half report

I’m working on a biochemistry report and need the explanation and answer to help me learn.Help guide for team work
student#1
1. Cover page







Title of the experiment
Date of the experiment
Name of this course
Department (Chemistry and Earth Sciences)
University
Full Names of the partners who worked on this report, ID numbers
Which student in the group worked on which part of the report
For the remaining sections of this report, some general guidelines are provided. However, students MUST
think of other important things to add and do not simply limit to these guidelines. You can include any other
aspects you think are important or information obtained from any other published articles you might have
accessed from scientific databases.
Your creativity will have assigned points
2. Abstract
This is an overall summary of the experiment:
➢ The aim of the experiment
➢ Highlights of methods
➢ important results
➢ And major conclusions.
➢ All about 150-200 words.
From Next Parts onwards, you must cite references in the paragraphs (= in the body of the text) for
any information is taken from publications, databases, or instructor’s theory and protocols)
Student #2
3. Introduction (Paragraph-1:)












In general, what are enzymes? By what mechanism do they make the reactions go faster?
Which are the 6 different major classes? Alkaline phosphate enzyme belongs to which major class?
What is the Subclass, and which type of reactions the subclass of enzymes catalyzes?
What is the sub-subclass, and which type of reactions for this sub-subclass?
What is the individual member number for alkaline phosphatase?
Now write the full E.C. Number for this specific enzyme based on the above descriptions.
(find it from Brenda, IUBMB or other databases).
In general, what types of reactions does the family of alkaline phosphatase enzymes catalyze?
What are examples of other individual members of this sub-subclass? Give examples and their
substrates.
Are there cofactors for any of these enzymes in the Main class, subclass, or sub-subclass?
In which organs and tissues the enzyme you worked on, is present in humans?
Although they are present in different tissues and organs, are they really different enzymes or isozymes?
What type of posttranslational modifications cause these differences?
What is the biological function of this enzyme?
Biochemistry Laboratory
Enzyme Catalysis: Enzyme Kinetics Experiment Week 1
Prepared by Dr. Rekha Ganaganur and Dr. Lakshmaiah Sreerama
Experiment
Materials Needed:
1) Master Mix solutions that contain the Tris Buffer (0.1M, pH 8.0) and also the substrate PNPP already
added at following final concentrations:
a) Master mix-1 of 0.020mM PNPP prepared in 0.1M Tris buffer
b) Master mix-2 of 0.025mM PNPP prepared in 0.1M Tris buffer
c) Master mix-3 of 0.033mM PNPP prepared in 0.1M Tris buffer
d) Master mix-4 of 0.050mM PNPP prepared in 0.1M Tris buffer
e) Master mix-5 of 0.100mM PNPP prepared in 0.1M Tris buffer
f) Master mix-6 of 0.200mM PNPP prepared in 0.1M Tris buffer
2) Tris Buffer 0.1M pH 8.0; (to be used for calibration blank of spectrovis, and for the one blank
kinetics reaction for the class).
3) Enzyme: Alkaline phosphatase enzyme
4) Inhibitor For Inhibition Studies: 10mM Sodium Phosphate stock solution.
Procedure
All students must bring and use their computer for entering all the data generated during the lab. If
you don’t have a computer, get it from the lab.
You must download the Excel file and be ready with your computer before you start the
experiment.
You will be divided into teams of 4 students (one team might have 3 or 5 as per how your instructor
makes your team, if we cannot do it equally 4 students in all teams).
Assuming Team of 4, here is how you should divide up your work:
Part A: Calibration of
Spectrophotometer
All students must learn it, as this step will appear in the
practical exam and every student in the exam must be able to do
it individually without any help from friends or instructor/TA
Part B: Blank Reaction
Nobody does this. Get the data from TA/instructor
Page 1 of 6
Part C (Table-1): Kinetic
reactions with No inhibitor
(= zero inhibitor)
Part D (Table-3): Kinetic
reactions with 0.067mM
phosphate inhibitor
Part E (Table-5): Kinetic
reactions with 0.133 mM
phosphate inhibitor
Part F (Table-7): Kinetic
reactions with 0.201 mM
phosphate inhibitor
Biochemistry Laboratory
Student-1: Conduct the set of reactions of Zero Inhibitor set:
Take help from Student-2, while measuring the absorbance, to
enter the values in Table-1 directly on the computer Excel file.
Student-2 Conduct the set of reactions of 0.067mM Inhibitor
Take help from Student-1, while measuring the absorbance, to
enter the values in Table-3 directly on the computer Excel file.
Student-3 Conduct the set of reactions of 0.133 mM Inhibitor:
Take help from Student-4, while measuring the absorbance, to
enter the values in Table-5 directly on the computer Excel file.
Student-4: Conduct the set of reactions of 0.201 mM Inhibitor
Take help from Student-3, while measuring the absorbance, to
enter the values in Table-7directly on the computer Excel file.
Part-A: Calibration of Spectrophotometer: Each pair of students learn to calibrate your
shared SpectroVis for 410 nm, using 0.1M Tris-HCl buffer as your calibration blank.
Part-B: Blank Reaction for Enzyme Kinetics: Get this Data from your TA. Your team
is not conducting this part of the experiment to save time. Blank reaction= Buffer + enzyme, no
substrate and no inhibitor) for absorbance at 410nm against time. Note the readings in the following
Table. This blank reaction data is required to ensure there is no interference by the buffer and
enzyme, during the time course of kinetics. There should not be any change in absorbance with time.
Blank Reaction Data:
Time, min
Absorbance at 410nm
0
0.5
1.0
1.5
2.0
2.5
3.0
Page 2 of 6
Biochemistry Laboratory
Based on the readings posted by your TA, what is your interpretation? Is there any
interference? Write your interpretation in your lab notebook.
From Part-C through Part-F General Steps for All Students:
Solutions Preparation:
• Every student in the team, take 6 clean and dry cuvettes and Label them as 1 through 6. Make
sure they are clean and dry by wiping with tissue paper inside the cuvette also.
• Every student is required to pipette 1.45mL of each of the Master Mix solutions into these separate
cuvettes. A good planning to save time could be as follows as an example:
o You set your P1000 micropipette to 1mL (1000uL) and let your lab partner set their P1000
pipette to 0.450 mL (450 µL).
o You pipette 1mL of Master-Mix-1 (that contains the 0.02mM PNPP substrate) into your
cuvette-1 and also to your lab partner’s cuvette-1.
o Let your lab partner pipette 0.450 mL to your cuvete-1 and their cuvette-1 as well.
o Next both of you use new tip and pipette the Master Mix-2 to cuvette-2 as above to get the
1.45 mL to each.
o Repeat with other Master Mix solutions, to corresponding new cuvettes to get 1.45 mL of the
different Master Mix solutions into the different cuvettes.
• Student-1 who is working on zero inhibitor, do not add any phosphate inhibitor to any of your
cuvettes.
• Student-2 working on 0.067mM inhibitor set of reactions, add 10uL of the phosphate inhibitor,
using new tip each time, to all your cuvettes and mix well, by pipetting up and down a few times
as shown by your instructor.
• Student-3 working on 0.133mM inhibitor set of reactions, add 20uL of the phosphate inhibitor
using new tip each time, to all your cuvettes and mix well, by pipetting up and down a few times
as shown by your instructor.
• Student-4 working on 0.201mM inhibitor set of reactions, add 30uL of the phosphate inhibitor
using new tip each time, to all your cuvettes and mix well, by pipetting up and down a few times
as shown by your instructor.
Nobody should add the enzyme yet!


