Investigating the Kinetics of Enzyme Catalysed Reaction: Lab Report

This lab report investigates the kinetics of an enzyme-catalyzed reaction, specifically the hydrolysis of an ester by alpha-chymotrypsin. The experiment aimed to determine the rate constants by measuring the absorbance of the product, 4-nitrophenol, at different time intervals. The study involved varying substrate concentrations and analyzing the impact of temperature on the reaction rate. The report includes detailed methods, results, and data analysis, including calculations of product concentrations and error analysis. The discussion section compares the determined and estimated product concentrations, substrate concentration with Km values. The conclusion summarizes the findings, emphasizing the effect of substrate values on reaction rates. The results were presented in tables and graphs, along with the calculated rate constants, providing a comprehensive analysis of the enzyme's catalytic activity. The report also includes references to relevant scientific literature.

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Introduction
Enzymes are frequently depicted as 'organic impetuses which increment the rate of reaction of a
bio-synthetic reaction' (David Nelson et. al 2008). Enzymes enhance the rate speed of different
reaction that happen in a natural system, for example, the mammalian stomach related
framework. Proteins can have capacities including exchange, amalgamation or breakdown of
particles. Mention that catalysts are proteins which accelerate the rate of response without being
spent themselves for example they are reusable. Catalysts have an unmistakable dynamic site
which is corresponding to a particular substrate 3 dimensional structure (Küchler et al., 2016).
The explicitness is because of the reciprocal hydrophilic/hydrophobic charge, electrical charge
and state of dynamic site on the protein (Bansode, Hardikar and Rathod, 2017). The specificity
of an enzyme with a particular substrate creates a chemical substrate complex (ES).The rate at
which the protein substrate complex is framed is drastically expanded or diminished in changes
in substrate focus, temperature, pH and nearness of an aggressive inhibitor the impact of these 4
factors on the catalyst action is known as protein energy (Jeremy M. Berg etal 2006).Enzymes
work on the idea of change states. Progress condition of proteins is where the substrate is not a
yet an item and not a substrate (von Sperber et al., 2017). Enzymes decrease this stage .The
contrast between the free vitality of the reactants and the free energy of the progress state is the
activation energy (Ea).The least vitality required for a triumph full response to happen. Catalysts
accelerate the rate of response by bringing down the initiation energy hindrance (Kamp et al.,
2018).
This examination concerns the impact of temperature on the rate of response. I would exepcet to
find that an expansion in temperature would result in an increment in the rate of response. The
explanation behind this wonder is that as there is increment in dynamic vitality being connected

to chemicals and substrates it expands the odds of crash happening so more item (PNP) is shaped
per unit time. Anyway I likewise trust that temperatures over 50 - 70 degrees Celsius would
denature the proteins dynamic site and the 3 dimensional structures. At this stage the compound
won't be integral fit as a fiddle to the substrate (Khan, Jadhav and Rathod, 2015).
This would imply that no response can be finished so the rate of response will diminish. State
that the catalyst will have an ideal temperature at which the ES buildings and items are made at
the quickest speed. The expansion in temperature increment the measure of atoms which have
higher vitality than the Ea hindrance this thus builds the measure of particles which can respond
expanding the rate of response or starting speed. I trust the ideal temperature is going to extend
between 20-40 degrees Celsius (Juneidi et al., 2017).
Aim
To determine the rate constants for the -chymotrypsin catalysed hydrolysis of an ester
Methods
The 10 mL of 3.8 x 10-3M arrangement of substrate, 4-nitrophenyl trimethylacetate, and
acetonitrile was moreover added and weakened to make a stock arrangement. After 50 mg of
alpha chymotrypsin was broken down in a 1.0 mL of 4.6 pH acetic acid derivation cushion. The
TRISS powder was weighed for 0.12114 grams and was put in a 200 mL container and 100 mL
of deionized water was utilized to break up the TRIS powder; after when all powder is broken up
HCL was included and estimated with a pH meter until pH stretched around 8 pH (P1). Later 10
mL of 4-Nitrophenol Solution (2.8 x 10-5 M) was set up in the TRIS cushion that was arranged
already, as the arrangement will be utilized to decide the molar absorptivity. Thereafter, the
spectrophotometer was set up at 400 nm (Which P1 is hydrolysis item); the TRIS support was

