exercise 4: enzyme activity and environmental effects enzyme activity and...exercise 4: enzyme...
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Exercise 4: Enzyme Activity and
Environmental Effects
Learning Objectives
• Explain the function of a spectrophotometer
• Use a spectrophotometer to measure enzyme activity
• Determine the relationship between substrate concentration and the rate of the enzymatic reaction
• Determine the relationship between other environmental variables (temperature) and the rate of the enzymatic reaction
• Design and conduct an experiment to explore the effect of another variable on the rate of the enzymatic reaction
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Enzyme Review
Enzymes are proteins that catalyze (facilitate) reactions.
Catalase and α-Amylase
CatalaseHydrogen
peroxide
H2O (water)
and
O2 (oxygen)
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A demonstration
Carrot and Hydrogen Peroxide
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• Substrates or reactants bind to the enzyme’s active site.
Hydrogen peroxideWater and Oxygen
Catalase (in carrot)
Why no bubbles before the carrot?
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What does catalase NOT change?
What DOES catalase change?
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Enzymes bind reactants
and stress reactants to
catalyze reactions.
This reduces the
activation energy.
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What does this graph tell us about pepsin and trypsin?
Figure 8.18
Ra
te o
f re
ac
tio
n
(b) Optimal pH for two enzymes
Optimal pH for pepsin
(stomach enzyme)Optimal pH
for trypsin(intestinalenzyme)
10 2 3 4 5 6 7 8 9
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What does this graph tell us about human and thermophilic
bacteria?
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• Some enzymes require cofactors to function optimally. These are
either metal ions or small organic molecules called coenzymes.
• So enzymes are regulated by molecules that are not part of the
enzyme itself.
Calcium
α -Amylase
Metal ion cofactors: Na+, K+, Mg++,
Zn++, Cu++, Fe++, Fe+++
CHELATORS: bind metal ions
What affect would a chelator
have on amylase?
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Substrate Concentration [S]
• The rate of product formation during an enzyme-
catalyzed reaction increases linearly at low [S].
• The reaction rate reaches maximum speed at high [S]
called Vmax.
Vmax = active sites on enzyme are all occupied!
Vmax
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Exercise Four- Enzyme Activity
Monitor the activity of Tyrosinase (an enzyme)
1. Substrate concentration
2. Temperature
And then either:
Enzyme concentration
Chelator (EDTA) or
Sodium chloride or
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Tyrosinase is an enzyme that catalyzes
the biosynthesis of the pigment
melanin from the amino acid tyrosine.
• Skin coloration
• Browning of fruits
Copper ionsOxygen
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In the presence of oxygen (O2), tyrosinase (E) catalyzes the
hydroxylation of tyrosine into 3,4-dihydroxyphenylalanine
(DOPA).
Tyrosinase then catalyzes DOPA into dopaquinone (which
spontaneously converts into dopachrome).
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You can monitor the activity of tyrosinase
by looking at the color of the solution.
Over time, what do you think will happen to the color of a
DOPA solution after the addition of tyrosinase?
a. Solution begins clear and then turns brown
b. Solution begins brown and turns clear
Dopachrome is a orange/brown
colored compound…..
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As the reaction proceeds, more substrate is converted to product
(dopachrome) by tyrosinase and the solution will become darker.
Time
How do you measure the change in color??
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Spectrophotometer
Cuvettes (containing the solution) are inserted into the
spectrophotometer.
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•You figure out which wavelength of light is absorbed by your
product
•Select wavelength and shine it through sample.
•Start your reaction
•Measure how absorbance changes over time
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You’ll be given the following to explore tyrosinase activity:
1) DOPA
2) A solution containing potato
3) SpectroVis
Where do you find the enzyme?
Where do you find the substrate?
How will absorbance values change over time?
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4.1 and 4.2: Turn on Spec and Calibrate
Turn LoggerPro on.
Page 64: Prepare a blank to calibrate or ‘tare’ the spectrophotometer:
Blank has everything EXCEPT reactant (no DOPA).
Buffer
Enzyme
dH2O (in place of DOPA)
QUESTIONS:
1) Why do you need to blank the spectrophotometer?
2) Why dH2O?
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4.3 Absorbance Spectrum
You have to figure out which wavelength to use to monitor
the reaction.
So you need the absorbance spectrum for dopachrome (the
product).
What is on the X axis? Y axis?
Explain why MAX around 470
nm and not 700 nm.
Select λ MAX and use it to
monitor change in absorbance
over time.
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4.4 Tyrosinase activity monitored over
time
What is on the X axis? What is on the Y axis?
How is this different from the absorbance spectrum?
Why is absorbance
increasing over time?
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4.5 Exploring Substrate Concentration
You will complete a serial dilution to prepare samples from a
stock solution of DOPA.
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Exploring Substrate Concentration
Prepare cuvettes with a range of [DOPA].
After adding enzyme, you will measure change in color over
time (Absorbance vs. Time) and determine the rate of the
reaction for EACH concentration.
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How will the rate of the reaction change with increasing
substrate concentration?
Why?
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You will design and conduct an experiment to
determine the effect of the following on
tyrosinase:
Enzyme concentration
EDTA (chelator)
Sodium chloride
What do you expect the effect increasing enzyme
concentration will be on absorbance values? EDTA?
Sodium chloride?
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How does enzyme concentration affect the reaction?