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AP Biology Lab #2

Enzyme Catalysis
Overview: In this lab you will observe the conversion of hydrogen peroxide (H2O2) to water and oxygen gas by the
enzyme catalase and then measure the amount of oxygen generated! "ou will also calculate the rate of an enzyme#
catalyzed reaction!
Ob$ectives:
%efore doing this lab please review the following from your text and&or notes:
'tructure function of enzymes
How temperature pH enzyme concentration and substrate concentration affect the initial
reaction rates of enzyme#catalyzed reactions
(eview the terms:
Catalyst
Catalase
Catalysis
)fter doing the lab you should be able to:
*easure the effects of changes in temperature pH enzyme concentration and substrate
concentration on reaction rates of an enzyme#catalyzed reaction in a controlled experiment!
+xplain how environmental factors affect the rate of enzyme#catalyzed reactions!
Introduction:
In general enzymes are proteins produced by living cells which act as catalysts in biochemical reactions! ) catalyst
affects the rate of a chemical reaction! One conse,uence of enzyme activity is that cells can carry out complex
chemical activities at relatively low temperatures!
In an enzyme#catalyzed reaction the substance to be acted upon the substrate binds reversibly to the active site of the
enzyme! One result of this temporary bond is a reduction in the energy re,uired to activate the reaction of the substrate
molecule so that the products of the reaction are formed more easily!
-ote that the enzyme are not changed in thee reactions and can be recycled to brea. down additional substrate
molecules! +ach enzyme is specific for a particular reaction because its amino acid se,uence is uni,ue and causes it to
have a uni,ue three#dimensional structure! /he active site is the portion of the enzyme that interacts with the substrate
so that any substance that bloc.s or changes the shape of the active site affects the activity of the enzyme! )
description of several ways enzyme action may be affected follows:
0! 'alt 1oncentration! If the salt concentration is close to zero the charged amino acid side chains of the
enzyme molecules will attract each other! /he enzyme will denature and form an inactive precipitate! If on the
other hand the salt concentration is very high normal interaction of charged groups will be bloc.ed new
interactions will occur and again the enzyme will precipitate! )n intermediate salt concentration such as that of
human blood (2!34) or cytoplasm is the optimum for many enzymes!
2! pH! pH is the logarithmic scale that measures the activity or H
5
concentration in a solution! "ou are
familiar with the scale! )mino acid side chains contain groups such as 61OOH and 6-H2 that readily gain or
lose H
5
ions! )s the pH is lowered an enzyme will tend to gain H
5
ions and eventually enough side chains will
be affected so that the enzyme7s shape is disrupted! 8i.ewise as the pH is raised the enzyme will lose H
5
ions
and eventually lose its active shape! *any enzymes perform optimumly in the neutral pH range and are
denatured at either an extremely high or low pH! 'ome enzymes such as pepsin which acts in the human
stomach where pH is very low have a low pH optimum!
9! /emperature! :enerally chemical reactions speed up as the temperature is raised! )s the temperature
increases more of the reacting molecules have enough .inetic energy to undergo the reaction! 'ince enzymes are
catalysts for chemical reactions enzyme reactions also tend to go faster with increasing temperature! However if
the temperature of an enzyme#catalyzed reaction is raised very high a temperature optimum is reached; above
this value the .inetic energy of the enzyme and water molecules is so great that the structure of the enzyme
molecules is disrupted! /he positive effect of speeding up the reaction is now more than offset by the negative
effect of changing the structure of more and more enzyme molecules! *any proteins are denatured at
temperatures around <2#=2
o
1 but some are still active at >2#?2
o
1 and a few may even withstand boiling!
<! )ctivators and inhibitors! *any molecules other than the stubstrate may interact with enzymes! If such a
molecule increases the rate of the reaction it is an activator and if it decreases the reaction rate it is an inhibitor!
