CSU Sacramento, Department of Chemistry

Chem 162 -Introductory Biochemistry Lab

Laboratory Manual

EXPERIMENT 3
-Enzyme Kinetics-

I. LEARNING OBJECTIVES

• To learn about the theory regarding Michaelis-Menten (M-M) kinetics

• To study the rate of an enzymatic reaction, and to determine the effects of modifying one
variable at a time, while holding all others constant
• To learn how to manipulate M-M kinetics data, perform the necessary calculations, and
generate both M-M and Lineweaver-Burk plots
• To learn how to design an experiment involving blanks and controls

II. INTRODUCTION

Enzymes are proteins that catalyze biological reactions. (A small number of RNA catalysts
have also been identified, but the vast majority of biological catalysts are proteins.) Enzymes can
speed up the rate of reactions by a million-fold or more, which makes them very powerful
catalysts. However, they are also subject to denaturation and are thus sensitive to pH,
temperature, detergents, and other factors that can cause denaturation of proteins.
Much of biochemistry today is involved with identifying and understanding the catalytic
mechanisms of the multitude of enzymes found in different cell types (E. coli cells contain at least
3000 different enzymes; other cell types may contain more). Whenever a biochemist purifies a
new enzyme, a first step in characterizing the enzyme is to study the kinetics of the reaction the
enzyme catalyzes.

Michaelis-Menten Kinetics

Enzyme kinetics, the study of reaction rates, was first developed by Michaelis and Menten
in the early 1900's. Michaelis-Menten kinetics begins with the assumption that an enzyme-
substrate [ES] complex is formed (the substrate being the molecule that is converted to product
by the enzyme):

k1 k2
E+S ES E+P
k-1 k-2
 CSU Sacramento, Department of Chemistry
Chem 162 -Introductory Biochemistry Lab
Laboratory Manual

Michaelis and Menten also realized that enzymes display saturation kinetics meaning
that, at high enough substrate concentrations, all of the enzyme molecules are bound to
substrate and the maximum velocity of the reaction has been reached. This concept is displayed
graphically as follows:

Using these concepts, an equation describing the reaction velocity of an enzyme-

catalyzed reaction was developed. This equation is referred to as the Michaelis-Menten equation
(a complete derivation of this equation can be found in most standard biochemistry texts):
V max [ S ]
V0 =
KM + [ S ]

where vo is the initial velocity of the reaction, Vmax is the maximum velocity of the reaction, [S] is
the substrate concentration and KM is the concentration of substrate at which the velocity of the
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reaction is 1/2 Vmax. For many enzymes, KM is a measure of the affinity of the enzyme for the
substrate (specifically, when k2 <<< k-1).
A first step in exploring the kinetics of a newly isolated enzyme is to determine the KM and
Vmax of the enzyme. This is done by varying the substrate concentration and determining the
velocity of the reaction over a short time period. A short time period is used to avoid the build-
up of too much product and conversion of some of the product back to substrate. Otherwise, the
reaction is approaching equilibrium, which complicates the kinetic analysis. Enzyme kinetics is
the study of the forward reaction (from ES to E + P) only. The velocity is usually measured as the
amount of product formed per minute. The velocity is then plotted against the substrate
concentration, to give a plot similar to that shown above.
 CSU Sacramento, Department of Chemistry
Chem 162 -Introductory Biochemistry Lab
Laboratory Manual

Notice that it is difficult to determine exactly when Vmax is reached from this type of plot
because Vmax is approached asymptotically. Since Vmax cannot be determined accurately, 1/2Vmax
and therefore KM also cannot be determined accurately. Several different types of plots have
been developed to avoid this problem, the most common one being the Lineweaver-Burk plot,
also called a double-reciprocal plot because the reciprocal of both sides of the Michaelis-Menten
equation is taken (followed by a simple rearrangement of terms) to give the following equation:

1 KM 1
= +
v0 V max [ S ] V max

This is a linear equation (y = mx + b), with slope = KM/ Vmax, and y-intercept = 1/ Vmax. The
data are plotted as 1/vo versus 1/[S].
€
Prior to determining KM and Vmax, a biochemist working with a new enzyme must first
ascertain what amount of enzyme, substrate and time of reaction will give the initial velocity of
the reaction (i.e. when the product formation over a given time period is linear). To determine
this takes some trial and error but it can be accomplished by varying the amount of enzyme and
the incubation time of the reaction.

