the discussion section

26 Jun the discussion section

Question
INTRODUCTION
THE FUNCTION OF ENZYMES:
DEVELOPING A TESTING A SCIENTIFIC HYPOTHESIS FOR INHERITED MUTATIONS IN THE ENZYME KINESIN

Before coming to Lab:

OVERVIEW

1. Read the entire lab
entitled The Function
of Enzymes

Enzymes are active proteins that catalyze the conversion of one biomolecule or
protein into another. Two critical enzyme catalyzed reactions are illustrated below:
Initiating glycolysis:

Read
Essential
Biology text, pg. 104105 (How do We Know2.

Measuring
performance)

enzyme

3. Watch the Enzyme
Kinetics tutorial (click
here) viewing Parts 1-6
only.
4. Complete PreLab
worksheet
5. Complete any
experimental design
needed. These sections
will be highlighted in
GREEN in the Lab
Activities section.

Transport of proteins from ER to cell surface:
There are 2 terms to become familiar with in this lab. These are enzyme activity
and rate of conversion of substrate to product. In effect these both mean the
same thing. The activity of an enzyme is directly related to the rate at which it
catalyzes the conversion of a reactant (ie. glucose or ADP) to a product (ie.
glucose-6-phosphate or ATP).
One major reason scientists study enzymes is because their affinity for a protein
is often related to the development of disease. For example, mutations in kinesin
reduce the ability of the enzyme to hydrolyze ATP, which may prohibit transport of
proteins in vesicles along neurons. Inhibited transport can lead to loss of motor
function and neurological disease. As another example, the cancer drug Gleevec
(Essential Cell pg. 721-724) is an inhibitor of the enzyme ABL, which binds
proteins that activate cell proliferation and tumor growth.
Studying disease is, thus, often directly linked to studying enzyme affinity for a
substrate.
In order to learn and practice skills in enzyme affinity studies that will be
applicable to careers in bioscience, ecological research, and/or healthcare, you
will prepare reactions between an enzyme and a substrate and examine the
conversion to product using both visual and analytical cues. You will learn how to
use a very important research instrument called a spectrophometer and you will
practice applying simple mathematical models to the raw data you generate.

LEARNING OBJECTIVES
Understand the function, mechanism, and data output of the spectrophotometer, and describe

how it is used in biochemical analysis of enzyme function.
Design and implement an experiment to measure the rate of conversion of a reactant to a
product in an enzyme catalyzed reaction.
Apply mathematical model to raw data, using Michaelis-Menton and Lineweaver-Burke
relationships.
Extract enzyme kinetic parameters, KM and Vmax, from those plots.
Compare and contrast the kinetic parameters between normal and mutant kinesin and discuss
how mutations in kinesin may lead to neuropathy and loss of motor control.

PRELAB ASSIGNMENT
THE FUNCTION OF ENZYMES:
DEVELOPING A TESTING A SCIENTIFIC HYPOTHESIS FOR INHERITED MUTATIONS IN THE
ENZYME KINESIN
A) Before you start:

Read the entire lab exercise

Read pages 104-105 in Essential Biology text.

View the Enzyme Kinetics tutorial (click here) viewing Parts 1-6 only.

Download PLoS ONE Kinesin article from BbLab Information Enzyme Kinetics Lab. You will
only read the abstract.
Peter Fuger, et al. Spastic Paraplegia Mutation N256S in the Neuronal Microtubule Motor KIF5A Disrupts
Axonal Transport in a Drosophila HSP Model. PLoS Genetics, 2012. 8(11): 1-20.

B) Answer the questions.
***

Remember that lab preparation is weighted more heavily than lab reports in this course. Most of your
time in any research experience will be planning and experimental design, and thus, a major focus in
BIOL 2081C lab is your prelab work.
Do not wait until the day of your lab to begin this pre-lab. Inability to access a computer is not a valid
excuse for not completing this assignment on time.