/hese molecules can regulate how fast the enzyme acts! )ny substance that tends to unfold the enzyme such as
an organic solvent of detergent will act as an inhibitor! 'ome inhibitors act by reducing the disulfide ('#')
bridges that stabilize the enzyme7s structure! *any inhibitors act by reacting with side chains in or near the
active site to change its shape! *any well#.nown poisons such as potassium cyanide and curare are enzyme
inhibitors that interfere with the active site of critical enzymes!
/he enzyme used in this lab catalase has four polypeptide chains each composed of more that =22 amino acids! /his
enzyme is ubi,uitous (present everywhere) in aerobic organisms! One function of catalase within cells is to prevent the
accumulation of toxic levels of hydrogen peroxide (H2O2) which is formed as a byproduct of metabolic processes!
1atalase also ta.es part in the oxidation reactions that occur in cells!
/he primary reaction catalyzed by catalase is the decomposition of H2O2 to form water and oxygen:
2 H2O 2 H2O + O2 (gas)
In the absence of catalase this reaction occurs spontaneously but very slowly! 1atalase speeds up the reaction! In this
experiment a rate for this reaction will be determined!
*uch can be learned about enzymes by studying the .inetics (particularly the changes in rate) of enzyme#catalyzed
reactions! @or example it is possible to measure the amount of product formed or the amount of substrate used from
the moment the reactants are brought together until the reaction has stopped!
If the amount of product formed is measured at regular intervals and this ,uantity is plotted on a graph a curve li.e the
one in @igure 2!0 is obtained!

'tudy the solid line on the graph of this reaction!
)t time 2 there is no product! )fter 92 seconds = micromoles (moles) have been formed; after one minute 02
moles; after two minutes 22 moles! /he rate of this reaction could be given as 02 moles of product formed per
minute for this initial period! -ote however that by the third and fourth minutes only about = additional moles of
product have been formed! Auring the first three minutes the rate is constant! @rom the third minute through the
eighth minute the rate is changing; it is slowing down! @or each successive minute after the first three minutes the
amount of product formed in that interval is less than in the preceding minute! @rom the seventh minute onward the
reaction rate is very slow!
In comparing the .inetics of one reaction with another a common reference point is needed! @or example suppose you
wanted to compare the effectiveness of catalase obtained from potato with that obtained from liver! It is best to
compare the reactions when the rates are constant! In the first few minutes of an enzymatic reaction such as this the
number of substrate molecules is usually so large compared with the number of enzyme molecules that changing the
substrate concentration does not (for a short while at least) affect the number of successful collisions between substrate
and enzyme! Auring this early period the enzyme is acting on substrate molecules at a nearly constant rate! /he slope
of the graph line during this early period is called the initial rate of the reaction! /he initial rate of any enzyme#
catalyzed reaction is determined by the characteristics of the enzyme molecule! It is always the same for any enzyme
and its substrate at a given temperature and pH! /he initial rate assumes that the substrate is present in excess!
The rate of reaction is the slope of the linear portion of the curve! /o determine a rate of reaction pic. any two points
on the straight#line portion of the curve! Aivide the difference in the amount of product formed between theses two
points by the difference in time between them! /he rate is then:
moles # moles
t2 6 t0
or from the graph
y
x
In the illustration in @igure 2!0 the rate between two and three minutes is calculated:
92 6 22 B 02 B 2!0> moles&sec
0?2 6 022 C2
/he rate of a chemical reaction may be studied in a number of ways including the following:
0! *easuring the rate of disappearance of substrate (in this example H2O2)
2! *easuring the rate of appearance of product (in this case O2 which is given off as a gas)!
9! *easuring the heat released (or absorbed) during the reaction!
General Procedre!
In this experiment the disappearance of the substrate is measured as follows (see @ig! 2!2)
0! ) purified catalase extract is mixed with substrate (HsO2) in a bea.er! /he enzyme catalyzes the conversion of
H2O2 to H2O and O2 (gas)!
2! %efore all of the H2O2 is converted to H2O and O2 the reaction is stopped by adding sulfuric acid (H2'O<)! /he
H2'O< lowers the pH denatures the enzyme and thereby stops the enzyme7s catalytic activity!
