Computer lab
Enzyme kinetics and characterization of reaction intermediates
ÅKR 2003
The heme peroxidases utilize a heme cofactor and H2O2 (hydrogen peroxide) to create a
radical on the substrate. A radical is an unpaired electron, and such species are usually
highly reactive. Radicals are usually illustrated with the symbol  . Substrate/product
radicals will immediately react with each other and form stable products.
Horseradish peroxidase (HrP) is an enzyme purified from the plant horseradish. It has
affinity for a range of substrates, and performs important oxidative reactions in this plant.
The catalytic mechanism is shown below (Figure 1).
A
Figure 1, Horseradish (A) and the peroxidase catalytic cycle (B).
In this lab we will study and compare the enzymes Horseradish peroxidase (HrP) and
Myoglobin (Mb). The main function of Mb is to bind and transport oxygen in muscle
tissue. However, after comparing the heme groups and active site environment, it was
proposed that Mb could carry out significant peroxidase activity in muscle tissue.
The main purpose of this exercise is to analyze kinetic data obtained by stopped flow
spectrophotometry to characterize the hypothesized peroxidase activity of Mb.
B
Summary of experiments:
1) Determine the Vmax and kcat for HrP and Mb at pH = 6.5 and T = 20 C using
different concentrations of the substrate ortho-phenylenediamine (OPD).
2) Investigate the rapid stopped flow kinetics (in ms time scale) for the reaction of
enzyme and H2O2.
3) Discuss which properties that influence the catalytic efficiency.
NB! SAVE ALL FIGURES YOU MAKE DURING THE EXERCISES
Determination of kinetic constants for HrP and Mb using OPD as substrate
Ortho-phenylenediamine is an excellent substrate for HrP, and because a colorless
substrate is converted to an orange-brown product, the reaction progress can easily be
monitored by spectrophotometry.
We will use the Michaelis –Menten formalism to extract the Vmax parameter and
subsequently calculate the turnover number, kcat, that describes the efficiency of the
enzymes.
Description of Michaelis – Menten experiment:
1)
2)
3)
4)
Import data from CSV files.
Determine initial rates from the absorption at 450 nm.
Make a Michaelis-Menten plot (initial rate versus [OPD]).
Fit data, calculate kcat and answer questions.
Import of data
Start Origin 7.0.
File -> Import -> ASCII Options
Do this only
once? You
may have to
do it each time
due to user
restrictions
Press Update Options, then
File ->Save Template As -> OK
If you want to import a single data set press
If you want to import multiple data sets press
NB! To import a single data set, select one worksheet. Before importing multiple data
sets, close all worksheets!
Format of imported data
Click to select
column with
wavelength data
(nm)
Click to select
column with
absorbance data
(AU)
Click to select
column with
wavelength data
(nm)
Click to select
row with time
data (s)
Click to select row
with absorbance
data at a single
wavelength (AU)
Remember, by right clicking a cell, column, or row, you can either insert or delete an
item.
Example: To isolate absorbance values only, delete the first row and first column.
Exercise 1
Make a 3D plot from data showing the production of DAP and calculate the extinction
coefficient of this product:
1)
2)
3)
4)
Select an imported data set (HRP_0100.CSV)
Delete row 1 and column 1 from the imported data.
To convert row to column press: Edit -> Transpose -> Yes
While imported data window is selected, press: Edit -> Convert to matrix ->
Direct. A yellow matrix containing all absorption data turns up.
5) Then set the matrix dimensions, press: Matrix -> Dimensions
8) Set X axis as wavelength (low- high)
9) Set Y axis as time in seconds (start --- end)
10) Plotting:
While matrix window is selected press: Plot, and then
 3D Color fill surface, to look at mesh surface
 3D X constant with base, to look at time lines
 3D Y constant with base, to look at single absorption spectra.
Use this toolbar to manipulate view
Use this button to read values
from plot (navigate with keyboard arrows)
At which wavelength is the product (DAP) absorption maximum found?
To calculate the extinction coefficient open the Project “epsilondetermination.opj ”.
Plot the data in the worksheet. This is an spectrum recorded when all substrate have been
converted to product. The initial substrate concentration was 0.1 mM.
Calculate the extinction coefficient 450 (mM-1cm-1) of the product DAP at 450 nm by
using Beers law; A450 = 450  [DAP] 1 cm . Remember that 2 OPD  DAP.
What is the extinction coefficient 450 of DAP? Why do we use the last spectrum to
calculate extinction coefficient?
Exercise 2
Determination of Vmax and kcat
Different concentrations of substrate have been added to Mb and HrP. Stopped flow data
have been recorded and stored as CVS files.
Data files
HrP
[OPD] mM
0.05
0.1
0.15
0.2
0.3
0.5
0.8
1.2
Mb
Filename
HRP_0050
HRP_0100
HRP_0150
HRP_0200
HRP_0300
HRP_0500
HRP_0800
HRP_1200
[OPD] mM
0.02
0.03
0.05
0.15
0.2
0.3
-
Filename
MB_0020
MB_0030
MB_0050
MB_0150
MB_0200
MB_0300
-
Find initial rates (v0) for all reactions
Import data file (see procedure above). Create a new worksheet. Copy first row of
imported data into new worksheet (time). Then find the row containing data at 450 nm.
Copy this row into the second row of the new worksheet.