Pre-Lab (2 pt each)
1. Consider the reaction: Glucose + hexokinase glucose-6-phosphate
Assign the molecule as the enzyme, substrate, and product
Glucose = __________________
Hexokinase = _______________
Glucose-6-phosphate= ________________
2. Which of the above reaction components corresponds to A, B, and E in the online tutorial?
Glucose = _________________
Hexokinase = ______________
Glucose-6-phosphate= ____ ___________
3. Fill in the blank:
When the reaction between glucose and hexokinase reaches equilibrium, the reaction rate, v, is
________________ because the reaction is moving ______________________.

4. How would you describe the relative proportions of substrate and enzyme in a biological reaction?

5. What is the ceiling that limits the rate at which an enzyme can convert a substrate into a product?

***

PRELAB ASSIGNMENT
THE FUNCTION OF ENZYMES:
DEVELOPING A TESTING A SCIENTIFIC HYPOTHESIS FOR INHERITED MUTATIONS IN THE
ENZYME KINESIN
_______________________ ____
6. What does the Michaelis-Menton (M-M) curve describe? Why do scientists care about the M-M curve?

7. What does the flattening of the Michaelis-Menton curve signify with the respect to the enzyme active
site and substrate concentration?

8. Referring to Section 5 of the tutorial:
If the Km and Vmax of Enzyme 1 are 16uM and 120uM/min, respectively, and Enzyme 2 has
Km=11.8 microM, which enzyme has a stronger affinity for a given substrate?

If Vmax stays the same and Km decreases, does the reaction rate reach Vmax faster or slower?

9. What does hyperbolic mean in regards to the Michaelis-Menton plot?
10. True or False? When a reaction reaches equilibrium, product is no longer formed.
11. True or False? The Michaelis-Menton plot is a graphic representation of the product formed over time.
12. If you answered FALSE to questions 10 or 11, explain why.
Question 10. _____________________________________________________

Question 11. _____________________________________________________

13. Lysozyme is an enzyme that breaks down bacterial cell walls. Its Km is 6 mM. Chymotrypsin is a
digestive enzyme that breaks peptide bonds and its Km is 5000 mM.
a) Insert the Km values for the two enzymes in the M-M curves below. Use a line to indicate where Km
crosses the y- or x-axis.

PRELAB ASSIGNMENT
THE FUNCTION OF ENZYMES:
DEVELOPING A TESTING A SCIENTIFIC HYPOTHESIS FOR INHERITED MUTATIONS IN THE
ENZYME KINESIN

b) Which of these two enzymes has the higher affinity for its substrate and why do you think so?
c) Which enzyme(s) will be operating at maximum efficiency at the following concentrations of substrate?
Write L, C, B, or N in the box to the right of the substrate concentration, where lysozyme only (L),
chymotrypsin only (C), both (B) or neither (N).
1 mM
100 mM
50,000 mM
6 mM
5000 mM

Questions on article:
14. Make a schematic which includes the general shape of the motor protein kinesin, including
the cellular components that bind to kinesin. (Will need to look up the function of kinesin in your
text book.) Hint: Kinesin is a polar molecule, meaning that each end has a specific function.
What are those functions?

15. Why is kinesin considered an enzyme?

PRELAB ASSIGNMENT
THE FUNCTION OF ENZYMES:
DEVELOPING A TESTING A SCIENTIFIC HYPOTHESIS FOR INHERITED MUTATIONS IN THE
ENZYME KINESIN

16. How does mutant kinesin promote muscle weakness?

17. NOW complete the Experimental Design sections in Lab Activities (6pt)

LAB ACTIVITIES
THE FUNCTION OF ENZYMES:
DEVELOPING A TESTING A SCIENTIFIC HYPOTHESIS FOR INHERITED MUTATIONS IN THE ENZYME
KINESIN

Specific Information for this lab
In this lab you will first practice recording enzyme kinetic data using the enzyme -galactosidase as a
model enzyme. You will then apply this knowledge to study the enzyme kinetics of kinesin, a cellular
motor protein. -galactosidase offers analytical and visual information to study the conversion of a
substrate to a product, because the substrate, ONPG, is clear, but the product , o-nitrophenol, is yellow.
-galactosidase is a bacterial enzyme which has been studied extensively for decades. The basic
reaction catalyzed by this enzyme in bacteria is:
You will be using a colorimetric assay to measure -galactosidase activity.

