL of water, was added to each solution. The Effect of Substrate Concentration on Enzyme Activity.
absorbance of each solution was measured immediately at Using 0.2% starch solution and 0.1 M phosphate buffer (pH
620 nm, and the starch concentration vs absorbance was 6.7), a 4.5 mL starch solution with the following starch
plotted, generating a standard curve. concentrations were prepared in separate test tubes:
0.10%, 0.08%, 0.05%, 0.02%, and 0.01%. To each test
Effect of Incubation Time on Enzyme Activity. A 10 mL tube, 0.5 mL saliva solution was added before incubating
of 0.1% starch solution was prepared using 0.2% starch each for 5 minutes. After 5 minutes of incubation, 1 mL of
solution and phosphate buffer pH 6.7. A saliva solution was each solution was placed in separate test tubes, and 100
made by mixing 28.5 mL phosphate buffer and 1.5 mL µL of concentrated HCl was added. The absorbance of
human saliva. In a 20 mL test tube, 9 mL of the previously each test tube was then measured at 620 nm after adding
stated starch solution was mixed with 1 mL of the prepared 20 µL of I2 in KI solution and 5 mL of distilled water to each
saliva solution. An mL of this starch-saliva solution was test tube. The reciprocals of average reaction velocity and
substrate concentration were then graphed, and the Km
and Vmax were determined.
Table 2. Absorbances at Varying Incubation Times
RESULTS
A. Preparation of Starch Standard Curve Time Absorbance Concentration
Different starch concentrations were prepared for the (minutes)
standard curve. To get the diluted version as shown in
Table 1, each starch concentration was multiplied by 1 mL 0 0.462 -0.016927
and divided by 6.02 mL by the M1V1 = M2V2 formula. From
3 0.240 -0.02158
this table, the absorbance, with a range of 1.348 to 2.859,
was seen to increase as the concentration increased. 5 0.237 -0.02164
40 0.621 0.0136
60 0.298 0.02036
80 N/A N/A
Figure 5. Starch Sample with 80 ° C Incubation 0.05% 0.130 0.03676 -0.011942 0.0097414
Temperature 471 5 5
The concentrations, as shown in Table 4, were obtained 0.01% 0.104 0.00735 -0.012896 0.0040498
from Equation 3 as the calculated x-values. 294 1 1
The plot obtained from these data, as shown in Figure 6 Figure 7. Lineweaver-Burk Plot
had a relative minimum value in between pH 6.7 and 7.4,
indicating that starch concentration is lowest in between y = 21.212x + 1897.6
these two points. Equation 4. Lineweaver-Burk Equation
From this linear graph, the Vmax value was 5.27*10-4 and the
KM value was 0.011.
DISCUSSION
Enzymes are biological catalysts responsible for
accelerating biochemical reactions that would otherwise
take a very long time. Enzymes lower the activation energy
of the reaction by providing an easier path for the
substrates to react on, making the formation of products
Figure 6. Starch Concentration vs pH faster. They have what is called an active site, or cleft,
where the substrate binds and sits through the reaction.
E. Effect of Substrate Concentration on Enzyme Activity The clefts will then position the substrates in the right
orientation or wherein the bond breaking and bond forming
The initial concentration of substrate, [S], was obtained happen more obligingly to form the transition state. They
by first taking dilution into account. As a sample calculation, can also control the Each enzyme only acts on a certain
0.10% was multiplied by 4.5 mL aliquot and divided by the specific substrate and this is due to the characteristic of the
final volume, 6.12 mL. The final concentration of [S] was active site which is given by the amino acids making it up -
obtained from Equation 3. The average reaction velocity their acidity/basicity, 3-D orientation, hydrophilic/
was obtained by subtracting the final [S] from the initial [S] hydrophobic tendencies, and more, altering the shape,
and divided by the total incubation time, 5 minutes, as
size, and charge of the cleft, as shown in Figure 8. [12]
presented in Table 5.
by α -helices. The highly conserved amino acid residues,
which are involved in catalysis and substrate binding, are
found in loops of the “C-termini of β − strands ” in the
aforementioned domain. The B-domain protrudes between
the β -sheet #3 and the α -helix #3, and ranges 44 to 133
amino acid residues. It also plays a role in substrate or Ca2+
Figure 8. Induced Fit Model[12] binding. Calcium binding to the amylase, whose structure is
shown in Figure 10, is important to the amylase as it may
Starch is a polymer of glucose linked through the C1 affect its activity and stability. The domain C is folded into
oxygen in a “glycosidic bonds”. This bond is stable at high an antiparallel β -barrel and has no known function.
pH, but hydrolyzes at low pH. A latent aldehyde group may
be found at the end of this polymeric chain, and is called
the reducing end. In starch, two kinds of glucose polymers
are present: amylose, a linear polymer consisting of around
6000 glucose units, and amylopectin, which consists of
significantly less glucose units. It may be noted that
amylopectin is soluble in water, but amylose and the starch
itself is insoluble in cold water, both of whose structures are Figure 10. Three Dimensional Structures of α -amylases [15]
shown in Figure 9. [13]
In these sites, the confirmed active sites of glycosyl
hydrolases are made of multiple binding sites, and formed
between domain A and B so domain B may participate in
substrate binding. They also contain many amino acid
residues which form hydrogen bonds to the substrate,
either directly or via water molecules. These substrate
binding sites are commonly aligned with aromatic residues,
making hydrophobic stacking interactions with the sugar
Figure 9. Amylose and Amylopectin [14a]
rings. The glutamic acid residue is said to be the proton
donor, while the first conserved aspartic acid is the
Amylases are enzymes capable of digesting the nucleophile. Below is the reaction of α -amylase and starch.
glycosidic linkages of starch. In humans, they may be found [13]
There are two types
in both saliva and in the pancreas. [14]
of amylase: endoamylase and exoamylase. Both types are
capable of hydrolysing starch, and are classified by their
manner of attacking the glycosidic bond. Endoamylases
can cleave α ,1-4 glycosidic bonds found in the inner part of
both amylose and amylopectin chain. One popular example
of an endoamylase is α - amylase. Exo-amylases, on the
other hand, either exclusively cleave α , 1-4 glycosidic
bonds, or cleave both α ,1-4 and α , 1-6 glycosidic bonds.