9! )fter the reaction is stopped the amount of substrate (H2O2) remaining in the bea.er is measured! /o assay this
,uantity potassium permanganate is used! Dotassium permanganate (E*nO<) in the presence of H2O2 and H2'O<
reacts as follows:
" H2O2 + 2 #$nO% + & H2'O% #2'O% + 2 $n'O% + ( H2O + " O2
-ote that H2O2 is a reactant for this reaction! Once all of the H2O2 has reacted any more E*nO< added will be in
excess and will not be decomposed! /he addition of excess E*nO< causes the solution to have a permanent pin. or
brown color! /herefore the amount of H2O2 remaining is determined by adding E*nO< until the whole mixture stays a
faint pin. or brown permanently! )dd no more E*nO< after this point! /he amount of E*nO< added is a
proportional measure of the amoung of H2O2 remaining (2 molecules of KMnO4 reacts with 5 molecules of H2O2 as
shown in the equation)
E)ercise 2A! *est o+ Catalase Acti,ity
0! /o observe the reaction to be studied transfer 02 m8 of 0!=4 (2!<< *) H2O2 into a =2 m8 bea.er and add 0 m8
of freshly made catalase solution! /he bubbles coming from the reaction mixture are O2 which results from the
brea.down of H2O2 by catalase! %e sure to .eep the freshly made catalase solution on ice at all times!
a !hat is the en"yme in this reaction#
$ !hat is the su$strate in this reaction#
c !hat is the pro%uct in this reaction#
% Can you fi&ure out a way to %emonstrate that the &as evolve% is really O2#
e !hy was there a %ifference $etween the reaction usin& fresh catalase an% $oile% catalase#
2! /o demonstrate the effect of boiling on enzymatic activity transfer = m8 of purified catalase extract to a test tube
and place it in a boiling water bath for = minutes! /ransfer 02 m8 of 0!=4 H2O2 into a =2 m8 bea.er and add 0
m8 of the cooled boiled catalase solution! How does the reaction compare to the one using the unboiled
catalaseF +xplain the reason for the difference!


9! /o demonstrate the presence of catalase in living tissue cut 0 cm
9
of potato macerate it in a mortar&pestle and
transfer it into a =2 m8 bea.er containing 02 m8 of 0!=4 H2O2! Ghat do you observeF Ghat do you thin.
would happen if the potato or liver was boiled before being added to the H2O2F


E)ercise 2C! *-e .ncatalyzed /ate o+ H2O2 0ecom1osition
/o determine the rate of spontaneous conversion of H2O2 to H2O and O2 in an uncatalyzed reaction put a small ,uantity
of 0!=4 H2O2 (about 0= m8) into a bea.er and set it out uncovered at room temperature for 2< hours! /itrate it with
+xercise 2% to determine the amount of H2O2 remaining! (@or ease of calculation assume that 0 m8 of E*nO< used in
the titration represents the presence of 0 m8 of H2O2 in the solution!) (ecord your results in the table below!
Hncatalyzed H2O2 Aecomposition
@inal reading of burette m8
Initial reading of burette m8
)mount of E*nO< titrant m8
)mount of H2O2 spontaneously decomposed
(m8 baseline # m8 E*nO<) m8
Ghat percent of the H2O2 spontaneously decomposes in 2< hoursF
I3m8 baseline 6 m8 2< hours)&m8 baselineJ x 022 4
E)ercise 2B! *-e Base Line Assay
E)ercise 20! An Enzyme2catalyzed /ate o+ H2O2 0ecom1osition
%ac.ground: Having done some initial investigations on the action of catalase you now need to run a
Ktime#courseL determinationMwhich will give you a KsnapshotL view of how the reaction proceeds over
time! "ou are going to set up a series of cups in each of which you will run the same procedure! /he only
difference will be the amount of time you allow the H2O2&catalase reaction to run! "ou will stop the
reaction by adding H2'O<! Ghen you have finished timing and stopping all the reactions you will run a
titration to determine how much H2O2 remains! Ge are going to run your Kbase#lineL and /ime 1ourse
assays at the same time! /his is a lot of wor. so you need to .now what you are doing and be organizedN
It is possible to get all of this done but you must wor. ,uic.ly and efficientlyMas a teamN
Drocedure:
0! 8abel ? clean cups as follows: %ase#line assay 02 seconds 92 seconds C2 seconds 32 seconds 022
seconds 0?2 seconds 9C2 seconds!