Watch the video How does a spectrophotometer work? to see how the spec instrument is used
detect color change in a colorimetric assay.
In this assay, a colorless substrate (ONPG, or o-nitrophenyl–D-galactopyranoside) is converted to a
colored product (o-nitrophenol), which absorbs light at wavelength 420 nm and can be measured using
a spectrophotometer. The molar absorptivity of o-nitrophenol =0.0045 OD/nmol. You can use this
information to calculate the amount of product formed in your reaction.

LAB ACTIVITIES
THE FUNCTION OF ENZYMES:
DEVELOPING A TESTING A SCIENTIFIC HYPOTHESIS FOR INHERITED MUTATIONS IN THE ENZYME
KINESIN

Activity 1: Investing the Activity of β-galactosidase
By monitoring the appearance of the product o-nitrophenol (yellow color) over time you will
quantitatively examine the rate at which -galactosidase catalyzes the conversion of ONPG to
o-nitrophenol.
****Read through the procedures and create outlines for your data tables and/or figures before
coming to lab. Your group will compile table/figures before beginning the procedure to make
sure everyone is on the same page****
1. Construct the framework of a data table that will allow you to record the activity of βgalactosidase every minute over a 10 minute time period. To do this, consider what data you will
collect and what your column headings should be.

2. Add experimental data to the table you generated above using the reaction between 3 ml of 0.2
mM ONPG mixed with 100uL enzyme.

3. Plot your results on the axes below. Add appropriate units to each axis.

LAB ACTIVITIES
THE FUNCTION OF ENZYMES:
DEVELOPING A TESTING A SCIENTIFIC HYPOTHESIS FOR INHERITED MUTATIONS IN THE ENZYME
KINESIN

4. Label the part of the above curve where the rate of the reaction is highest.
5. Label the part of the above curve where the rate of the reaction is lowest.
6. The highest rate is ___________________________ (estimate value, include units)
7. The lowest rate is ____________________________(estimate value, include units)
8. Now, convert your raw OD versus time data into rate (OD/min) versus time data, by calculating the
rate of the reaction for each 1 minute interval:
Enter Table of data for rate of o-nitrophenol production at 0.2mM ONPG here:

9. Plot the rate of the reaction as a function of time, using the axes below. Include units on each axis:

10. What do you conclude from this experiment? Do all time intervals provide equally reliable
measurements of the reaction rate between beta-galactosidase and 0.2 mM ONPG? Why or why not?

LAB ACTIVITIES
THE FUNCTION OF ENZYMES:
DEVELOPING A TESTING A SCIENTIFIC HYPOTHESIS FOR INHERITED MUTATIONS IN THE ENZYME
KINESIN

LAB ACTIVITIES
THE FUNCTION OF ENZYMES:
DEVELOPING A TESTING A SCIENTIFIC HYPOTHESIS FOR INHERITED MUTATIONS IN THE ENZYME
KINESIN

Activity 2: Effect of Substrate Concentration on the
Reaction Rate
For this section, you will measure the activity of β-galactosidase at several different substrate
concentrations and construct Michaelis-Menton curve from your data. This is the only way to
ESTIMATE the Km and Vmax of the enzyme -galactosidase for the substrate ONPG.
1. Write the protocol that you will follow to measure the rates of the reactions set up in the table below.
Measure enzyme activity over a 20 minute period, and decide what time intervals will you use to
determine the rate of the reaction in each tube. Also construct a data table to record your results.
Enter protocol here:

Enter Data table here:

2. Set up four -galactosidase/ONPG reactions as follows (DO NOT ADD enzyme until you are ready to
put the reaction into the spec. When ready, add 100uL enzyme to tube, invert to mix, pour in cuvette,
and place in spec):
2 mM ONPG
0.2 mM ONPG
Buffer

Rxn 1
3.0ml
—

Rxn 2
1.5ml
-1.5mL

Rxn 3
-3.0mL
—

Rxn 4
-1.5mL
1.5mL

3. What is the initial substrate concentration in each of the reaction mixtures?
Rxn 1
Initial [ONPG]

Rxn 2

Rxn 3

Rxn 4

LAB ACTIVITIES
THE FUNCTION OF ENZYMES:
DEVELOPING A TESTING A SCIENTIFIC HYPOTHESIS FOR INHERITED MUTATIONS IN THE ENZYME
KINESIN

(units?)

