Biology 120
Enzyme Function
Exercise 4
www.celltech.com/resources/vt/enzymes.html
What you need to Know
• Understand the experiment being preformed today
• What is: Enzyme / Catalyst / Active Site / Substrate
• Answer all questions in the lab handout
• Understand the effect of pH, temp, and concentration
has on an enzyme
• Understand the scientific processes that took place
Terminology
• Metabolism
– Definition: The sum total of all biochemical activity that
takes place in a living organism
• Catabolic Metabolism – break down
– AB = A & B
• Anabolic Metabolism – build up
– A + B = AB
• In vitro – out of the body (or in a tube)
• Substrate - The material being acted upon
Introduction
• Catalyst – speeds up the rate of a reaction
• Enzyme – a protein that is a organic catalyst
– Hint: Most of the time ends in –ase
• Active Site – a pocket at the outside of the enzyme
specific to a substrate (here is where it is taken apart or put together)
Bubbles
H2O2
Catalase
2H2O2
Catalase
H2O2
Catalase
(organic)
2H2O +O2
(Hydrogen peroxide)
(Water & Oxygen)
Substrate
**Oxygen will bubble in vitro**
How Enzymes Work
• Biological Catalysis (usually proteins) – Make a reaction faster
• Enzymes are Proteins – built to work “active site”
• Proteins are Happy’ist in their natural Temp and pH
–
–
–
–
Usually ~370C or 980F & Usually a neutral pH (Function)
What would happen if you boil it? Guesses -___________
What would happen if you put it in acid? ________________
What would happen in the cold?________________
• Enzymes can only work so fast……..
– If you keep adding substrate will the
speed of the Rxn
a)
H2O2
•Poison
What does cyanide do?
Arsenic?
O2 & H2O
b)
or
Catalase
Catalase
How Enzymes Work
• Biological Catalysis (usually proteins) – Make a reaction faster
• Enzymes are Proteins – built to work “active site”
• Proteins are Happy’ist in their natural Temp and pH
–
–
–
–
Usually ~370C or 980F & Usually a neutral pH (Function)
What would happen if you boil it? Guesses -____denature________
What would happen if you put it in acid? _______denature_________
What would happen in the cold?________inhibit________
• Enzymes can only work so fast……..
– If you keep adding substrate will the
speed of the Rxn
a)
H2O2
•Poison
What does cyanide do?
Arsenic?
O2 & H2O
b)
or
Catalase
Catalase
p. 35?
Temperature
-cold temperatures inhibit (prevent) enzyme activity
-hot temperatures denature enzymes
High activity
Denature: enzyme
unravels/unfolds and
loses structure and
function
Extreme
heat
Low activity
COLD
HOT
Best
temperature
Normal
enzyme
Denature
d enzyme
p. 35?
pH = 0
Acidic –less
than 7
pH
Neutral = 7
Basic (alkaline) –
greater than 7
pH = 14
Enzymes have a
certain pH at
which they work
best
-above or below
optimum pH
inhibits activity
Your Experiment
The reaction:
2 H2 O 2
Catalase
2 H2O + O2
Hydrogen peroxide
water + oxygen
What’s the substrate? Product?
Catalase: enzyme (organic catalyst) that converts
hydrogen peroxide into water and oxygen
H2O2
H2O + O2
catalase
(gas)
The Experiment
• Close the buret valve & fill with water (bucket also)
• Invert the buret and clamp
(make sure the water level is blow the 50 ml mark)
• Add 3 Grams of plant tissue and stopper the flask
***Put the tubing into the buret***
• Put the rubber stopper on the flask an put the hose
into the buret (careful not to lose the water)
• Add 3 ml 2H2O2 to the flask (do NOT remove pipette)
Record water level and Time!!
• Swirl the flask for 30 sec. Record the Level (Table 1)
• To Remove – take out the hose and then uncork
• Add Tap Water to the plant tissue (Clean Flask)
Water Level at:
T0 = Time Start
• Repeat with Blood (animal)
Tf = Time Finish
• Test the Effects of Temperature (Hot and Cold)
– Do Not Leave The Flask Unattended when on the hot plate.***
– Follow the instructions – pg 43 & Record on Table 2
• The Effects of pH (3 different flasks)
Pre-incubate
– Follow instructions on pg 47&48 – Record on Table 3
– You MUST ware safety equipment when near the HCL & NaOH
• The Effects of [Concentration]
– Read the instructions Carefully
– Be as accurate as possible
– No exact time limit (until oxygen is no longer produced)
• When ever you remove the Stopper……
– Where should the tube be? What would happen?
• Calculate the amount of oxygen produced
– Oxygen will displace the water (water level will go )
– Simple subtraction (bigger # from the smaller #)
• Put answers on the board
•
•
•
•
•
THE CLEAN UP!!!
Clean all Flasks and other glass stuff completely
(Tap water)
Points can (and will be)
Empty all water
deducted for messes left
Wipe up the counter and any spills
Check the center counter – Wipe up
Put equipment all on the side counter
Experimental Setup
p. 36-37?
2 H 2 O2
2 H2 O + O2
Buret tube
Tubing
Water trough
-Set up apparatus as
described on p. 36.
-Place tissue in flask
-Record exact water level in
buret tube
-Add hydrogen peroxide
-Swirl flask 30 seconds
-Record exact water level
after 30 seconds
-Test under other conditions
Rubber stopper
Flask
Ring Stand
Why does the water level change? What specific molecule
caused it to change? Is this a substrate or product?
What was in the tissue that caused the reaction to occur?
-To keep the swirling consistent,
designate one group member to
swirl gently for all activities!
Activities:
Plant tissue
Animal tissue
1) Activity #1: Presence
of catalase
For #2, #3, #4 use plant tissue only!!!
2) Activity #2: Effect of Temperature
-Keep on ice entire time!
Ice
At ~4:20,
we’ll stop to
discuss your
observations
& take roll
-Remove once
it bubbles or it
will burn!
Hot plate
3) Activity #3: Effect of pH
-Let sit 10 min with HCl or NaOH
before running the experiment HCl
Buffer
NaOH
4) Activity #4: Effect of Enzyme concentration
and Substrate concentration
Once you’ve completed the tables, answer
questions on p. 42 and understand the objectives in
the lab on p. 35
Critical Thinking
Hummm
What to do
…. But think
• Take a look at the board
Brain in a jar
– Do all the results match or show a trend?
– Are there a few results that don’t make sense
(why do you think this happened?)
– What do the results lead you to conclude?
– Why is it good to replicate experiments?