moved into a 1 cm spectrophotometer cell and set in the spectrophotometer cavity, and
instrument was focused with TRISS support, the cell is then evacuated and put with a 4 –
nitrophenol arrangement in a 1 cm cell in the spectrophotometer and the absorbance was
recorded. 3 mL of TRIS support was included another clean test cell and 100 mL of the 4-
nitrophenyl trimethylacetate was included. A short time later, the example is set in a
spectrophotometer and was recorded at the absorbance of 400nm for 30 minutes at 3- minute
interim; likewise, temperature is recorded. 3 mL of TRIS cushion was added to the
spectrophotometer cell and set in spectrophotometer hole and was focused. 20 mL of substrate
stock arrangement was included and blended completely. 1 mL of arrangement is put in the cell
and recorded in spectrophotometer as well as the absorption is measured each minute for about 3
minutes. The process is done repeatedly for 40ul, 60 ul as well as 8 ul
Results
Absorption of P1
(catalysed only)
Measured [P1] Calculated [P1] Error
0.3772 2.20954E-05 6.32121E-07 -2.1E-05
0.3748 2.19548E-05 1.04512E-05 -1.2E-05
0.4762 2.78946E-05 2.01813E-05 -7.7E-06
0.533 3.12218E-05 2.97786E-05 -1.4E-06
0.5732 3.35766E-05 3.91779E-05 5.6E-06
0.6042 3.53925E-05 4.82817E-05 1.29E-05
0.6254 3.66343E-05 5.69448E-05 2.03E-05
0.644 3.77238E-05 6.49504E-05 2.72E-05
0.6556 3.84033E-05 7.1975E-05 3.36E-05
0.6666 3.90477E-05 7.75363E-05 3.85E-05
0.6818 3.99381E-05 8.09145E-05 4.1E-05
0.6872 4.02544E-05 8.10359E-05 4.08E-05
0.6956 4.07464E-05 7.62988E-05 3.56E-05
0.7034 4.12033E-05 6.43137E-05 2.31E-05
0.7088 4.15197E-05 4.15157E-05 -4E-09
0.7132 4.17774E-05 2.58684E-06 -3.9E-05
0.7186 4.20937E-05 -6.04064E-05 -0.0001
0.7224 4.23163E-05 -0.0001593 -0.0002

0.7258 4.25155E-05 -0.000311749 -0.00035
0.7306 4.27967E-05 -0.000544095 -0.00059
0.733 4.29372E-05 -0.000895633 -0.00094
0.7322 4.28904E-05 -0.001424984 -0.00147
0.7404 4.33707E-05 -0.002219602 -0.00226
0.7428 4.35113E-05 -0.00340995 -0.00345
0.7446 4.36167E-05 -0.00519066 -0.00523
0.7474 4.37808E-05 -0.007852084 -0.0079
Sum of error -0.0223
Time
(MINS)
Calculated [P1] 1/t LN[P1]
0 0.000632121 0 -7.36643
1 0.001631752 1 -6.4181
0.000001 2 0.002631384 0.5 -5.94025
10 3 0.003631015 0.333333 -5.61824
10 4 0.004630646 0.25 -5.37506
-1.10E+06 5 0.005630277 0.2 -5.1796
-3.53E+05 6 0.006629907 0.166667 -5.01616
-2.83E-05 7 0.007629536 0.142857 -4.87573
0.000001 8 0.008629166 0.125 -4.75261
9 0.009628795 0.111111 -4.643
10 0.010628423 0.1 -4.54422
11 0.011628052 0.090909 -4.45433
12 0.012627679 0.083333 -4.37186
13 0.013627307 0.076923 -4.29568

14 0.014626934 0.071429 -4.22489
15 0.015626561 0.066667 -4.15878
16 0.016626187 0.0625 -4.09678
17 0.017625813 0.058824 -4.03839
18 0.018625439 0.055556 -3.98323
19 0.019625064 0.052632 -3.93095
20 0.020624689 0.05 -3.88127
21 0.021624313 0.047619 -3.83394
22 0.022623938 0.045455 -3.78875
23 0.023623561 0.043478 -3.74551
24 0.024623185 0.041667 -3.70407
25 0.025622808 0.04 -3.66427
TIME
(MINS)
Absorptio
n of P1
(total)
Absorption of P1
(catalysed only)
Measured
[P1]
Calculated
[P1]
Error
0 0.189 0.1586 9.29038E-06 0 9.29038E-06
1 0.451 0.4094 2.39816E-05 0.25591168
7
-0.25588771
2 0.53 0.4796 2.80937E-05 0.36332943
2
-0.36330134
3 0.566 0.51 2.98745E-05 0.40851866 -0.40848879
4 0.587 0.5246 3.07297E-05 0.42763013
5
-0.42759941
5 0.601 0.5346 3.13155E-05 0.43581335 -0.43578204