Acid Phosphatase
Before beginning any enzyme kinetic experiment, the biochemist must have some way of
determining the velocity of the reaction. This is usually accomplished by monitoring either the
disappearance of substrate or the appearance of product. The enzyme you will be using in this
experiment is an acid phosphatase which catalyzes the following general reaction:

ROPO3H- + H2O ® ROH + HOPO3H

Acid phosphatases are found in seeds, where they are believed to play a role in
germination by removing phosphate groups from inactive metabolic enzymes, converting them
to active enzymes. Most metabolic enzymes in seeds are responsible for breaking down the
stored fats and converting them to sugars which are then used to make leaves and stems. In this
experiment, the substrate you will be using, p-nitrophenylphosphate, is converted to the product
p-nitrophenol. Both of these substances are
colorless; in order to determine the amount
of product formed, p-nitrophenol is
converted to p-nitrophenolate by raising the
pH, resulting in a strong absorbance at 405
nm.
 CSU Sacramento, Department of Chemistry
Chem 162 -Introductory Biochemistry Lab
Laboratory Manual

The velocity of the reaction is determined by calculating the µmoles of product formed
divided by the time of incubation in which the product was formed. (The amount of product can
be calculated using the extinction coefficient at 405 for p-nitrophenolate, 18,700 M-1cm-1.) In this
experiment, you will determine the KM and Vmax of acid phosphatase, the pH and temperature at
which the enzyme works best, and the effects of metabolic inhibitors on the activity.

III. PROCEDURE

Overview

Each reaction will be conducted in a microplate with a reaction volume of 150µl at an

incubation temperature of 30oC. Each well will consist of the following components:

1. Enzyme (0.25 mg/mL WGAP in 0.1 M succinate buffer, pH 4.8)

2. Buffer (0.1M succinate, pH 4.8)
3. Substrate (0.03 M p-nitrophenylphosphate).
4. Water (added to achieve the same final concentration of enzyme in each well).
5. 0.5 M NaOH (usually added last to stop the reaction and drive pNP to pNP-).

Each reaction will be performed by adding buffer, water, and either enzyme or substrate
to the well. If enzyme is being varied, then this is added to the well before substrate. If substrate
is being varied, then the substrate is added to the well. (The reason for this will be become clear
when you do the experiment – the goal is to simplify the addition of the final reaction component,
either enzyme or substrate, under timed conditions). In all cases, the reaction is stopped by the
addition of NaOH.

Each series of reactions will also contain a blank. Here, the NaOH is added before the
final component of the reaction. We do not want product to form in the blank, so we prevent
product formation by adding NaOH to the well before the final component is added.

The general outline of each experiment will be as follows:

1. While keeping the plate on ice, add buffer, water, and either enzyme or substrate to each
well.
2. Add the final component of the mixture (either enzyme or substrate), and quickly mix the
contents of the well.
3. Float the plate in the 30˚C water bath, and start timing the reaction.
4. After the appropriate time (usually between 2 and 5 minutes; you will determine the
optimal time), add 0.5 M NaOH to stop the reaction. Mix the contents well.
5. Dry the bottom of the plate, and read the absorbance at 405 nm.
 CSU Sacramento, Department of Chemistry
Chem 162 -Introductory Biochemistry Lab
Laboratory Manual

First Lab Period

Materials and Equipment

0.1 M succinate buffer, pH 4.8
0.25 mg/ml WGAP enzyme solution
0.5 M NaOH
0.03 M pNPP substrate
Microplate Reader
Water bath
Microplate
Stopwatch

Determining Linear Reaction Conditions for Kinetic Analysis

As discussed in the Introduction, before the kinetics of the enzyme can be analyzed, it is
first necessary to determine approximately how much enzyme should be used and how long the
reaction should be incubated. Remember that the goal is to obtain a measurable amount of
product while still measuring initial velocity (when accumulation of product is linear with respect
to time). In this part of the experiment, you will vary both the amount of enzyme to be used and
the incubation time to establish the conditions that generate enough product to measure, but
not so much that the reverse reaction becomes significant and the accumulation of product is no
longer linear with respect to time. We will vary the time and the enzyme separately and then plot
all of the data to determine the optimal conditions.

