Competitive Inhibition
of
Lactase by Galactose
Lab Experiment Protocol
by
Mireille A. Captieux
Michael J. McCarthy, Timothy J. Dobbs,
Moses V. Lleva, Dominic A. Chirico,
Hannah J. Barrett, Josiah Barrett, Tomeka L. Byrd, Matthew L. Eure,
Paula S. Hahn, Trakelia L. Hamlin, Anna E. Joyner, Joshua S. Stacey, Savanaha R. Toothman, Carl W.
Vermeulen*
The Numerous biochemistry lab exercises
revolve around the "on/off" characteristics of the lac-operon,
the gene that deals with the metabolism of lactose.
However as any production-line manager can tell us,
it takes more than an "on/off" switch to run an operation.
A speed control is also necessary.
In the complex world of gene expression,
there exist at least two possible rate regulators.


This current work focuses on devising a laboratory
exercise that reveals an example of feedback competitive
inhibition as the regulator of the enzyme lactase's efficiency.
In a following submission we shall consider regulation
of the reading of the gene itself.
Enzymes
are proteins that act as biological catalysts
(speeding up reactions and allowing them to occur at or near room
temperature).
As do all catalysts, they work by lowering activation energy so that substrates
(what you start with) can readily be split into products (what you end with).

Without enzymes most of the reactions needed to fuel
our body would occur too slowly or possibly not at all.

However, the body must still have control
over the production of these enzymes
so that they can also be turned off.

Just like the temperature inside your home acts
on the thermostat to turn off your heat or air,
enzymes can work to turn off their own production
when necessary in the process of inhibition.
There are three main types of inhibition:
Non-competitive
Uncompetitive
Competitive.
Non-competitive inhibition:
(aka allosteric inhibition)
the inhibitor binds to another place on the enzyme
causing the enzyme to warp and be less effective.
Un-competitive inhibition:
the inhibitor removes the enzyme from the reaction - such as a heavy metal
would do by precipitating the enzyme.
Competitive inhibition:
the inhibitor binds to the same site as does the substrate, thus competing for
the enzyme's active site.
After one of us (Mireille A. Captieux) discovered that
galactose was a feedback inhibitor of lactase,
we then sought to ascertain which type of inhibition was
being exerted upon lactase by galactose.
(But, as Captieux found, not glucose or other hexoses)
A lactose molecule's being hydrolyzed in the forked arrow
by the enzyme lactase into two monosaccharides
glucose and galactose.
Galactose is then normally converted to glucose by another enzyme
(NAD:galactose-glucose epimerase), and then onwards to glycolysis.
This protocol will allow you to identify the type of inhibition
that galactose has on lactase.
Galactose is a hexo-pyranose
(carbohydrate with a six-member ring of 5 carbons and an oxygen)
and is less sweet than glucose.
When galactose is covalently bonded to glucose forming a disaccharide
called lactose.
Lactose is found most notably in milk products.
Once lactose is enzymatically hydrolyzed (split) in our bodies,
the glucose and galactose products are readily metabolized.
However, after infancy some of our bodies turn off the gene
that codes for the making of lactase that allows us to break down lactose.
Lactose Tolerance: The ability to consume milk or cheese products
containing lactose without becoming ill.
Lactose Intolerance:
The inability to consume milk or cheese products containing lactose.
those who are, usually feel ill or become sick after consuming these products.
Fortunately, for the lactose intolerant adults, lactase in a pill form can be
ingested and assist in the breakdown of lactose so that dairy products mostly - will not have to be forever forsaken.
The Lab Exercise
The lactase pill, which can be purchased at any drugstore or supermarket,
will supply us with our enzyme.
The first hurdle we face is that there is no easily observable change
seen when lactase reacts upon lactose.
For this exercise a chromogenic CHEMICAL ANALOG of lactose,
ONPG, will be used instead.
ONPG
(ortho-nitrophenyl-beta-D-galacto[purano]side)
is similar in structure to lactose
except instead of having
glucose as one of its subunits
it has an o-nitro-phenyl group,
ONP
ONPG molecule's being hydrolyzed
by the enzyme lactase into galactose and yellow ONP (o-nitro-phenol)
when ONPG is split by Lactase it yields galactose and ONP
in the form of a bright yellow dye
(i.e.: chromogenic - producing color).
Following the products and inhibitor
being combined:
the speed of the reaction (rate at which the solution turns yellow)
can be measured in a spectrophotometer set at Abs420nm.
Different levels of ONPG
can then be added and the inhibitory
effects of added galactose can be observed
as different reaction rates result.
MATERIALS AND METHODS:
Preparation of Reagents:
ONPG (o-nitrophenyl-β-D-galactopyranoside)
Start this first because ONPG dissolves very slowly –
crushing it very finely helps speed dissolution!
Make 10 mL of 0.5% ONPG using DISTILLED water.
(Hint: 50 mg ONPG +10 mL H2O.)
( The reason for using distilled water is that tap water contains a
few living organisms and thus possibly lactase.
We don't want any lactase in our ONPG reagent!)
Lactase
A pill of Lactaid® (bought from any drugstore/supermarket)
After crushing into powder and then dissolved into 100mL of tap water
yielding a turbid solution as some of the fillers in the tablet are insoluble.
(By the way tap water is used as it may provide any needed mineral cofactors.)
Centrifuge the solution at about 3,000 rpm for about 20 seconds
to separate any filler from the lactase.
Descant the top lactase solution and dispose of the large sediment particles
that remain stuck to the bottom of the centrifuge tube.
Test the activity of this supernate:
First, put a drop of ONPG into a test tube
WARNING! It is very important that you do not contaminate your stock
ONPG with any lactase.
Second, using another dropper, add a couple of drops of your supernate
If the mix turns yellow in a minute or so, continue.
If no yellow, your lactase preparation is inactive.
Next Dilute the lactase with 7 more volumes of tap water .
This dilution will lower the concentration of lactase so that the reaction
will occur at a reasonable rate with the ONPG so that it can be observed.
(Unrefrigerated this lactase solution is stable for several days at least.)
Galactose
Dissolve the galactose powder in tap water making a 2% solution (0.111M)
Dissolve 2 grams of galactose in 75 mL tap water, and then add more water up to 100 mL.
Making 2-fold serial dilutions of the 2% galactose
Start with three flasks labelled 2%, 1% and 0.5%.
Put 50 mL tap water into the 1% and 0.5% flasks.
Mix 50 mL of your 2% into the 1% flask to make 100 mL of 1%.
Mix 50 mL of the 1% into the 0.5% flask to make 100 mL of 0.5%.
In this way, by merely adding 1 mL of the desired one in Step 1
in the following procedure.
At your bench, and using the lactase solution and the solutions of
galactose, set up a rack of 5 test tubes (0→4) according to this table:
TUBE
Lactase
H20
O
4mL
1mL
1
4mL
½%
gal
1%
gal
2%
gal
Actual
Working
Concentrations
2
4mL
3
4mL
ONPG
1mL
mM
ONPG
gal
mM gal
25 mL
0.09
½%
5.5
35 mL
0.13
1%
11
50 mL
0.18
2%
22
100 mL
0.35
1mL
1mL
ONPG volume

