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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
Enzyme Review
Enzymes are proteins that catalyze (facilitate) reactions.
Catalase and α-Amylase
CatalaseHydrogen
peroxide
H2O (water)
and
O2 (oxygen)
A demonstration
Carrot and Hydrogen Peroxide
• Substrates or reactants bind to the enzyme’s active site.
Hydrogen peroxideWater and Oxygen
Catalase (in carrot)
Why no bubbles before the carrot?
What does catalase NOT change?
What DOES catalase change?
Enzymes bind reactants
and stress reactants to
catalyze reactions.
This reduces the
activation energy.
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
What does this graph tell us about human and thermophilic
bacteria?
• 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?
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
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
Tyrosinase is an enzyme that catalyzes
the biosynthesis of the pigment
melanin from the amino acid tyrosine.
• Skin coloration
• Browning of fruits
Copper ionsOxygen
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).
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…..
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??
Spectrophotometer
Cuvettes (containing the solution) are inserted into the
spectrophotometer.
•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
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?
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?
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.
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?
4.5 Exploring Substrate Concentration
You will complete a serial dilution to prepare samples from a
stock solution of DOPA.
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.
How will the rate of the reaction change with increasing
substrate concentration?
Why?
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?
How does enzyme concentration affect the reaction?