Computer lab
Enzyme kinetics and characterization of reaction intermediates
ÅKR 2004
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 in OPJ 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.
Exercise 1
Make a plot from data showing the production of DAP and calculate the extinction
coefficient of this product:
1) Open the file Epsilon determination.OPJ
2) Plot the spectra recorded at 3, 30, 55, and 60 seconds in the same graph.
At which wavelength is the product (DAP) absorption maximum found?
The last spectrum (60 s) was recorded when all substrate had 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
at 450 nm have been recorded and imported into Origin OPJ files. HRP_MM.OPJ and
MB_MM.OPJ contains the HrP and Mb Michaelis –Menten data, respectively.
Data files
HrP
[OPD] mM
0.05
0.1
0.15
0.2
0.3
0.5
0.8
1.2
Worksheet name
HRP0050
HRP0100
HRP0150
HRP0200
HRP0300
HRP0500
HRP0800
HRP1200
Mb
[OPD] mM
0.02
0.03
0.05
0.15
0.2
0.3
-
Worksheet name
MB0020
MB0030
MB0050
MB0150
MB0200
MB0300
-
Find initial rates (v0) for all reactions
Open OPJ file for HrP or Mb. Plot all the curves in one graph using
to see the
effect of increasing substrate concentration. Include this graph in the report. Do not
include all individual linear fittings. (Tips; plot the first worksheet and drag the other
Y-data into the graph).
To determine the initial velocity (v0) for each substrate concentration, do a linear fit of
the first 5-10 data points, it should be a relatively straight line:
Select the first 10 data points. Press
to make a graph.
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. If the fitting fails, use the
horizontal asymptote of the plot as a approximation for B.
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.OPJ”
and “Rapid_kinetics_Mb.OPJ”
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)
 75 ms (HrP0075, first spectrum)
 15 s (HrPH15, last spectrum)
2) Make similar plot for Mb
 0 s (Mbresting)
 5 ms (Mb0005, first spectrum)
 500 ms (Mb05, 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/decrease in the first time interval (0 s –
75 ms, this is resting HrP to first rapid kinetics spectrum) and then increase/ decrease
in the second time interval (75 ms – 15 s)?
Which spectrum corresponds to which intermediate in Figure 1? *
For each wavelength (496, 543, 578, and 650 nm), 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.
The rate constant k = 1/ t1 (the value for t1 can be found in the result box in the graph
after fitting). A high rate constant indicates a fast reaction.
Insert the calculated rate constants in the table below.
Wavlelength
75ms – 15s
First Order Rate Constants for HrP
496 nm
543 nm
578 nm
650 nm
B)
Repeat the procedure above for Mb, however, the interesting and corresponding
wavelengths are now 503, 550, 588, and 640 nm. 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).
Wavlelength
5 – 500 ms
503 nm
First Order Rate Constants for Mb
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.
Compare HrP and Mb and consider:
1) How fast they react with H2O2.
2) The rate of product (DAP) formation.
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?