Elissia Vecere

2/22/21
Enzyme Kinetics
Introduction
Enzyme kinetics is the study of enzyme rates and how these rates are affected by
enzyme concentration substrates, and any inhibitors or activators. The kinetics of an enzyme
can tell the experimenter a lot about the nature of the enzyme and the metabolism that it is
involved in. By observing how reaction rates are affected by the concentration of enzyme,
substrate, inhibitors, and activated, we are able to understand the nature of the reaction that Is
taking place between the enzyme and the substrate. This information can be very useful in
understanding the biological reactions that take place within our bodies. The ability to apply
and practice these concepts is completed within the lab.
Throughout the experiment, the student will study enzyme kinetics via the use of
mushroom tyrosinase. The enzyme and substrate volumes will vary in separate portions of the
experiment and will demonstrate the different nature of the effects of each reaction velocity.
From the data collected, the student will determine the values for Km, Kcat, and Vmax. With
the data and overall understanding of the concepts present in this lab, the student will make
use of different plots.

Objectives
The objectives throughout this lab consist of observing and analyzing the effects of
changing the concentration of an enzyme or substrate and how this effects initial reaction rates.
Students also work with constructing v versus [S] plots and how to estimate Km and Vmax,
constructing Lineweaver-Burk plots and determining Km and Vmax, and calculating Kcat and
given enzyme concentrations. Finally, students will learn how to design protocols to determine
parameters and explain the significance of Km, Vmax, and Kcat values.

Procedure
To begin, the student set up a protocol for each determination of the correct tyrosinase
concentration. Each tube had a total of 3 mL of solution and were 5 mM in concentration of L-
DOPA. The volume of each tube was controlled by the phosphate buffer, and the amount of
varied enzyme. 5 tubes were made ranging from 0.1 to 0.5 mL of tyrosinase. Next, the student
pipetted the L-DOPA and phosphate buffers into each tube. The student then pipetted the
chosen amount of enzyme into one tube and mixed via inversion. The change in absorbance
was immediately read a 475 nm. This value was measured for 2 minutes at 15 second intervals.
These steps were then repeated for the other 4 testing tubes with different enzyme
concentrations. The student then determined the rate for the five different tyrosinase levels.
The student then chose a value that would give a change in absorbance per minute that was
between 0.2-0.3/min (the volume we chose was 0.5 mL).
Beginning Part B: Determining Kinetic Constant of Tyrosinase, the student first set up a
protocol using the level of tyrosinase that was chosen at the end of part A. Each tube had a
total of 3 mL of solution and contained varying levels of L-DOPA. One tube had one of the
following values in it of L-DOPA: 0.05, 0.10, 0.2, 0.4, 0.8, and 1 mL. Each tube contained the
level of tyrosinase that was determined to have a change in absorbance of 0.2-0.3/min. Next,
the student pipetted the designated L-DOPA volume and phosphate buffer into each tube. The
tubes were mixed via inversion. The change in absorbance was immediately read a 475 nm. This
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Enzyme Kinetics
value was measured for 2 minutes at 15 second intervals. These steps were then repeated for
the other 4 testing tubes with different L-DOPA concentrations.

Observations
Throughout the lab, the student noticed that in part A, there was a constant amount of
substrate (L-DOPA) whereas in part B, the amount of L-DOPA varied. The solution L-DOPA was
clear and had no color to it, similarly to the phosphate buffer. When recording the data, the
time intervals was 15 seconds, but on the provided table from the machine increased in a
fraction of a minute. For example, 0.25 minutes represented 15 seconds and so on. After adding
the enzyme in part A and the inversion of each cuvette, the cuvette had to be immediately
placed into the spectrophotometer in order to get an instantaneous read on the enzyme
kinetics taking place. Over time, the solution inside the cuvette, that was once clear became a
darker yellow/orange/brown color. Not all of the cuvettes had the same shade, but each was
darker than their initial look. In regard to the slopes calculated in the spectrophotometer, we
were able to see them upward sloping due to the fact that as time increased, the level of
absorbance also increased. Finally, in part A, the volume of 0.5 mL of the tyrosinase was
efficient to give a good change in absorbance and was used in part B.
All the cuvettes in part B contained 0.5 mL of the tyrosinase enzyme. After following the
procedure, the student noticed that the color in the cuvettes also changed here. The cuvette
with little substrate value, although not colorless, was extremely light in comparison and could
have very easily been mistaken as no change in color. As the volume of the substrate increased,
the degree of color increased. Along with this, the longer the cuvettes sat out, the darker they
became due to the continuous interaction of the enzyme and substrate. Similarly, to A, for the
slope calculated in the spectrophotometer, we were able to see the upward slop within the
graph due to the fact that as time increased, the level of absorbance also increased.
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Enzyme Kinetics
Results
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Enzyme Kinetics
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Enzyme Kinetics
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Enzyme Kinetics
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Enzyme Kinetics
Discussion

Michaelis-Menton Plot
0.16
0.14 f(x) = 0.0283154471544715 x + 0.0137463414634146

0.12
Vo (umol/min)

0.1
0.08
0.06
0.04
0.02
0
0 1 2 3 4 5 6
L-DOPA mM

Lineweaver-Burk Plot
90
80
f(x) = 19.7524379654816 x + 2.79688040078456
70
Velocity 1/(umol/min)

60
50
40
30
20
10
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
L-DOPA (1/mM)
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Enzyme Kinetics
Conclusion
Throughout this lab, the student addressed the concept of enzyme and substrate
binding in the presence of inhibitors. The student did this throughout the lab via two parts that
consisted of variable enzyme volumes and variable L-DOPA (substrate) volumes. The student
was able to use the data derived from the lab to work with both Michaelis Menten and
Lineweaver-Burk plots to determine the Km and Vmax values from the two. This data allows the
student to numerically represent and understand the reaction between the enzyme and the
substrate. Overall, the student was able to analyze the effects of the changing enzyme and
substrate concentrations and their effects on the reaction rates, with the use of Km, Vmax, and
Kcat values.