! Disposal of Materials: Place all solutions containing p-nitrophenol in the “aqueous waste” jar
in the middle hood.

! Precautions: Avoid skin contact with 0.5 M NaOH. Promptly clean up any NaOH spills.

A. Time Study

1. Prepare five wells containing 50µl of 0.10 M succinate buffer, pH 4.8, 10µl of enzyme
solution, and 75µl of water.
2. To well 1, add 100µl of 0.5 M NaOH. This is your blank.
3. Start the reaction by quickly adding 15µl of substrate (0.030 M pNPP) to each well by
pipetting up and down. Immediately place the plate into the water bath and note the
exact time. (You may want to use a stopwatch or timer.)
4. Stop the reactions in well 2-5 by adding 100µl of 0.5 M NaOH after 1, 2, 3, and 5 minutes
of incubation, respectively.
5. Read the plate absorbance at 405 nm.
 CSU Sacramento, Department of Chemistry
Chem 162 -Introductory Biochemistry Lab
Laboratory Manual

B. Varying Enzyme Concentration

Repeat the experiment in part A twice, the first time using 20µl of enzyme, and the second
time using 30µl of enzyme. Adjust the volume of the water accordingly and keep the buffer and
substrate the same as in part A. Make sure all samples have the same total volume. You may
find it useful to construct a table of reaction components before conducting the experiment. For
efficiency, you may choose to set up and incubate all experimental variations at the same time.
Plot the [P] versus incubation time for the three different enzyme concentrations.
Examine your data and choose the volume of enzyme and time of incubation that gives a linear
accumulation of product and an absorbance between 0.5 and 0.9.

Second Lab Period

Materials and Equipment

0.1 M succinate buffer, pH 4.8
0.025 M WGAP enzyme solution
0.5 M NaOH
0.03 M pNPP substrate
Microplate Reader
Water bath
microplate
Stopwatch

A. Varying Substrate Concentration

1. Prepare according to a table such as the one below. Use the volume of enzyme
determined to be optimal in the first lab period and an amount of water that, when
combined with the amounts of substrate and buffer given in the table below, will give a
total volume in each well of 150µl. The table shown below is an EXAMPLE. It shows the
correct volumes of each substance when 10µl of enzyme is used. Construct your own
table, using the volume of enzyme that you determined in part B, first lab period, and/or
vary the amount of substrate.
Note: Do not add ENZYME until step 3!

Volume of Solution (µL)

Reagent Well 1 Well 2 Well 3 Well 4 Well 5 Well 6 Well 7 Well 8
Buffer 50 50 50 50 50 50 50 50
Water
(to 150µl) 75 90 89 87 85 80 75 70
Substrate 15 0 1 3 5 10 15 20
Enzyme* 10 10 10 10 10 10 10 10
 CSU Sacramento, Department of Chemistry
Chem 162 -Introductory Biochemistry Lab
Laboratory Manual

This is This is
the Blank! negative ctrl.
Add NaOH
before enzyme!
*NOTE: DO NOT ADD ENZYME UNTIL STEP 3!

2. Add 150µl of NaOH to well #1 (the blank). Then add the enzyme.
3. Add enzyme to each of the remaining wells (2-8).
4. Incubate the plate in the 30˚C water bath. Mix and record the exact time, or start the
stopwatch.
5. Add 100µl of NaOH to each well after the appropriate incubation time (as determined in
part B, first lab period), except well 1 (already has NaOH), and mix thoroughly.
6. Dry the bottom of the plate, and read the absorbance at 405 nm.
7. Repeat steps 1-7 until you have obtained two sets of data that are consistent. Check
with your instructor if you are unsure.
8. Plot the data using both Michaelis-Menten (initial velocity vs. [substrate]) and
Lineweaver-Burk plots (1/vo vs. 1/[S]). From each graph, determine the KM and Vmax of
your enzyme. Velocity should be calculated as µmoles of product formed per minute.
(See your instructor if you are unsure how to perform this calculation). How do the two
curves compare?

