INTRODUCTION

Objectives of the lab report

o To introduce the concepts of data analysis and interpretation.

o To consolidate your research and investigation skills.
o To demonstrate that you can interpret and gather scientific data.

The study of the rates of enzyme-catalyzed chemical reactions is known as enzyme

kinetics. The reaction rate is monitored in enzyme kinetics, and the consequences of
changing the reaction conditions are explored (Alberty, R. A. (2011a)). The catalytic
mechanism of an enzyme, its role in metabolism, how its activity is controlled, and
how a drug or a modifier (inhibitor or activator) might influence the rate can all be
revealed by studying its kinetics in this way (Bisswanger, H. (2017)).

Michaelis-Menten kinetic analysis is an important tool for analyzing enzyme kinetics.

This study shows how the rate of an enzyme-catalyzed reaction is related to the
maximum rate, affinity, and substrate concentration (Segel, I. H. (1975)). The
Michaelis-Menten saturation plot can be used to visualize these connections and
determine important parameters (Km, Vmax, and Kcat) at a fixed enzyme
concentration (Plowman, K. M. (1972) Enzyme).

The Michaelis-Menten equation.

The Michaelis-Menten equation is a mathematical model that is used to analyze

simple kinetic data. The model has certain assumptions, and as long as these
assumptions are correct, it will accurately model your experimental data (Nagar, S.,
Argikar, U. A. and Tweedie, D. J. (2014)). The derivation of the model will highlight
these assumptions.

Vmax [ S]
V=
Km+[S ]
Where,

V is the rate of reaction, Vmax is the maximal rate of reaction, [S] is the substrate
concentration, and Km is the Michaelis-Menten constant (Marangoni, A. G. (2003)).

In an enzyme catalysed reaction, the substrate initially forms a reversible complex

with the enzyme (i.e. the enzyme and substrate have to interact for the enzyme to be
able to perform its catalytic function) (Lorente-Arevalo, A., Ladero, M. and Bolivar, J.
M. (2021)).

Figure 1: A Michaelis-Menten saturation curve.

The Beer-Lambert law.

The relationship between a sample's absorbance and an analyte's concentration is

shown by this equation (C). For xanthine oxidase at a wavelength of 290 nm, this
constant = 11.6 M-1cm-1.

Absorbance=ϵCl

Where,
ϵ is molar extinction coefficient

L is path length travelled by light

Figure 2: The Lineweaver-Burk plot.

Methods

The rate of change in absorbance was converted into a rate of product formation.
The above was repeated in the presence of 1 µM Allopurinol and several other
substrates. The rate of enzymatic activity (change in absorbances over 10 minutes)
was recorded. This rate of change in absorbance was converted into a rate of
product formation. The above was repeated in the presence of 1 µM Allopurinol and
the rate of enzymatic activity was recorded over 10 minutes.

For each data point, the Beer-Lambert law was used to determine the concentration
of uric acid. In this data point, the calculated concentrations were converted into an
amount (i.e., number of moles). The initial linear rate of reaction was determined at
each substrate concentration by plotting a graph for each one of amount of uric acid
produced versus time and the gradient determined.

The calculated rates, and the substrate concentrations was used to do the Michaelis-
Menten analysis for both treatments.
Results

Table 1: Data for the first substrate concentration

Table 2: Data for the second substrate concentration

Table 3: Data for the third substrate concentration

AVG represents the rate (micromoles per minute) of reaction for each substrate.

Concentration of uric acid

For the following calculations to find the concentration of an analyte using the
equation below for each data point.

Absorbance=ϵCl

For 0.05mM

C=absorbance ∈ L

0.05
C= =0.00521 μM
9600 ×1

CONCENTRATION OF URIC ACID

CONCENTRATION (mM) C( micromoles)

0.05 5.21E-06
0.1 1.04E-05
0.2 2.08E-05
0.4 4.17E-05
0.6 6.25E-05
0.8 8.33E-05
1 0.000104

Table 4: Concentration of uric acid

Number of moles

moles=molarity ×litres of solution

For 0.05mM

Moles=0 , 0431× 1000=4 . 31 E−5 μmoles

Number of moles
concentration (mM) Moles (micromoles)
0.05 4.31E-05
0.1 2.08E-05
0.2 4.17E-05
0.4 3.15E-05
0.6 0.000625
0.8 0.000083
1 0.000104

A GRAPH OF THE AMOUNT OF URIC ACID

AGAINST TIME
2.5

2 f(x) = 0.212837662337662 x + 0.0891883116883121

Amount of uric acid

1.5

0.5

0
0 2 4 6 8 10 12
Time

Figure 3: A graph for the first substrate concentration

The gradient (the reaction rate for the first substrate concentration)
0.2187mM/minute

0.2128mM/minute
 A GRAPH OF THE AMOUNT OF URIC ACID
AGAINST TIME

2.5
Amount of uric acid

2 f(x) = 0.208705627705628 x + 0.0679870129870128

1.5

0.5

0
0 2 4 6 8 10 12

Time

Figure 2: A graph for the second substrate concentration

The gradient (the reaction rate for the second substrate concentration)
0.2087mM/minute.
 A GRAPH OF THE AMOUNT OF URIC ACID
AGAINST TIME

2.5

2
f(x) = 0.185146103896104 x + 0.110438311688312
Amount of uric acid

1.5

0.5

0
0 2 4 6 8 10 12

Time

Figure 4: A graph for the third substrate concentration

The gradient (the reaction rate for the third substrate concentration)
0.2087mM/minute

is 0.1851mM/minute.