To create X and Y columns for plotting press: Edit -> Transpose, and click Yes.
Delete the first row (select row, right click and press delete).
Select X and Y column and press the
button.
Is the curve linear? If not, select only 6-10 points at the beginning of the curve (skip the
first 1-3 points) and plot them again.
To find the initial rate do a linear fit of the first data points:
Analyzis -> Fit linear (the result of the fit shows up in the box at the lower right corner
of the screen, scroll and find the slope B).
V0 = B/ 0.5 * 450, DAP Calculate v0 for all data sets, and write them down.
Michaelis- Menten plot
For each protein, create a worksheet with [OPD] in the X-column and v0 in the Y-column.
Plot each worksheet with
and fit using the Advanced fitting tool.
Press: Analysis -> Non linear curve fit -> Advanced fitting tool.
A new dialog box appers:
Press: Function -> Select
Choose:
Category = Pharmacology
Function = OneSiteBind
This function resembles the Michaelis –Menten
equation:
B = Vmax
K = Km
X = [OPD]
Press: Action -> Fit, Active dataset.
Choose all initial fitting parameters = 1
and press 100 Iter.
Write down the best fitting parameters.
The enzyme concentrations used are [HrP] = 1.0 *10-5 mM and [Mb] = 5.8 *10-3 mM
For both HrP and Mb, calculate
kcat = Vmax / [Enzyme]
What does the kcat constants tell you about the of the reactions? How many times faster
is HrP than Mb catalyzing the reaction 2 OPD  DAP ?
Exercise 3
Introduction
Rapid stopped flow kinetics detection of intermediates
The reaction cycle of HrP is shown in Figure 1. Two intermediates, compound I (A) and
compound II (B), can be detected when the enzyme reacts with H2O2.
A
B
In this experiment the ferric (Fe3+) form of the two enzymes HrP and Mb are allowed to
react with hydrogen peroxide (H2O2) in absence of the substrate OPD.
We want to study the reaction of H2O2 with the two enzymes HrP and Mb. Perhaps we
can gain some insight that will explain the difference in catalytic efficiency between the
two enzymes?
Soret () band
 and  bands
350
400
450
500
550
600
650
700
Wavelenght (nm)
Figure 2. Visible spectrum of ferric (Fe3+) HrP
p  p*
(, Low intensity)
p  p*
(, High intensity)
p  p*
(, Low intensity)
350
400
450
500
550
600
650
Charge Transfer
Low intensity
700
Wavelength
Wavelength (nm)
450
500
550
600
650
700
Wavelength
Figure 3. Visible spectrum of ferric (Fe3+) Mb
Experimental
The data files used in this exercise are stored in the catalogs “Rapid_kinetics_HrP” and
“Rapid_kinetics_HrP”.
1) Make a plot where you compare the “resting” ferric HrP with the following
stopped flow data (2D-graph). The interesting region is 450 – 700 nm.
 0 s (Hrpresting.opj)
 75 ms (HrPH20215s.CSV, first spectrum)
 15 s (HrPH20215s.CSV, last spectrum)
2) Make similar plot for Mb
 0 s (Hrpresting.opj)
 5 ms (MbH2O2.CSV, first spectrum)
 500 ms (MbH2O2.CSV, last spectrum)
A)
From the plot for HrP at different times, make a table of the absorbance values at 496 nm,
543 nm, 578 nm and 650 nm at 0 s, 75 ms, and 15 s.
Are there any peaks that first seem to increase in the first time interval (0 s – 75 ms)
and then decrease in the second time interval (75 ms – 15 s)?
Which spectrum corresponds to which intermediate in Figure 1? *
Make a 3D plot for HrPH202.CSV and for HrPH20215s.CSV.
For each wavelength, calculate first order rate constants k using: Analysis -> Fit
exponential decay -> First order.
TIPS!!
If the fitting is bad (e.g. t1 > 10 ), use the Advanced fitting tool, select the exponential
category and use the function ExpDec1 with the parameter A1 < 0.
Another trick if the fitting is bad is to exclude data that does not “look exponential” or
restart the Origin program.
The rate constant k = 1/ t1 (compare the function used by Origin to the integrated first
order function at page 7, line 4 in the compendium from A.V. Agasøster). A high rate
constant indicates a fast reaction.
Insert the calculated rate constants in the table below.
Wavlelength
5 – 2000 ms
75 ms – 15 s
First Order Rate Constants for HrP
496 nm
543 nm
578 nm
650 nm
B)
Repeat the procedure above for Mb. Since the Mb + H2O2 reaction is finished in 500 ms,
there is only one data file (MbH2O2.CSV). Skip the question above marked with “*” and
answer this one instead:
In the Mb + H2O2 reaction only one of the intermediates in Figure 1 is observed.
Which one? (Hint: look for similar spectral features).
Make a 3D plot for MbH2O2.CSV as well.
Wavlelength
5 – 500 ms
First Order Rate Constants for Mb
503 nm
550 nm
588 nm
640 nm
Both compound I and compound II are very reactive. Consider the results from the
Michaelis- Menten experiments (calculation of the turnover number kcat) and the rapid
stopped flow studies of reaction intermediates in the absence of substrate.
Discuss the seemingly contradictory results. What do you think determine the
formation of the product DAP from OPD?
Considering the low substrate affinity of H2O2 activated Mb; do you think Mb can
carry out significant peroxidase activity in human tissue?