Third Lab Period

Materials and Equipment

0.1 M succinate buffer, pH 4.8
0.025 M WGAP enzyme solution
0.5 M NaOH
0.03 M pNPP substrate
Inorganic phosphate solution
Copper acetate solution
Microplate Reader
Water bath
Microplate
Stopwatch

Enzyme Inhibition

Enzyme inhibition is an important mechanism for regulating enzyme activity and is a tool
used by cells and pharmaceutical companies alike for this purpose. Enzyme inhibition falls into
two major categories, reversible and irreversible. We will be dealing with reversible inhibitors in
this experiment which can be further divided into competitive, non-competitive and
uncompetitive inhibition. In competitive inhibition, the inhibitor structurally resembles the
 CSU Sacramento, Department of Chemistry
Chem 162 -Introductory Biochemistry Lab
Laboratory Manual

substrate and so competes with the substrate for the active site on the enzyme. Competitive
inhibition results in a higher apparent KM for the enzyme but Vmax remains unchanged. Reversible
inhibitors can also bind at a site distinct from the active site. In mixed inhibition, the inhibitor
binds to both free enzyme and the enzyme-substrate complex, and Vmax decreases whereas KM
can increase, decrease, or remain unchanged. A special type of mixed inhibition is non-
competitive inhibition, when the inhibitor binds to free enzyme and the enzyme-substrate
complex with equal affinity so that Vmax is reduced but KM is unchanged. In uncompetitive
inhibition, the inhibitor binds only to the enzyme-substrate complex. Both KM and Vmax decrease
in this type of inhibition but KM/Vmax remains the same.
In this experiment, you will conduct velocity versus substrate reactions to determine KM
and Vmax, just as you did in the second lab period of this experiment, but now you will conduct
the reactions in the presence of inhibitor. Each group will carry out the experiment in the
presence of inorganic phosphate and one other inhibitor (copper acetate).

A. Inhibition Study

You will be carrying out three experiments, one with no inhibitor, one with phosphate as
an inhibitor, and one with copper acetate as the inhibitor. For the reaction containing no
inhibitor, set up plate as you did in the second lab period. For the two sets of reactions containing
inhibitor, use 10µl of inhibitor solution and adjust the water accordingly. It is strongly
recommended that you construct a table showing the reaction components and their volumes
(in fact, your instructor may require you to do this) before doing the experiment.

IV. CONCLUSIONS

Your report should include the data listed below:

1. Plot of [Product] vs. time at each enzyme volume (all on the same graph).
2. Michaelis-Menten plot of initial velocity vs. substrate concentration from second lab
period (both trials on the same graph).
3. Lineweaver-Burk plot of initial velocity -1 versus [substrate]-1 from second lab period (both
trials on the same graph). Determine KM and Vmax values from each line. Report the
average KM and Vmax from the two trials.
4. Plot of initial velocity (vo) versus temperature.
5. Plot of initial velocity (vo) vs. pH.
6. Plot of enzyme inhibition data from fifth lab period by both Michaelis-Menten and
Lineweaver-Burk curves. Plot data from uninhibited and both inhibitors together on one
graph. Use your graphs to determine KM and Vmax for each trial. Compare these values to
determine what type of inhibition is involved with each of the inhibitors.
7. A table with KM and Vmax for no inhibition, phosphate inhibition and copper acetate
inhibition.
 CSU Sacramento, Department of Chemistry
Chem 162 -Introductory Biochemistry Lab
Laboratory Manual

Your report should include a thorough discussion of the data. Here are some things to
ponder. What are the linear conditions for the assay in terms of [E], [S]? What were the optimum
temperature and pH for the enzyme? Discuss the effect of the various temperatures and pH on
the enzyme. What types of inhibitors are represented by inorganic phosphate and copper
acetate? How can you tell? What do your KM and Vmax values mean? How do the values obtained
without inhibitor compare to the KM and Vmax results from each type of inhibitor studied? As
discussed, make sure you relate your results to the published literature.