Make sure your spectrovis is calibrated for 410 nm using the Tris buffer.
And make sure you have logged on to your computers, download the Spreadsheet File and open
the specific Data Table in the file which you are supposed to use.
Planning for the kinetic reactions.
• Caution! Very important! Do not add enzyme to all the cuvettes at the same time. If you
make mistake and add it to all the tubes, the reactions will all be over within 3 minutes in all the
cuvettes and your whole set of expt you will have start over,
• One student in the pair start the reactions as follows, and the lab partner will help in entering
all the absorbance data on the computer. After completing all 6 reactions, the other student in
the pair will conduct their reactions and you help.

Set your mobile phone timer or the timer given in the lab.
Page 3 of 6


Biochemistry Laboratory
To only your cuvette-1, add 50µL(0.05mL) of alkaline phosphatase enzyme. Quickly
Mix by pipetting up and down a couple of times.
Immediately start absorbance measurement at 410nm in intervals of 30 sec (0.5min), up to
3 min. Take help of your team-mate student-2 to enter all the readings on the computer
Excel File in your assigned data Table, for the first cuvette reaction.
Make sure you “Save” the file.









Only after completing cuvette-1 reaction for 3 minutes, start with next sample in Cuvette-2. Add
50µL(0.05mL) enzyme to cuvette-2, mix well and immediately start measuring absorbance data
again at 30 seconds interval for 3 min.
Make sure the readings are entered in the Data Table. Remember to “Save” the file again.
One by one, complete all other remaining reactions, for each of the remaining cuvettes, adding
the enzyme to only the particular cuvette you are working with.
Make sure all the data entered in the assigned data Table. Save the file.
Check if your readings are making sense as per enzyme kinetics theory! Can get help from
TA and/or instructor to get this verified. Remember! For each substrate concentration reaction,
the absorbance should increase with time. Likewise with each higher concentration of substrate,
the absorbance should increase when you compare your six reactions with each other.
If any of the sets do not show proper readings, repeat those and enter the data on the computer
file.
If you worked on the lab computer or friend’s computer, make sure to email the file to
yourself.
Before coming to next week’s lab, everyone must have all their data Tables entered and
ready on your computers, for data analysis lab. Remember to bring your computer.
Before coming to the lab next week, read the theory related to Progression Curves, MichaelisMenten theory/curve, LB-plots, and types of inhibition. Watch the recorded lecture and videos
posted.
Table-1: Absorbance Data of Part-C with Zero inhibitor: With 50 µL enzyme and various
concentrations of PNPP Substrate:
Absorbance at 410nm
Time
Cuvette#1
Cuvette#2 Cuvette#3 Cuvette#4 Cuvette#5 Cuvette#6
(min)
0.020mM
0.025mM 0.033mM 0.050mM 0.100mM 0.200mM
PNPP
PNPP
PNPP
PNPP
PNPP
PNPP
0
0.5
1.0
1.5
2.0
2.5
3.0
Page 4 of 6
Biochemistry Laboratory
Table-3: Absorbance Data of Part-D with 0.067 mM inhibitor: With 50 µL enzyme and various
concentrations of PNPP Substrate:
Time
(min)
Cuvette#1
0.020mM
PNPP
Absorbance at 410nm
Cuvette#2 Cuvette#3 Cuvette#4 Cuvette#5 Cuvette#6
0.025mM 0.033mM 0.050mM 0.100mM 0.200mM
PNPP
PNPP
PNPP
PNPP
PNPP
0
0.5
1.0
1.5
2.0
2.5
3.0
Table-5: Absorbance Data of Part-E with 0.133mM inhibitor: With 50 µL enzyme and various
concentrations of PNPP Substrate:
Absorbance at 410nm
Time
Cuvette#1
Cuvette#2 Cuvette#3 Cuvette#4 Cuvette#5 Cuvette#6
(min)
0.020mM
0.025mM 0.033mM 0.050mM 0.100mM 0.200mM
PNPP
PNPP
PNPP
PNPP
PNPP
PNPP
0
0.5
1.0
1.5
2.0
2.5
3.0
Page 5 of 6
Biochemistry Laboratory
Table-7: Absorbance Data of Part-F with 0.201 mM inhibitor: With 50 µL enzyme and various
concentrations of PNPP Substrate:
Absorbance at 410nm
Time
Cuvette#1
Cuvette#2 Cuvette#3 Cuvette#4 Cuvette#5 Cuvette#6
(min)
0.020mM
0.025mM 0.033mM 0.050mM 0.100mM 0.200mM
PNPP
PNPP
PNPP
PNPP
PNPP
PNPP
0
0.5
1.0
1.5
2.0
2.5
3.0
Page 6 of 6
Biochemistry Laboratory