3
6 0.61 0.5412 3.17021E-05 0.43941692
9
-0.43938523
7 0.617 0.545 3.19247E-05 0.44110134 -0.44106942
8 0.623 0.5478 3.20887E-05 0.44198149
7
-0.44194941
9 0.629 0.5538 3.24402E-05 0.44252462 -0.44249218
10 0.634 0.5564 3.25925E-05 0.44292650
5
-0.44289391
11 0.638 0.5596 3.27799E-05 0.44326920
1
-0.44323642
12 0.642 0.5628 3.29674E-05 0.44358709
3
-0.44355413
13 0.647 0.5662 3.31665E-05 0.44389459
1
-0.44386142
14 0.652 0.5704 3.34126E-05 0.44419773
3
-0.44416432
15 0.657 0.5746 3.36586E-05 0.44449905 -0.44446539
16 0.662 0.5788 3.39046E-05 0.44479960
2
-0.4447657
17 0.667 0.5822 3.41038E-05 0.44509983
3
-0.44506573
18 0.672 0.5864 3.43498E-05 0.44539993 -0.44536558
19 0.676 0.5888 3.44904E-05 0.44569997
1
-0.44566548
20 0.681 0.593 3.47364E-05 0.44599998
8
-0.44596525
21 0.686 0.5956 3.48887E-05 0.44629999
5
-0.44626511
22 0.691 0.6022 3.52753E-05 0.44659999
8
-0.44656472
23 0.695 0.6054 3.54628E-05 0.44689999
9
-0.44686454
24 0.701 0.6098 3.57205E-05 0.4472 -0.44716428
25 0.706 0.6132 3.59197E-05 0.4475 -0.44746408
Sum of error -10.7792723
0.0003 0.44
0.86973079
8
[S]0 0.0000567
K 3.498
[E] Y ≈ [E]0act 0.00009
kcat X/[E]0act ≈ kcat 3.33333333

3
k3 ≈kcat
3.33333333
3
k2
≈ B=(k2/1+(K/[S]0))
+k3 -1.52E+05
k2/K -4.35E+04
KM .=K3*K/(k2 + k3) -1.77E+06
p[E] .=[E]0act 0.00009
Time
(MINS)
Calculated
[P1]
1/t LN[P1]
0 0 0 0
1 0.001001 1 -6.90676
2 0.002001998 0.5 -6.21361
3 0.003002996 0.333333333 -5.80814
4 0.004003992 0.25 -5.52046
5 0.005004988 0.2 -5.29732
6 0.006005982 0.166666667 -5.115
7 0.007006976 0.142857143 -4.96085
8 0.008007968 0.125 -4.82732
9 0.00900896 0.111111111 -4.70954
10 0.01000995 0.1 -4.60418

11 0.01101094 0.090909091 -4.50887
12 0.012011928 0.083333333 -4.42186
13 0.013012916 0.076923077 -4.34181
14 0.014013902 0.071428571 -4.26771
15 0.015014888 0.066666667 -4.19871
16 0.016015873 0.0625 -4.13418
17 0.017016856 0.058823529 -4.07355
18 0.018017839 0.055555556 -4.01639
19 0.019018821 0.052631579 -3.96233
20 0.020019801 0.05 -3.91103
21 0.021020781 0.047619048 -3.86224
22 0.02202176 0.045454545 -3.81572
23 0.023022738 0.043478261 -3.77127
24 0.024023714 0.041666667 -3.72871
25 0.02502469 0.04 -3.68789
Discussion
Looking at the charts of figure 3, for determined [P1] and with estimated [P1], they are close yet
due to the utilization of exceed expectations making it difficult to make the qualities close as

conceivable to one another; and contrasting the figure 5, estimations of determined [P1] and
estimated [P1] are a long way from one another or having similar qualities for focuses.
Contrasting the substrate focus with Km esteems; the qualities for 60 microlitres are the
equivalent however for 80 microlitres the qualities are far various to one another; thusly, the
substrate is bigger than value of Km (Penders-van Elk, van Aken and Versteeg, 2016).
Conclusion
The study was attained through a change in the substrate values for instance 40 uL, 60 uL and 80
uL which was attainable through the injection of a solution of enzyme stock into
spectrophotometer cell as when the reaction was going on, there was an increase in concentration
with time.

References
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Juneidi, I., Hayyan, M., Hashim, M.A. and Hayyan, A., 2017. Pure and aqueous deep eutectic
solvents for a lipase-catalysed hydrolysis reaction. Biochemical engineering journal, 117,
pp.129-138
Kamp, M.W., Prentice, E.J., Kraakman, K.L., Connolly, M., Mulholland, A.J. and Arcus, V.L.,
2018. Dynamical origins of heat capacity changes in enzyme-catalysed reactions. Nature
communications, 9(1), p.1177
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von Sperber, C., Lewandowski, H., Tamburini, F., Bernasconi, S.M., Amelung, W. and Frossard,
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revealed by Raman spectroscopy. Journal of Raman Spectroscopy, 48(3), pp.368-373