A GRAPH OF RATE PER MINUTE AGAINT TIME

0.16
RATE (MICROMOLES PER MINUTE)

0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0 2 4 6 8 10 12
S

Figure 5: A Michaelis-Menten saturation curve for the first substrate

 A GRAPH OF RATR PER MINUTE AGAINT TIME
0.16
RATE (MICROMOLES PER MINUTE)

0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0 2 4 6 8 10 12
S

Figure 6: A Michaelis-Menten saturation curve for the second substrate

A GRAPH OF RATR PER MINUTE AGAINT TIME

0.18
RATE(MICROMOLES PER MINUTE)

0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0 2 4 6 8 10 12
S

Figure 7: A Michaelis-Menten saturation curve for the third substrate

Vmax [ S]
V=
Km+[S ]

For the first substrate concentration

Vmax= 0.152857 µM/s

 0.152857
0.2187=
Km +0.152857

Km=0.5461

For the second substrate concentration

Vmax= 0.142857µM/s

0.142857
0.2087=
Km +0.142857

Km=0.54165

For the third substrate concentration

Vmax= 0.152857 µM/s

0.152857
0.1851=
Km+ 0.152857

Km=0.6730

Discussion
The maximum rate of activity was found to be 2.223571mM, 2.157619mM and
1.936786mM for the first substrate, second substrate and third substrate
concentration. It was found that the first substrate has the highest activity rate
followed by the second substrate and then the third substrate concentration.

From figure 5, Vmax was estimated as 0.152857 µM/s. After this point, the curve
decreased until if flattened which implied that the enzyme concentration was used
all. The same relationship was observed in figure 6 and 7 having Vmax as 0.142857
µM/s and 0.152857µM/s respectively.

At 1/2Vmax km for the first, second and third substrate were found as 0.2730,
0.27064 and 0.34442 respectively. Therefore, third the substrate has the highest
affinity since it has lowest value of km followed by the first substrate and then the
second one.

Conclusion

To conclude, the first and the second substrates have same Vmax of 0.152857 µM/s
and the second substrate has 0.142857 µM/s. Therefore, all the substrates became
fully saturated after 1s of the reaction.

It was also found that the third substrate concentration (km= 0.34442 ¿ has the
highest affinity followed by the second substrate concentration ( km=0.2730 ¿ then the
first substrate concentration (km=0.27064 ¿. The graph of amount of uric acid against
time is a linear relationship.

References
Alberty, R. A. (2011a) Enzyme kinetics: Rapid-equilibrium applications of
Mathematica. 1st ed. Nashville, TN: John Wiley & Sons.

Baici, A. (2015a) Kinetics of enzyme-modifier interactions: Selected topics in the

theory and diagnosis of inhibition and activation mechanisms. 2015th ed. Vienna,
Austria: Springer.

Bisswanger, H. (2017) Enzyme kinetics: Principles and methods. 3rd ed. Weinheim,
Germany: Wiley-VCH Verlag.

Cornish-Bowden, A. (2012) Fundamentals of enzyme kinetics. 4th ed. Weinheim,

Germany: Wiley-VCH Verlag.

Cornish-Bowden, A. and Wharton, C. W. (1988) Enzyme Kinetics. London, England:

Oxford University Press.

Engel, P. C. (1981) Enzyme kinetics: The steady-state approach. 2nd ed. London,
England: Chapman and Hall.

Lorente-Arevalo, A., Ladero, M. and Bolivar, J. M. (2021) “Framework of the kinetic

analysis of O2-dependent oxidative biocatalysts for reaction intensification,” Reaction
chemistry & engineering, 6(11), pp. 2058–2074. doi: 10.1039/d1re00237f.

Marangoni, A. G. (2003) Enzyme Kinetics: A Modern Approach. 1st ed. Nashville,

TN: John Wiley & Sons.

Nagar, S., Argikar, U. A. and Tweedie, D. J. (2014) “Enzyme kinetics in drug

metabolism: fundamentals and applications,” Methods in molecular biology (Clifton,
N.J.), 1113, pp. 1–6. doi: 10.1007/978-1-62703-758-7_1.

Plowman, K. M. (1972) Enzyme Kinetics. New York, NY: McGraw-Hill.

Schulz, A. R. (1994a) Enzyme kinetics: From diastase to multi-enzyme systems.

Cambridge, England: Cambridge University Press.

Segel, I. H. (1975) Enzyme kinetics: Behaviour and analysis of rapid equilibrium and
steady-state enzyme systems. Nashville, TN: John Wiley & Sons.

(08/02/2022) Teachmephysiology.com. Available at:

https://teachmephysiology.com/biochemistry/molecules-and-signalling/enzyme-
kinetics/ (Accessed: February 13, 2022).a