4. Before you actually do the experiment, predict what will happen in the reactions from step 2 by
plotting points that you expect on the axes below. Add the appropriate units to the axes.

5. Run the reactions according to your protocol. Plot results on the axes provided. Add the appropriate
units to the axes.

6. If you think that your data are unreliable, alter your protocol and make the measurements again.
7. Label the parts of your curve where the reaction rates are highest and lowest.
8. Is the reaction rate equally sensitive to all changes in substrate concentration? Why or why not?
9. Where the rate is highest, estimate the fraction of enzyme molecules that are in complexes with
substrate?

LAB ACTIVITIES
THE FUNCTION OF ENZYMES:
DEVELOPING A TESTING A SCIENTIFIC HYPOTHESIS FOR INHERITED MUTATIONS IN THE ENZYME
KINESIN

10. Does this rate approximate Vmax for beta-galactosidase? Why or why not?

Activity 3: Mutations in the enzyme kinesin lead to loss of
motor control and neuropathy
NOTE: This portion of the lab is performed virtually on computer.
Scenario
You are an undergraduate researcher in a neuroscience lab that studies disorders in muscle motor
control. Tamara, a postdoc in your lab, studies inherited genetic mutations in kinesin, a motor protein
that walks along microtubules to deliver proteins and vesicle cargo down the length of neurons
(Essential Cell, 577-578). To introduce you to the kinesin protein, Tamara tells you to check out these
videos: here (video 2min) and here (video: 22 sec). Tamara is excited about a recent study she read
that characterizes different mutants of kinesin and discusses the role of kinesin mutants in hereditary
spastic paraplegias (HSPs), a group of neurodegenerative disorders characterized by spastic
weakness of the lower extremities. She has acquired a sample of mutant kinesin from the authors of
this paper and is performing a number of biochemical experiments on it.
Your project
Your project in the lab is to determine the enzyme kinetics, Vmax and Km, of normal and mutant kinesin
using ATP as the substrate. You are excited because if you succeed you will be included as an author
on Tamaras manuscript. This would be a great resume booster and you want the lab to hire you as a
summer intern, so you are trying to do your very best.
Tamaras exciting reference
Peter Fuger, et al. Spastic Paraplegia Mutation N256S in the Neuronal Microtubule Motor KIF5A
Disrupts Axonal Transport in a Drosophila HSP Model. PLoS Genetics, 2012. 8(11): 1-20.
Find on Bb under Lab Documents.
Procedure
Access the Virtual Kinetics Experiment in the Enzyme Kinetics tutorial and follow the prompts to
perform the kinesin kinetics experiment virtually.

BACKGROUND READING
THE FUNCTION OF ENZYMES:
DEVELOPING A TESTING A SCIENTIFIC HYPOTHESIS FOR INHERITED MUTATIONS IN THE ENZYME
KINESIN

Introduction to Enzyme Kinetics
The vast majority of reactions occurring in cells are catalyzed by enzymes. The number of different
enzymes at work at any given time is very large, as are the number of substrates consumed and
products produced. Yet the fundamental mechanism of catalysis is similar for all enzymes. Enzymes do
not alter the equilibria between substrates and products, nor do they alter Ghey simply make the
reactions go much faster than they normally would- sometimes by a factor of a million or even a billion.
Enzymes accomplish this by first binding to the substrate in a highly specific manner (Essential Cell 9093) and then by altering the substrate molecule in such a way as to favor the formation of, and then
stabilize, the transition state. The transition state is usually an unstable intermediate that can quickly
break down. The spontaneous break down of the transition state molecule can lead to the reformation
of the original molecule OR it can lead to the formation of a new molecule, the product. The ability to
form and stabilize the transition state is the key to enzyme catalysis. It leads to a lower activation
energy of the conversion from substrate to product, and this causes the overall reaction rate to increase
dramatically.
There are a number of ways to study the properties of enzymes and enzyme substrate complexes.
Biochemical and biophysical approaches tend to concentrate on the amino acids of the protein,
particularly those that make up the active site and those that interact with cofactors and prosthetic
groups. Another approach, called enzyme kinetics, focuses on the reactions themselves. These
methods involve measurements of the rate of the reaction under different experimental and natural
conditions.

