Download 1 ENZYMES Introduction Enzyme Kinetics

ENZYMES
Introduction
Enzymes are biological catalysts that carry out thousands of chemical reactions in living cells. They are
generally large proteins made up of several hundred amino acids. Enzymes often contain a nonproteinaceous group called the "prosthetic group" that is important in the reaction. Prosthetic groups
include substances such inorganic cofactors (calcium, copper, magnesium, zinc, and manganese ions) and
organic compounds (called co-enzymes) such as some B vitamins (niacin, flavin).
In an enzyme-catalyzed reaction, the substance acted upon, or substrate, binds to a specialized region of
the enzyme called the active site. The enzyme and substrate are held together in an "enzyme-substrate
complex" by hydrophobic bonds, hydrogen bonds, and ionic bonds. The enzyme then converts the
substrate to the reaction products in a process that often requires several chemical steps, and may involve
forming or breaking covalent bonds. Finally, the products are released and the enzyme is ready to form
another enzyme-substrate complex. The enzyme is not consumed as it carries out the reaction but is
recycled again and again. One enzyme molecule can carry out thousands of reaction cycles every minute.
The general "equation" summarizing enzyme action is:
Enzyme + Substrate - Enzyme-Substrate complex  Product + Enzyme
……or, simply E + S  ES  P + E
The rate of reaction will depend upon the concentration of enzyme, the concentration of the substrate, and
"environmental" factors such as the presence of inhibitors, the pH of the medium, and temperature.
Enzyme Kinetics
If we start with a fixed concentration of enzyme and measure the rate of enzyme activity with different
substrate concentrations [S], enzymes typically show what is called “first order kinetics.” At first there is
a linear relationship between the rate (velocity, V) of the reaction and the amount of substrate present
(point A below), but with higher levels of substrate, the reaction rate levels off, reaching a maximal
velocity, or Vmax ( C ). At Vmax, the enzyme is working as fast as it can and adding more substrate will
not matter. The substrate concentration that produces a rate that is 1/2 of Vmax (B) is called the Km
(Michaelis-Menton constant) and is characteristic of the enzyme and the substrate. Km is a measure of
the affinity of the enzyme for the substrate – the smaller the Km, the greater the affinity.
Km
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Each enzyme is specific for a certain reaction because its amino acid sequence is unique and causes it to
have a unique three-dimensional structure. The "business" end of the enzyme molecule, the active site,
also has a specific shape so that only one or a few of the thousands of compounds present in the cell can
interact with it. If there is a prosthetic group on the enzyme, it will form part of the active site. Any
substance that blocks or changes the shape of the active site will interfere with the activity and efficiency
of the enzyme.
Objectives
In this exercise you will study the enzyme catalase, which accelerates (catalyzes) the breakdown of
hydrogen peroxide (a common end product of oxidative metabolism) into water and oxygen, according to
the summary reaction:
2H2 O2 + catalase  2H2O + O2 + catalase
This catalase-mediated reaction is extremely important in the cell because it prevents the accumulation of
hydrogen peroxide, a strong oxidizing agent that tends to disrupt the delicate balance of cell chemistry.
Catalase is found in animal and plant tissues. You will measure its rate of activity under different
conditions. A paper disc will be immersed in the enzyme solution, and then placed in the hydrogen
peroxide substrate. The oxygen produced will become trapped in the disc and will give it buoyancy. The
time measured from the moment the disc enters the substrate to the time it reaches the surface of the
solution is a measure of the rate of the enzyme activity.
Keep the catalase in the ice bath throughout the experiment.
General Procedure: Use a hole-punch to generate a number of small discs from the filter paper and
place them in a plastic petri dish. Using forceps, immerse a filter disc "punch" into the catalase solution.
Allow the disc to absorb the enzyme solution for a few seconds, remove excess enzyme solution by
touching the edge to a paper towel for a few seconds. Drop the disc into the first substrate solution (use
forceps to place the disc in the fluid). The disc will sink rapidly into the solution. The oxygen produced
from the breakdown of the hydrogen peroxide by catalase becomes trapped in the fibers of the disc
causing the disc to float to the surface of the solution.
Rate of Enzyme Reaction = 1 / disc rising time. The time (t) in seconds, from the instant the disc enters
into the solution to the time it floats back to the surface is inversely proportional to enzyme activity: So,
the longer it takes to rise, the slower the reaction rate. Repeat the procedure twice for each test and
average the results. Record your results in the tables provided and then plot your data.
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Test 1: Effect of Substrate Concentration
To determine the effect of substrate concentration on enzyme activity obtain six 50-ml plastic
beakers and label them as follows: 0%, 0.5%, 1.0 %, 1.5% 2.0% and 3% H2O2 (Note: They may
already be labeled).
Add 30 mls of the proper (as outlined above) H2O2 solution to each beaker. Using the filter disc
procedure described above, determine the rate of the reaction at the various substrate
concentrations. Record your results in the Table below. Repeat the procedure twice for each
substrate concentration and average the results.
Substrate
% H2O2
0
Time 1
(Seconds)
Time 2
(Seconds)
Time 3
(Seconds)
Average
Time
Rate
(1/Ave Time)
0.5
1.0
1.5
2.0
3.0
(Note: if the disc doesn’t rise within a few minutes, consider that it takes an infinite amount of
time. 1/infinity is “0” for our purposes)
Plot your results (Make sure you label the y-axis and estimate the Km and Vmax).
0
0.5
1.0
1.5
Substrate Concentration (% H2O2)
2.0
3.0
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Test 2: Effect of Enzyme Inhibitor
Hydroxylamine attaches to the iron atom of catalase (iron is a cofactor that is part of the active
site) and thereby interferes with the formation of enzyme-substrate complex. Add 1 drop (50
microliters) of 0.1% hydroxylamine into each beaker of hydrogen peroxide substrate. Stir or
swirl the solution to make sure the hydroxylamine is well diffused, and then measure the activity
as above and record your data below.
Plot your data on the graph on the previous page. Indicate the inhibited rates with a different
style point. For example, if you used filled circles ● to indicate the normal results, use a box
❑ or asterix * to note the inhibited rates.
Rates with Inhibitor
Substrate
% H2O2
0
Time 1
Time 2
Time 3
Average
Time
Rate
(1/Ave Time)
0.5
1.0
1.5
2.0
3.0
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Lab Report Questions
NAME: _______________________
Lab: Mon 4:00, 7:00, Tues. 4:00, 7:00
What are the key features of an enzyme?
Define active site.
What are the two working models of enzyme action? Which one is believed to be more
accurate?
How is the rate of enzyme activity affected by increasing the concentration of the substrate? Was
there any evidence of enzyme saturation? What do you think would happen if you tested a
substrate concentration of 10%?
What was the effect of the hydroxylamine on the enzyme reaction rates? Did higher amounts of
substrate overcome the inhibition?
What are some of the uncontrolled variables in this experiment and how might they have
affected your results?
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Data Sheet
Effect of Substrate Concentration on Reaction Rates
Substrate
Time 1
Time 2
Time 3
% H2O2
0
Average
Time
Rate
(1/Ave Time)
0.5
1.0
1.5
2.0
3.0
Rates With Inhibitor
Substrate
Time 1
% H2O2
0
Time 2
Time 3
Average
Time
Rate
(1/Ave Time)
0.5
1.0
1.5
2.0
3.0
Plot your data below. (Make sure you label the y axis and indicate the “Km” and Vmax).
0
0.5
1.0
1.5
2.0
Substrate Concentration (% H2O)
3.0
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