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)

2H2O2 2H2O +O2

Catalase

(organic)(Hydrogen peroxide)

Substrate

(Water & Oxygen)

**Oxygen will bubble in vitro**

Bubbles

Catalase

Catalase

H2O2

H2O2

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

Catalase

H2O2 O2 & H2O

Catalase

or

a) b)

•Poison What does cyanide do? Arsenic?

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

Catalase

H2O2 O2 & H2O

Catalase

or

a) b)

•Poison What does cyanide do? Arsenic?

Temperature

Denature: enzyme unravels/unfolds and loses structure and function

COLD HOT

Low activity

High activity

-cold temperatures inhibit (prevent) enzyme activity

-hot temperatures denature enzymes

Best temperature

Normal

enzyme

Extreme heat

Denatured

enzyme

p. 35?

pHAcidic –less than 7

Basic (alkaline) –greater than 7Neutral = 7

Enzymes have a certain pH at which they work best

-above or below optimum pH inhibits activity

pH = 0 pH = 14

p. 35?

Your Experiment

The reaction:

Catalase: enzyme (organic catalyst) that converts hydrogen peroxide into water and oxygen

2 H2O2Hydrogen peroxide water + oxygen

2 H2O + O2

What’s the substrate? Product?

Catalase

H2O2

catalase

H2O + O2(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)

• Repeat with Blood (animal)

• 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)– 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)

Water Level at:

T0 = Time Start

Tf = Time Finish

Pre-incubate

• 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) • Empty all water• Wipe up the counter and any spills• Check the center counter – Wipe up• Put equipment all on the side counter

Points can (and will be) deducted for messes left

Buret tube

Flask

Rubber stopper

Tubing

Ring Stand

Water trough

Experimental Setup-Set up apparatus as described on p. 36.-Place tissue in flask

-Add hydrogen peroxide-Swirl flask 30 seconds-Record exact water level after 30 seconds

-Record exact water level in buret tube

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?

-Test under other conditions

2 H2O2 2 H2O + O2

p. 36-37?

Activities:

2) Activity #2: Effect of Temperature

3) Activity #3: Effect of pH

1) Activity #1: Presence of catalase

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

At ~4:20, we’ll stop to discuss your observations & take roll

Plant tissue Animal tissue

For #2, #3, #4 use plant tissue only!!!

Ice Hot plate

HCl Buffer NaOH-Let sit 10 min with HCl or NaOH before running the experiment

-Remove once it bubbles or it will burn!-Keep on ice entire time!

-To keep the swirling consistent, designate one group member to swirl gently for all activities!

Critical Thinking

• Take a look at the board

– 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?

Hummm

What to do …. But think

Brain in a jar