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PBHS AP Biology
STEVENSON
2009-10
Lab #2: Enzyme Catalysis
 Introduction: This the first of 12 AP Labs designed to
illustrate the themes of this class. These labs are very
important as the AP Test will have least one essay question
and several multiple choice questions based on these labs.
Lab #2: Enzyme Catalysis
 Introduction
 Enzymes are proteins produced by living cells that act as catalysts in
biochemical reactions
 Substance to be acted on is called the substrate (S)
 The substrate binds reversibly to the active site of the enzyme (E)
 Reduces the energy required to activate the reaction so that products
(P) can be formed
 E + S  ES  E + P
Lab #2: Enzyme Catalysis
 Introduction
 The enzyme is not changed in any way and so can be recycled to
break additional substrate molecules
 The active site is the portion of the enzyme that reacts with the
substrate
 Any substance that blocks or changes the active site can affect the
activity of the enzyme
Lab #2: Enzyme Catalysis
 Salt concentration
 If the salt concentration is very low, the enzyme will denature and
form an inactive precipitate
 If the salt concentration is very high, new interactions will occur and
again an inactive precipitate is formed
 Intermediate salt concentrations such as human blood (0.9%) is the
optimum for many enzymes
Lab #2: Enzyme Catalysis
 pH
 As the pH is lowered (solution becomes acidic), the side chains will
attract H+ ions and the enzymes shape is disrupted
 As the pH goes up, the enzyme will lose H+ ions and again, the
shaped is altered
 Optimum pH is in the neutral range
 At very low or high pH, the enzyme denatures (breaks down)
Lab #2: Enzyme Catalysis
 Temperature
 Increasing temperature cause enzyme reactions to go faster… up to a
point
 At very high temps, the enzymes structure is broken down
 Many enzymes function well up to 40-50 C, and some are active up
to 70-80 C
Lab #2: Enzyme Catalysis
 Activators and Inhibitors
 Many molecules other than the substrate may interact with an
enzyme
 If such a molecule increases the rate of reaction, it is called an
activator; if it decreases the rate of reaction, it is an inhibitor
 Many well know poisons such as potassium cyanide and curare are
inhibitors that interfere with the active sites of critical enzymes
http://en.wikipedia.org/wiki/Catalase
 Catalase was first noticed as a substance in 1811 when Louis Jacques
Thénard, who discovered H2O2 (hydrogen peroxide), suggested that its
breakdown is caused by a substance. In 1900, Oscar Loew was the first
to give it the name catalase, and found its presence in many plants and
animals. In 1937 catalase from beef liver was crystallised by James B.
Sumner and the molecular weight worked out in 1938.
 In 1969 the amino acid sequence of bovine catalase was worked out.
Then in 1981, the 3D structure of the protein was revealed.
 Hydrogen peroxide is a harmful by-product of many normal metabolic
processes: To prevent damage, it must be quickly converted into other,
less dangerous substances. To this end, catalase is frequently used by
cells to rapidly catalyze the decomposition of hydrogen peroxide into
less reactive gaseous oxygen and water molecules
Lab #2: Enzyme Catalysis
 Lab #2
 The enzyme used in this lab is Catalase
 Catalase has 4 polypeptide chains, each composed of more than
500 amino acids
 One function of Catalase is to facilitate the breakdown of Hydrogen
Peroxide (H2O2) to H2O and O2



2 H2O2 → 2 H2O + O2
In the absence of catalase this reaction occurs spontaneously but
proceeds very slowly
The rate of the reaction can be measured in one of three ways
Measuring the rate of disappearance of the substrate (H2O2)
 Measuring the rate of appearance of a product (O2 gas)
 Measuring the heat released or absorbed)

Lab #2: Enzyme Catalysis
 Lab #2: General Procedure
 Read p 22
 Exercise 2A: Test of Catalase Acvitity
 Exercise 2B: The Base Line Assay
 Exercise 2C: The Uncatalyzed Rate of H2O2 Decomposition
 Exercise 2D: An Enzyme-Catalyzed Rate of H2O2 Decomposition