Enzyme Catalysis: Data Analysis and Interpretation-Week-2
Prepared by Dr. Rekha Ganaganur and Dr. Lakshmaiah Sreerama
Theory and Principles
Main Goal of Week-2: Data Analysis from kinetics experiments of last week:
Objectives of Week-2:
1) Develop Progression Curves of each set of data in the absence and presence of inhibitor
2) Determine Initial Velocity Vo in each case, from the slopes of the linear progression curves, using
the trendline equations.
3) Understand nature of Michaelis-Menten curve.
4) Develop LB-plots and obtain K m and V max in the absence and presence of various Inhibitor
concentrations.
5) Determine the type of inhibition through combined graphs of all the LB-plots.
6) Determine Ki value for the inhibitor.
Nature of Progression Curves of Enzyme-Kinetics:
Progression curves of kinetics are graphs of amount of
product formed (or substrate converted), against time.
Absorbance against Time
The progression curve with any substrate concentration
will start off as almost linear curve, in the initial few
minutes (Figure 1). And with further increase in time
when most of the substrate is catalyzed, it levels off.
Progression Curves allow you to determine the initial
velocity (v o ) of the reaction. This can be obtained by
measuring the slope of the linear part of the graph and
then converting it to concentration. Because Vo is the
rate = concentration of product formed per minute.
Figure 1: Enzyme reaction progress curve
In your experiment, because you measured the kinetics
only in the initial 3 minutes of the reaction, you should be
able to obtain linear graphs.
Figure 2: Progression Curves
Each team member in the team, using the Data Table
you worked on last week, create “scatter plot” of your
Data Table, for absorbance against time (minutes) for all
the substrate concentrations. An example graph is
shown in Figure-2 for zero inhibitor set.
Zero Inhibitor set: Use Data from Table-1
0.067 mM Inhibitor: Use Data from Table-3
0.133 mM Inhibitor: Use Data fromTable-5
0.201 mM Inhibitor: Use data from Table-7
Page 1 of 7
Biochemistry Laboratory
And for each line you plot as per your Data Table, obtain Trendline equation.
From the trendline equation Determine the slope of the reaction.
For example, if the trendline is y = 0.1423x + 0.0964, then slope = 0.1423
For each of the lines, get these slope values, and enter them in the subsequent Table show in the Excel
file as follows:
Data from Table-1 gives slope values which should be entered in Table-2.
Data from Table-3 gives slope values which should be entered in Table-4.
Data from Table-5 gives slope values which should be entered in Table-6.
Data from Table-7 gives slope values which should be entered in Table-8.
What does the slope value represent? It is the rate of change of absorbance per minute=∆A/min
How to calculate Initial Velocity V 0 from slope?
Slope = ∆A/min is the rate of reaction = Initial Velocity Vo
In the above example, ∆A/min = 0.1423.
into concentration units as follows:
However, you need to convert this initial velocity (v o )
Beer’s law states A = εbc. If b= 1 cm (using 1cm cuvette), and by knowing ε value, you can calculate
c = concentration of product formed. Therefore c = A/ ε .
In the case of PNP product in your experiment, ε = 18000 M-1cm-1. Therefore, in the above example,
concentration c = ( 0.1423 x 106) /18000 = 7.9055 umols/min
Note that we have multiplied by 106, because V 0 units should be in µmols/min for enzyme activity
units.
All of these calculations formulas have been already set up in the Excel files that you have been
using. Therefore, as soon as you enter the slope value in your respective Data Tables, you should be
able to automatically get the above conversion calculations populated under the V values column.
The Data Tables you used, have also been set up to automatically calculate 1/V and 1/[S] values.
Michaelis-Menten Curve:
This curve represents the Relationship between V o and
Substrate Concentrations:
For most enzymes, when the initial reaction rate (v o )
is plotted as a function of the concentration of
substrate [S], a rectangular hyperbola is obtained