Figure 11. Reaction of α - amylase and starch[16]
The β − amylase is a good example of the former
exo-amylase. [13] Unlike typical organic and inorganic catalysts, enzymes
have an unusual behavior with regards to substrate
As mentioned before, α -amylase is an endoamylase concentration, as illustrated in Figure 12.
that may cleave the α ,1-4 glycosidic bonds of the amylose
or amylopectin chain. The end products of this amylase are
oligosaccharides with varying lengths. This amylase is
often divided into two types, saccharifying α -amylase and
liquefying α -amylase, depending on the degree of
hydrolysis of the substrate. Saccharifying α -amylases
hydrolyze 50 to 60%, while liquefying α -amylases only
cleave about 30-40% of the linkages of starch. [13]
The α -amylase is a starch hydrolase composed of Figure 12. Effect of Substrate Concentration on the Initial
several amino acid sequences. [14] They are monomeric, Velocity of an Enzyme-Catalyzed Reaction
calcium-containing enzymes, with a single polypeptide Source:
chain folded into three domains. The most conserved https://www.khanacademy.org/science/biology/energy-and-enzym
domain consists of eight parallel β − strands that are folded es/enzyme-regulation/a/basics-of-enzyme-kinetics-graphs
in a symmetrical manner, and arranged in a barrel encircled
At low substrate concentrations, the rate of the reaction because HCl can hydrolyze the enzyme and break it apart,
increases with increasing substrate concentration. causing it to lose its function.
However, the rate increases less at relatively higher One factor that may affect enzyme activity is incubation
substrate concentrations, up to a certain point where the time, that generally follows a linear trend and is directly
rate becomes constant (Vmax). In 1913, Michaelis and proportional to enzyme activity. Enzyme activity is
Menten derived a mathematical equation to describe this reversible, and the formation of products occurs while little
hyperbolic plot by first proposing the enzyme action where to no products have formed. As the reaction continues, and
enzymes, E, and substrate molecules, S, bind to each other a significant amount of product has been formed, the rate
to form an ES complex, as shown in Figure 1. Initial rates of back reaction becomes more significant, and the rate of
are measured so that the concentration of the product, P, formation of product slows down. Should the incubation
and therefore the rate constant, k-2, are both negligible. By time be too great, the enzyme activity may be measured as
assuming this as well as the steady-state condition of the falsely low. Thus, as a general rule, incubation must be
ES complex, where its concentration is held constant, long enough for a moderate amount of product to form and
Equation 2 was derived.[17] for error in timing to be insignificant, but not too long that
there is a noticeable levelling off of the curve. [19]
From Equation 2, it was determined that the Michaelis
constant, Km, is equal to the substrate concentration that Generally, enzyme activity increases over time. This is
yields an initial velocity that is half of Vmax. Another useful because the longer an enzyme is incubated with its
constant is the turnover number, defined as the amount of substrate, the more product is formed. However, this rate of
substrate that is transformed to products in a certain time formation of product is not always linear. Should the
period, as shown in Equation 5.[18] substrate be used up during the incubation time, the
formation of product will decrease, regardless of
concentration of substrate.[19]
[14] Dongyou Liu, Aeromonas, Molecular Medical [24] ‘Introduction to Enzymes’, Manual of Clinical Enzyme
Microbiology, 2015, Measurements (1972), accessed 9 October 2019
https://www.sciencedirect.com/topics/biochemistry-genetic http://www.worthington-biochem.com/introBiochem/Enzym
s-and-molecular-biology/alpha-amylase es.pdf
[14a] Muhammed Lamin Sanyang, Ilyas RA, SM Sapuan, [25] ‘Results - Drug Delivery’, iGEM Freiburg Nanocillus, 6
Ridhwan Jumaidin, 6 Starch amylose and amylopectin October 2019, http://2016.igem.org/Team:Freiburg/Delivery
structure, Research Gate, January 2018,
https://www.researchgate.net/figure/Starch-amylose-and-a [26] ‘Michaelis-Menten Equation Calculator’, Calistry, 6
mylopectin-structure_fig6_32122677 October 2019,
https://calistry.org/calculate/michaelis-menten-Equation
[15] Daniel Mihálik, Introduction of a synthetic
Thermococcus -derived α-amylase gene into barley [27] ‘Enzyme Turnover Example,’ 6 October 2019,
genome for increased enzyme thermostability in grains, http://wajimypi12.dragon.ru.net/829234/615002/gynebuz_e
Electronic Journal of Biotechnology, August 2017, nzyme-turnover-number-example_ymapihocaz/xygob.asp
https://www.researchgate.net/figure/Three-dimensional-str
[28] ‘Biomolecules: Enzymes,’ Chem Pages Netorials, 7
uctures-of-a-amylases-Protein-structures-were-predicted-u
October 2019,
sing-Phyre_fig1_319133552
https://www.chem.wisc.edu/deptfiles/genchem/netorial/mod
[16] Enzyme Kinetics (Activity), Biology OER, 6 October ules/biomolecules/modules/enzymes/enzyme4.htm
2019,
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