In this experiment, you will use kinetic techniques to explore the properties of the
enzyme beta-galactosidase.
You will then apply these skills in an online experiment to make predictions and
hypotheses about the kinetics of the normal and mutant motor protein, kinesin.

Enzyme kinetics
Consider a simple cellular reaction in which A is converted to B by enzyme E.
The rate of this reaction can be measured by determining the rate of formation of B over time. The
kinetic characteristics of the enzyme can be assessed by setting up a series of tubes and adding to
each of them a different concentration of substrate A, while adding the same amount of enzyme to
each, thus holding the enzyme concentration constant (Figure 2). In each tube, the initial linear rate of
the reaction is called the initial velocity and is abbreviated, v0. The value of each v0 is determined as
the slope of the line for each reaction; that is, the slope of each line shown in Figure 2. As the
concentration of substrate (in this case A) is increased, the v0 of the reactions increase (Figure 2).

BACKGROUND READING
THE FUNCTION OF ENZYMES:
DEVELOPING A TESTING A SCIENTIFIC HYPOTHESIS FOR INHERITED MUTATIONS IN THE ENZYME
KINESIN

Figure 2. The rate of reaction of A B can be measured by measuring the rate of
formation of B. The kinetics of the reaction can be assessed by holding the
concentration the enzyme, [E], constant and increasing the concentration of the
substrate, [A]. The slope of each line (change in B created / time) equals vo and it
increases as [A] increases in each panel, from left to right.
If these individual reaction rates (vo) are then plotted together on a graph of reaction velocity (vo) as a
function of substrate concentration [A] (or in Figure 3, [S]), a curve like that shown in Figure 3 is
produced. The amount of product increases linearly as substrate concentration increases up to a point,
where the curves begins to level out and then finally plateaus. The maximum catalytic rate of the
enzyme, i.e., the point at which the enzyme cannot work faster, no matter how much substrate is
present, is call the maximum velocity and is abbreviated Vmax.

Figure 3. A plot of the initial reaction rates (v0) of an enzyme-catalyzed reaction as a
function of substrate concentration [S] yields a characteristic curve, called the MichaelisMenton curve.
One of the simplest kinetic models used to explain these phenomena is the one described in 1913 by
Leonor Michaelis and Maud Menten. In the Michalis-Menton model (Karp text, Figure 3.17), it is
assumed that the enzyme and substrate briefly unite to form an enzyme-substrate complex that then
breaks down to form product and the original enzyme. Mathematically, this can be described in an
expression for defining the initial velocity (v 0) of an enzyme catalyzed reaction that can be used for all
enzymes:

BACKGROUND READING
THE FUNCTION OF ENZYMES:
DEVELOPING A TESTING A SCIENTIFIC HYPOTHESIS FOR INHERITED MUTATIONS IN THE ENZYME
KINESIN

The important terms in this expression are Vmax and Km, which are unique to each specific enzymesubstrate pair. Today we call Km the Michaelis constant.
Note that Km is a concentration, not a reaction rate. It, therefore, has units of moles/liter, just as does
substrate concentration. When v0 is equal to 1/2 Vmax, Km = [S]. Therefore, Km can be determined
graphically by simply determining the substrate concentration giving half-maximal velocity. Note that
enzymes with a small Km for a particular substrate will reach saturation at a low substrate
concentration. A large Km indicates that saturation is reached only at high substrate concentrations.
The Michaelis-Menten equation shown above (equation 2) can be linearly transformed. That is, it can
be mathematically manipulated so that it produces a straight line instead of a curve like that of Figure 3.
This allows Km and Vmax to be more easily determined by using linear regression techniques. Linear
plots also require fewer points (i.e., trials) to construct. One of the most common linear transformations
is the Lineweaver-Burke plot. The Michaelis-Menten equation can be arranged in the form:

Notice that this equation has the general form y = mx+b. A plot of 1/v 0 against 1/[S] yields a straight line
with a y-intercept of 1/Vmax. The intercept of the line on the x-axis is equal to -1/Km. These plots are
discussed in Karp page 103.
The two parameters that characterize the kinetics of an enzyme for a given substrate are Vmax and
Km. More information on each is provided below.
Vmax:
A single enzyme molecule can convert molecules of substrate into product multiple times during a given
time period. The maximum rate for a group of molecules is called Vmax. Imagine that the concentration
of substrate is so high that there is essentially no time when an enzyme molecule is not bound to
substrate. That is, as soon as a bound substrate molecule reacts to form product, it leaves the enzyme
and is instantly replaced by a fresh substrate molecule. This is what we call a "saturating" concentration
of substratebecause at this concentration, the active site is always filled (saturated) with substrate.
Under such conditions, the rate at which an individual enzyme molecule can turn substrate into product
is the highest it can possibly be. This maximum cycling rate per molecule is called the turnover number
(TON). A group of 100 enzyme molecules, each able to convert substrate to product at the same
maximum cycling rate, would produce an observed Vmax that was 100-fold higher than the turnover
number.
Km:

BACKGROUND READING
THE FUNCTION OF ENZYMES:
DEVELOPING A TESTING A SCIENTIFIC HYPOTHESIS FOR INHERITED MUTATIONS IN THE ENZYME
KINESIN

The Michaelis constant (Km) of an enzyme is a measure of the affinity of the enzyme for its substrate.
The value of Km for a particular enzyme is defined as the substrate concentration at which half of the
enzyme molecules are complexed with substrate. Under these conditions, at any instant, half of the
total enzyme molecules are capable of catalysis. Therefore, the observed reaction rate is half of the
maximum possible ratethe rate achieved when all the enzyme molecules are complexed with
substrate, Vmax.
The substrate concentration required to drive half of the enzyme molecules into enzyme-substrate (ES)
complexes depends on the ability of the enzyme to bind its substrate. Thus, an enzyme with a high
affinity for its substrate will have a low Km. That is, 50% of the enzyme molecules will have bound
substrate at a relatively low concentration of substrate. In contrast, an enzyme with a low affinity for its
substrate has a high Km valuebecause a relatively high concentration of substrate is required to drive
50% of the enzyme molecules into complexes with substrate.
Enzyme Inhibition (Essential Cell 106)
One of the major control mechanisms of enzymes is inhibition. There are two general categories of
inhibitory molecules, irreversible and reversible.
1. Irreversible inhibitors covalently bind to an enzyme and cannot dissociate from it.
a.i. Examples: Nerve gas, penicillin
2. Reversible inhibitors, on the other hand, do not bind covalently and can dissociate from the
enzyme. Two types of reversible inhibitors are competitive and non-competitive.
a. Competitive inhibitors bind to the active site, and thus compete with the substrate for
access to the enzyme. In theory, the effects of the inhibitor can be negated if enough
substrate is added; therefore, a competitive inhibitor should increase the Km of an
enzyme but not alter the Vmax.
a.i. Examples: ACE-inhibitors to decrease blood pressure
b. A non-competitive inhibitor binds to the enzyme at an allosteric site, or a location other
than the active site. The binding of the inhibitor alters the enzyme so that the catalysis
of substrate to product is inhibited and, as a result, the V max is decreased. A true noncompetitive inhibitor does not change the Km; however, many inhibitors that decrease the
Vmax also increase the Km.
b.i. Examples: Tipranavir, an HIV therapeutic drug
The figure below illustrates how the Lineweaver-Burke cu…