(Figure 3).
Figure 3: Michaelis-Menten Kinetics: Effect of
substrate concentration on enzyme activity.
Page 2 of 7
Biochemistry Laboratory
The rectangular hyperbolic plot is known as Michaelis-Menten Kinetic Curve. At low substrate
concentrations V o is directly proportional to [S], with increasing response, as substrate concentration
is increased, showing a linear relationship initially.
But when the active site of the enzyme is saturated, at high concentration of the substrate, the
maximum velocity (Vmax) is reached and the curve levels off.
The Substrate value on X-axis, when v = ½ Vmax, represents the Km value for the substrate.
For your Data Table you worked on, that as [S] and [V] values, Create the Michaelis-Menten
kinetics curve.
Michaelis-Menten Equation: Michaelis-Menten kinetic curve can be described by a mathematical
relationship, known as the Michaelis-Menten equation.
[ ]
+ [ ]
(Michaelis-Menten equation)
=
Michaelis-Menten theory:
K m is derived based on Michaelis-Menten theory. The enzyme E and its substrate S first associate
reversibly to form an ES complex to decrease the free energy of activation. At higher substrate
concentration, when enough ES accumulated, The ES complex then breaks down irreversibly to form
product P. The enzyme gets released to make it available again to react with another molecule of S.
With a single substrate (S) and single product (P) enzymatic reaction, the process can be described
as follows:
E + S
k1
k2
ES
k3
E + P
At low substrate concentration, the first step is reversible. At high saturating substrate conc., all the
enzyme is converted to ES complex and the active site is starting to get saturated. Therefore, the
catalytic reaction to form product now depends on second step of ES dissociating and releasing the
product P. Therefore, the second step becomes rate-limiting.
Hence overall reaction does Not depend on concentration of S at high substrate concentration.
k 1 , k 2 and k 3 are rate constants for each of the reactions shown above. The Michaelis constant K m can
be expressed as:
Km =
k2 + k3
k1
Significance of V max : In the Michaelis-Menten Equation, V max is the maximum velocity of the reaction
(at infinite substrate concentration); V max is a function of the amount of enzyme and is the appropriate
rate to use when determining the specific activity of a purified enzyme.
Page 3 of 7
Biochemistry Laboratory
Therefore, higher the Vmax, better is the enzyme activity, because it brings maximum rate of
reaction with less amount of enzyme.
Significance of K m : K m is the Michaelis constant. It is defined as the saturating substrate
concentration when V o = (½) V max . The active site of the enzyme is saturated by the substrate.
Therefore, lower the Km, better the substrate, because the enzyme has higher affinity for it and
has higher catalytic efficiency (affinity), as small amount of substrate is needed to saturate the enzyme
active site.
The K m is expressed in terms of substrate concentration (mol/liter) and is independent of enzyme
concentration. K m values can be used to select a substrate concentration that will give maximum reaction
velocity. It can be used to compare the affinities of different substrates for a given enzyme or the same
substrate with different enzymes.
Lineweaver Burke (LB) Equation and Construction of LB plots to determine Km
and Vmax.
Directly from Michaelis-Menten curve it is not possible to determine Km and Vmax easily in lab
experiments when V and [S] are plotted. Because it is hyperbolic curve it levels off, and becomes almost
parallel to x-axis at higher substrate concentration. Which means one has to do many experiments leading
to infinite substrate concentration to get the real Vmax (remember two parallel lines meet only at
infinity!).
However, the Michaelis-Menten equation can be transformed mathematically to derive a new
linear equation in the form of y= mx =b.
The Michaelis-Menten equation is
=
[ ]
+ [ ]
Mathematically by taking reciprocals on each side of the equation and rearranging, to arrive at a
new equation called Lineweaver-Burk Equation or Double-Reciprocal Equation.