Know what your group's assigned ONPG volume (concentration) is.
Your assigned ONPG volume will be one of these volumes:
0, 25 μL, 35 μL, 50 μL, 100 μL (i.e.: 100 μL = 0.1 mL)

While you are awaiting your turn at the spectrophotometer,
read the remaining steps before proceeding.
"Dry lab" the procedure in preparation for efficient,
error-free data acquisition.

When your turn comes, move your rack to the spectrophotometer
that is set at 420nm

To start each tube, use the appropriate micropipette
to add the required amount of ONPG into the tube.
Immediately dump the tube's contents into the cuvette.
(The mere dumping mixes it.).

Immediately zero the spectrophotometer


Beginning within 15 seconds of the zeroing,
take readings every 20 seconds
for about 2 minutes.

Repeat the procedure with varying amounts of galactose
DATA TREATMENT
GRAPHING:
Vo -Determination Graphs:
You must first make a graph like the one below for each "run" you make.
From this graph you determine the initial velocity (V0) of that particular reaction.
Note: Use only the early straight-line portion and discard the points if they show signs of plateauing.
Thus as you make three runs at different galactose concentrations but all at the same ONPG
concentration, you must make 4 graphs (perhaps all on the same sheet of graph paper!),
and from each best-fit line you have determined their individual Vo's.
Lineweaver-Burk Plots:
After collecting these Vo's, you then proceed to creating a [double-reciprocal]
Lineweaver-Birk plot (1/Vo versus 1/[ONPG]).
Your goup will contribute one line to the class's combined plots
on one piece of graph paper, compare them with the
following graphs for comparison.
The graph which best resembles the graph from your data
should then indicate what type of inhibition you observed
galactose having on lactase.
Such knowledge of this and other enzymes are important
when concocting new chemotherapeutics or in an industry that uses enzymes.
What do your graphs
Tell you?
If galactose acts as a competitive inhibitor
it means that not only does the substrate lactose fit into the enzymatically
active site on the lactase but so also does galactose itself.
This is analogized in the supermarket model in the case where a penniless
customer tries to check-out along with other customers having sufficient
money to pay for their purchases.
The cashier's time is wasted on the penniless person and the whole sales
process slows down the more impecunious people try to check out.
In brief, if galactose is competitive, its Lineweaver-Burk lines intersect at the
vertical axis (where [gal]=∞), which is also called Vmax.
noncompetitive inhibitor
A more modern name for this type of inhibition is called
"allosteric inhibition," which means "second-site inhibition."
Jacob and Monod were awarded their second Nobel Prize for
determining that the lac-operon's repressor protein acts that way.
Were the galactose to attach to a different portion of the lactase
enzyme molecule and in some way ”distorting" the enzyme slowing it down,
we would then call galactose a noncompetitive inhibitor.
In second-site inhibition, the repressor has two attachment sites. One site attaches
to the operator region on the gene and turns it "off." The other site attaches to any
lactose that is present, and upon such attachment the repressor protein has its shape
warped such that it can no longer attach to the operator, leaving the gene "on." Thus,
perhaps, galactose interfers with lactase by warping it into inactivity. This is
graphically seen by lines that converge but at a point to the left of the vertical axis (in
fact, on the horizontal axis!).
Uncompetitive inhibition
This can be visualized in the
supermarket as something that
comes along and breaks one or more
of the cash registers closing those
check-out lines in the supermarket .
Enzymatically
this is like the inhibitor's permanently inactivating an enzyme
molecule – irreversibly taking it out of service forever.
In effect it lowers the concentration of active enzymes
resulting in parallel lines.
Were galactose this type of inhibitor, it must bind to the
lactase and cause irreparable damage such as causing
peptide bonds to break and the molecule to fall apart.
RESULTS
The following Lineweaver-Burk plot (1/V versus 1/[ONPG]) is in constant revision. This is the first submission:
Future revisions will
narrow the variables:
[ONPG] = 25, 35, 50, 100 μL
[gal] = 0%, ½%, 1%, 2%