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Enzyme

Kinetics
Learning Objectives for Theme 2 Topic B Energy & Metabolic Pathways

3. Compare and contrast irreversible and reversible inhibition, and


competitive and noncompetitive inhibition of enzymes.

All diagrams and photos without attribution are MS Word


clip art or the authors (RA Edwards) own work/photo.

Whats Going On Today


Review the function of enzymes.
Find out how to measure the rate of an enzyme catalyzed
reaction in a test tube.
Discover how the rate of a reaction depends on three
different things: enzyme concentration [E], substrate
concentration [S], and presence of an inhibitor.

Review Enzymes
Nearly every reaction that occurs in living organisms is catalyzed
by an enzyme.
Enzymes vastly increase the rate of reaction by decreasing G*
(E*, EA, G0,).
Enzymes are quite specific and catalyze only one reaction.
Review of Enzyme function
http://www.youtube.com/watch?v=E-_r3omrnxw&feature=related
Many bio-reactions look like coupled reactions.
Enzymes catalyze the two coupled reactions simultaneously.
Often one of the coupled reactions is ATP + H2O ADP + Pi
Therefore: Enzymes look like energy coupling devices.
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Example Reaction
CPRG(aq) + H2O(aq) CPR(aq) + galactose
Lactaid, is an enzyme supplement (beta-glactosidase)

(CPRG = Chlorophenolred-galactoside)

How to Measure How Fast Enzymes Work


1. Add enzyme to substrate in buffer.
2. When enzyme is added to substrate start a timer.
3. After a shot time (1 or 2 minutes) measure the colour change by
the absorbance of the product (CPR) at 572 nm.

Calculating Rate in umol/min


1. Use the absorbance to calculate the concentration of CPR.
2. Use the conc. and the volume to calculate the amount (umoles) of CPR.
3. Divide the number of umoles of product by the time (in minutes) to get
the rate in umoles per minute.

Example:
A572 = 0.44 umoles of Product after 2.0 minutes.
Velocity = 0.44 umoles / 2.0 minutes = 0.22 umol/min.

Finding out how Rate Depends on [E]


1. Use the same procedure as already described (add substrate, add
enzyme, time to 1 minute and measure A572), but use several tubes.
2. Use _________________________
enzyme in the different tube.
3. Repeat to get duplicates and then repeat to get triplicate.
4. Repeat previous calculation procedure & average triplicate rates.

All of these
tubes have have
the same [S] but
they have
different [E].
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Plot Rate versus [E]

Finding out how Rate Depends on [S]


1. Use previous procedure, but add same amount of enzyme to
several tubes which have different concentrations of substrate.
2. Do step #1 in triplicate.
3. Repeat previous calculation procedure & average triplicate rates.

All of these
tubes have have
the same [E] but
they have
different [S].

Substrate

Plot Rate versus [S]


Vmax 15 umol/min

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Km is the [S] needed to get Vmax

Vmax 7.5 umol/min

Km 4.0 uM

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How does Rate Depend on Inhibitor


1. Use previous procedure, but add same amount of enzyme to same
concentration of substrate to two tubes one has an inhibitor the
other does not have the inhibitor.
2. Do step #1 in triplicate on several substrate concentrations (with or
without the same inhibitor concentration in pairs of tubes).
3. Repeat previous calculation procedure & average triplicate rates.

These two tube


all have the
same [E] and the
same [S].

No Inhibitor

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Plot Rate with & without Competitive Inhibitor


Vmax 15 umol/min is the same
with and without inhibitor

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Competitive Inhibitor
Inhibitor looks chemically somewhat like the substrate.
Inhibitor binds at the active site in competition with the substrate.
High concentrations of the substrate will out-compete the inhibitor.

Active
Site

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Noncompetitive Inhibition
Inhibitor is not chemically similar to the substrate.
Inhibitor binds at some other site on the enzyme (not the active site).
High concentrations of the substrate do not out-compete the inhibitor.
Active
Site

Inhibitor
Site

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Reversible Inhibition
Competitive Inhibition is one type of Reversible Inhibition because the
inhibitor can be released from the enzyme.
o High [S] will knock the inhibitor off of the enzyme Vmax is unchanged
Non-Competitive Inhibition is another type of Reversible Inhibition.
o High [S] will not knock the inhibitor from the E Vmax is decreased.

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Irreversible Inhibition
Inhibitor reacts with the enzyme so that the enzyme doesnt catalyze
properly.
The substrate can NOT out-compete so that Vmax is decreased.

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5.1 What is a common way to obtain the data needed to calculate the
velocity of an enzyme catalyzed reaction?
A. Measure the heat released by the reaction.
B. Measure the equilibrium constant after the reaction has stopped.
C. Measure the decrease in the substrate concentration.
D. Measure the absorbance of the products.

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5.2 How does the velocity (v) of an enzyme catalyzed reaction depend on [E]
and [S]?
A. v increases linearly with both increasing [E] and increasing [S].
B. v does not increase linearly with either [E] or [S].
C. v increases linearly with increasing [E], but reaches a plateau with
increasing [S].
D. v increases reaches a plateau with increasing [E], increases linearly with
increasing [S].

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