=

+

This is in the form of a linear equation y= mx+b. Therefore, a graph of 1/V against 1/[S] results in a
linear plot.
In the Lineweaver burke plot, y = 1/V; Slope = K m /V max ; x = 1/[s] and the y-axis intercept =
1/Vmax. The x-axis intercept will be -1/K m .
Page 4 of 7
Biochemistry Laboratory
Hence, an LB-Plot which is a graph of 1/v from
experiments with different 1[S], results in a linear curve
(Figure-4). Its y-intercept will give 1/Vmax. Hence
Vmax can be determined.
And since the x-intercept gives -1/K m , the K m can be
determined.
Or because slope value is Km/Vmax, substituting for
Vmax obtained from y-intercept, you can also
determine Km value.
Figure 4: LB Plot
However, small errors in the data points at lower concentration of
substrates are magnified in mathematical transformation and most data points aggregate to one end of
the line. Therefore, one has to be careful in selecting the substrate concentrations (x-axis scale)
such that the datapoints will be evenly spaced.
Enzyme inhibition
Inhibitor: A compound that decreases the rate of enzyme-catalyzed reaction
The velocity of the enzyme-catalyzed reaction is reduced by the formation of enzyme-inhibitor (EI)
complex, or enzyme-substrate-inhibitor (ESI) complexes
Inhibitor Constant Ki: The efficiency of inhibition is based on the Inhibition Constant Ki. It is the
concentration of the inhibitor that can bring about maximum inhibition. Smaller the Ki value,
higher the inhibition = more efficient is the inhibitor, because small concentration of inhibitor can
bring about maximum inhibition.
Investigating the inhibition of enzyme activities by specific molecules is very important because:

enzyme inhibition phenomenon is an important reaction control mechanism in biological
systems, e.g., regulation of metabolic pathways by negative feedback by a product(s),

many medications act by inhibiting enzymes, e.g., protease inhibitors used to treat HIV.
Therefore for a medical drug which is designed to be an inhibitor of an enzyme causing the
disease, the smaller the Ki, the better the drug.