2! @ill a glass bea.er with approximately 32 m8 of H2'O<! %e careful with H2'O<N If you spill it on your
s.in flush with 8O/' of water immediatelyN (/his is a dilute sulfuric acid but it will still burn if you
don7t get it off!) :et a 02 m8 syringe and reserve it for sulfuric acid! (One group member should be
in charge of sulfuric acid!)
9! :et another glass bea.er and put approximately 02 m8 of catalase into it! /a.e it bac. to your des.
and .eep it on iceN (/his is very importantMcatalase disintegrates ,uic.ly!) :et a 0 m8 syringe and
reserve it for catalase!
<! :et approximately 32 m8 of H2O2 in a plastic cup and then put 02m8 in each of the labeled plastic
cups except the baseline cup!
=! :et a stopwatch and figure out how to use itN
C! Dut 0 m8 of water in the base#line assay cup! )dd 02 m8 of H2'O< to the cup! 8eave the cup in place
and go on to the next cup!
>! Dut 0 m8 of catalase in the K02 secondL cup! 'wirl gently for 02 seconds! )t 02 seconds add 02 m8
of H2'O< and set the cup aside!
?! Dut 0 m8 of catalase in the K92 secondL cup! 'wirl gently for 92 seconds! )t the end of 92 seconds
add 02 m8 of H2'O< and set the cup aside!
3! (epeat O? for each of the remaining cups varying the amount of time you swirl the cup stopping the
reaction with H2'O< at the appropriate time! Ghen you have completed the reaction in each of the
cups remove a 5 ml sample and determine the amount of H2O2 present by running a titration in
which you add E*nO< from a burette one drop at a time to the sample stopping when a persistent
pin.&brown color is obtained! (-ote: If you overshoot the endpoint remove another = ml sample and
start over againN) 'o not %iscar% any of your solutions until your la$ is over( "ou also need to titrate
the uncatalyzed reaction that you left out yesterday!
02! )s you determine each value enter it in /able 2!0 in your lab noteboo.!
00! )raph the amount of H2O2 on graph paper in your noteboo.! (Aon7t forget that you need to title your
graph and label your axes appropriately! "ou need to ma.e your graph clear large enough that I can
read itN
02! 1omplete the )nalysis of (esults!
09! %e prepared to discuss your results on *onday (and maybe ta.e a ,uizFFFF)
/able 2!0
E*nO< (ml) /ime
02 92 C2 32 022 0?2 9C2
a!) %ase 8ine
d!) )mount of E*nO<
consumed (b#c)
e!) )mount of H2O2 used (a#
d)
Analysis o+ /eslts! (3ote! 1lease ans4er t-ese 5estions in yor lab noteboo67 8O/ *H9' O3E *9$E: 9 4ill
e)cse yo +rom 4riting a lab abstract; )
* +rom the formula %escri$e% earlier for rate ( y, -). %etermine the initial rate of the reaction an% rates $etween
each of the time points in your proce%ure an% recor% them $elow/
/ime
2 6 02 02 6 92 92 6 C2 C2 6 32 32 6 022 022 6 0?2 0?2 # 9C2
(ates
2 !hen is the rate the hi&hest#
!hy is it hi&hest here#
0 !hen is the rate the lowest#
!hy is it lowest here#
4 !hat was the inhi$itin& effect of sulfuric aci% on this reaction# !hat %i% the sulfuric aci% %o to the en"yme#
5 !hat woul% lowerin& the temperature in this reaction %o to the rate of en"yme activity#