the action of many toxins can be explained by enzyme inhibition, e.g., toxicity due to heavy
metals.
Inhibitions can be either Irreversible or Reversible
Irreversible Inhibitors: These are substances, usually not of biological origin, which react covalently
with an enzyme and prevent the substrate from binding to enzyme active site. The inhibition is
permanent = enzyme loses is activity permanently. The binding could be within the active site forming
part of the binding; or it may be further from the active site and affect the three-dimensional conformation
of the enzyme. For e.g.,The toxicity of heavy metal ions, e.g. Hg2+ is due to their irreversible effects on
enzyme activity.
Reversible inhibitors: Inhibition is reversible. Therefore, Reversible inhibitors do not react covalently
with an enzyme but rather through weak intermolecular forces.
Page 5 of 7
Biochemistry Laboratory
They rapidly and reversibly bind to, or dissociate, from the enzyme.
Types of Reversible Inhibition Mechanisms: Inhibitors interact with enzymes and inhibit the reactions
they catalyze in one of 3 ways:

competitive inhibition or

non-competitive Inhibition or

Un-competitive inhibition.
Competitive inhibition (Reversible)
• Competitive inhibitors bind to the enzyme E reversibly
to the active site. But not to the ES complex.
E
• Often the inhibitor resembles the substrate, and hence
competes with the substrate
• The occupation of the active site by an inhibitor
molecule prevents a substrate molecule from
effectively binding to the same active site
• The enzyme (E) is now bound to the inhibitor (I), to form
EI. Hence, A competitive inhibitor lowers the rate of
catalysis by reducing the proportion of enzyme molecules
bound to the substrate.;
• V max is unaffected, but Km increases (= 1/Km decreases).
• Hence in the LB-plots made for 1/Vo vs. 1/[S], the 1/Vmax
will still intersect on the y-axis. But 1/Km will NOT
intersect on x-axis.
Non-Competitive inhibition (reversible):
• The inhibitor binds to the free enzyme E as well as
the ES complex but not at the active site.


E
+
S
+
E
+
E
I
E
I
E
S
No Reaction
X
E
S
+
P
+
+
I
+ P
a: Competitive inhibition
I
I
The K m in this type of inhibition remains
unchanged and will intersect on the x-axis for
1/Km. Because the affinity of substrate molecules
that bind to the active site is unaffected.
E
S
I
E
c: Non-competitive inhibition
S
X
No Reaction
However, V max decreases (= 1/Vmax increases) due to the
concentration of active enzyme molecules being effectively
reduced by the presence of inhibitor.
Page 6 of 7
Biochemistry Laboratory
Uncompetitive inhibition the inhibitor binds to
the Enzyme -Substrate complex = after the
E + S
E S
substrate has bound.
I
Because the inhibitor binds to enzyme substrate
(ES) complex effectively decreasing ES from
forming the product. It is most common in
reactions involving more than one substrate.
I E
+ P
+
+
Hence both Km and V max values in this type of
inhibition decrease.
E
I
c: Non-competitive inhibition
I E S X
No Reaction
Dixon Plots to Determine Ki (Secondary plots)
Construct the Lineweaver-Burk plot for the Vo and [S] in the absence of presence of various inhibitor
concentrations. Obtain 1/Vmax and slope values.
Then generate another plot called Dixon Plots, as follows:
For Noncompetitive or Uncompetitive Inhibitions: Draw a graph of the 1/Vmax values against
Inhibitor Concentration. The Ki will be on the X-intercept.

For Competitive inhibition: Draw a graph of slopes of the lines from Lineweaver-Burk plots
against the inhibitor concentration. The Ki will be on the X-intercept.
Slope of LB plot

Dixon plot/Secondary Plot
0.02
y = 0.0838x + 0.0015
R² = 0.9893
0.01
-0.05
0
-0.01
0
0.05
0.1
0.15
0.2
0.25
Inhibitor, mM
Page 7 of 7
Biochemistry Laboratory
Enzyme Catalysis: Enzyme Kinetics Experiment Week 1
Prepared by Dr. Rekha Ganaganur and Dr. Lakshmaiah Sreerama
Theory and Principles
This experiment will be carried out in a period of two weeks.
Week-1: Conduct experiments to collect data
Week-2: Data Analysis and Interpretations.
The theory and principles used for week-1 is described here.
Objectives of Week-1:
1. Experiment for obtaining enzyme catalytic activity data
2. Experiment for obtaining enzyme inhibitor data with various concentrations of an inhibitor
Role of Enzymes
Enzymes (generally proteins) are biological catalysts that increase the rate of biochemical reactions. The
chemical it binds to, and catalyzes, is called Substrate (for example, see Figure 1).
Enzymes increase the rate of the reaction by decreasing the Free Energy of Activation required for
the Transition-State. (Figure2)
Figure 1: Succinate dehydrogenase (SDH)
catalyzed oxidation of succinic acid to fumaric acid.
Figure2: uncatalyzed vs. Enzyme-catalyzed Reaction
energy diagram
Each enzyme catalyzes the reaction of either a specific substrate, or a limited number of reactions with
substrates of similar structure.
Enzymes and their classification: Enzymes are classified according to the chemical reactions they
catalyze. Accordingly, there are six Main classes of enzymes (See Table 1). Further, the enzymes are
named based on type of reaction they catalyze, the substrate they use or the product they produce. And
the name ends with a suffix “ase”. Most enzymes also have recommended common names.
Page 1 of 6
Biochemistry Laboratory
Table 1: Main Classes of enzymes.
Enzyme class
Reaction catalyzed
1. Oxidoreductases (dehydrogenases) Catalyze oxidation-reduction reactions
2. Transferases
Catalyze reactions that involve transfer of functional
groups
3. Hydrolases
Catalyze hydrolysis reactions where water is the
acceptor of the transferred group
4. Lyases
Catalyze lysis (breaking) of bonds in a substrate,
generating a double bond in a non-hydrolytic, nonoxidative elimination
5. Isomerases
Catalyze isomerization reactions
6. Ligases (synthetases)
Catalyze ligation, or joining of two substrates.
(opposite of lyases)
Factors affecting enzyme activity:
The enzymatic catalyzed reactions in the living systems are affected by many factors such as enzyme
concentration, substrate specificity, availability of co-factors and conditions of temperature, pH,
salt concentrations. For example, enzymes have a narrow range of temperature and pH
stability where they are most active.
Fig. 3: Effect of temperature on enzyme
acti it
Fig 4: Effect of pH on enzyme activity
Hence enzymatic reactions should be carried out at the optimum temperature and at optimum pH
conditions using appropriate buffers. Many enzymes require appropriate concentrations of
specific cofactors for maximum activity. e.g., NAD+, FAD or ADP, or inorganic metal ions, e.g.
Mg2+ and K+. These cofactors actively participate in catalysis via accepting or donating specific
chemical groups or electrons.
Enzyme Kinetics expressed through Rates or Velocity of the Reaction:
Because catalysis is to increase the speed of the reaction, enzyme reactions are described in terms of
Kinetics through rates of reaction = velocity of reactions.
Rate or Velocity of reaction: how much reaction occurred per unit time. This is expressed as either
the amount of product formed or substrate utilized, per min or per second.
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Biochemistry Laboratory
International Unit (IU): It is the amount of enzyme required to catalyze one micromole of substrate
(or 1 micromol of product formed) per min = µmol/min; temperature, pH and which substrate or
product, must be specified. For example, 2 µmol of starch converted per minute, at 25°C, pH 7.2.
In SI Units: Katal or Kat: It is the amount of enzyme required to convert 1 mol of substrate (or 1 mol
of product formed) in 1 second (s) ; with specified reaction conditions.
It is very important that the units also clearly specify the substrate or product used, especially when the
enzyme transformations involve different number of substrates or product molecules.
In either case, note that the of amount of substrate catalyzed or product formed is expressed in
micromoles or mols, not as concentration (not molarity or mol/liter).
The SI unit is relatively large (1 kat = 6 x 107 U). Therefore, SI prefixes, e.g., nkat or pkat are often used.
For example 16.67 nkat = 16.67 nanomol/s; or 16.67 pkat = 16.67 pmol/s
Specific Activity: Enzyme activities when expressed as µmol/min/mg protein is known as specific
activity (amount of substrate consumed or product produced per min per unit mass of protein).
Because the amount of protein (remember enzymes are proteins!) is also included, it is useful for
comparing purity of different enzyme preparations. Or when comparing the activity of one substrate
with another, for the same enzyme.
Enzyme assays = Methods to Monitor Enzyme Activity
By measuring a characteristic property of the substrate or the product, the enzyme catalyzed reactions
can be monitored. In continuous assays, monitor the progress continuously with time.
For example, if product formed is colored = Absorbance in a certain wavelength. Then the absorbance
will increase with time as the reaction progresses and more product is formed. Therefore, if monitoring
the product formation, because more products is formed with time, the graph will show a positive
slope.
Or if the substrate is colored (= has absorbance), and gets converted to a colorless product, the
absorbance will decrease with time. Therefore, the graph will give a negative slope.
Figure 5 and 6: Type of graphs from monitoring
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Biochemistry Laboratory
Some Types of Assays:
Spectrophotometric assays: Many substrates or their
products formed, absorb Visible or UV light at a
particular wavelength. But when the reaction occurs,
there is change in absorbance due to change in the
concentration of substrate and product.
Hence
monitoring absorbance change with time using
spectrophotometry is a convenient assay method in such
cases.
Fig 7: Colored substrate or product
Fluorimetric assays: Certain substrates are converted to
fluorescent products = emit light in the form of photons.
Because number of photons released are measured, there
will be a large amount of photons per molecule. Hence this
is a highly sensitive method = useful when very small
amount of substrates or products involved. However, care
should be taken because even small amount of impurities
may produce background fluorescence, or quench (reduce)
the signal.
Fig 8: Fluorescent product
Today’s Experiment:
Which Enzyme, what is it’s role, which Main Class and which SubClass does it belong to?

You will be studying the Enzyme Kinetics for Alkaline Phosphatase Enzyme.

It hydrolyses phosphate group. Hence, it belongs to Hydrolase Main Family of enzymes.

It catalyzes the hydrolysis of monoesters type of molecules. Therefore, it belongs to the
Subclass of ester hydrolase.
Which specific groups attached to the esters are released?
Specifically, it releases the phosphate group attached to the monoesters upon hydrolysis.
Therefore, the enzyme belongs to the SubSubClass of Phosphomonoester hydrolase or
phosphatase.
And because the hydrolysis is brought about in alkaline pH conditions, the enzyme is called
Alkaline Phosphatase.
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Biochemistry Laboratory
Which Substrate in today’s experiment and what
is the product formed?:
para-nitrophenyl phosphate (PNPP) is the substrate.
Upon hydrolysis of the phosphomonoester, the
phosphate group of the ester is released, forming the
product para-nitrophenol (PNP).
Experimental Conditions: It has been established that alkaline phosphatase enzyme has optimum
activity at pH 8.0 and 25°C. Accordingly, you will perform alkaline phosphatase reactions under
these conditions.
The buffer to use for maintaining the pH is 0.1 M Tris-HCl Buffer
Monitoring of Product Formation: The para-nitrophenol (PNP) product formed is fully ionized at
pH 8.0, and is yellow in color in its fully ionized form. Due to this yellow color, it absorbs
maximally at 410 nm wavelength. Therefore, you will monitor the rate of enzymatic reaction, by
measuring absorbance of PNP formed using a spectrophotometer, over a course of 3 minutes.
Because remember! The velocities are linear only in the initial time periods in enzyme kinetics.
Inhibitor for Inhibition Studies: Inorganic Phosphate is the inhibitor used today.
accomplished by using sodium phosphate solution at appropriate concentrations.
This is
What does the experiment involve today?
You will be working in teams of 4 as assigned by your instructor. (If a team ends up with less than 4,
check with your instructor how to handle all the sets of reactions).
Each student in the team must attempt one full set of experiment and gather data. For example:
Student-1 works on “zero inhibitor” set of experiments. While Student-2 helps and enters the
absorbance values in Table-1.
Next, switch roles so that Student-2 works on “0.067 mM inhibitor” set of experiments. Student-1
will help and enter the absorbance values in Table-3.
Student-3 works on “0.133 mM inhibitor” set of experiments, while Student-4 will help and enter the
absorbance values in Table-5.
Next, Student-4 will work on “0.201mM inhibitor” set of experiments, while student -3 helps enter
the absorbance values, in Table-7.
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Biochemistry Laboratory
Make sure each student has saved their data files, and emailed to themselves.
A blank reaction that contains everything but no enzyme, should always be included to check for nonenzymatic background contribution to absorbance readings.
For the whole class one blank reaction can be conducted by a volunteer or your TA and shared
with the rest of the class, in the interest of time.
For next week, everyone must bring their computer and the Excel file of Data Table they worked
on today (with all data entered), and continue to work on further data analysis and interpretation.
Read the theory document for next week’s experiment on enzyme kinetics and inhibition
concepts before coming to the lab. Watch the recorded lectures posted.
Page 6 of 6

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