ChE 4013

ENZYME KINETICS

Objective: To determine the Michaelis-Menten constant and maximum

velocity for the enzyme -galactosidase with ortho-nitrophenyl -
galactopyranoside at two temperatures and compare them.

Equipment: spectrophotometer, pipettes, and cuvettes

Chemicals: enzyme -galactosidase, 1.50 mM ONPG (ortho-nitrophenyl -

galactopyranoside), 80 mM phosphate buffer (pH = 7.7), distilled water

Theory: The largest and most important group of proteins exhibiting

biological activity is the enzymes, which are the catalysts responsible for
accelerating the chemical reactions within a cell. Like inorganic catalysts,
enzymes do not affect the position of equilibrium of a reaction (that is, the
equilibrium constant is unaffected) but they do increase the rate at which
equilibrium is obtained
by decreasing the free
energy of activation of
the reaction. The free
energy of activation may
be defined as the energy
input required to convert
stable reactant(s) into a
metastable activated
complex, which may
then form product. The
activated complex is a
transitory state in which
the first stages of the
reaction are taking
place, where bonds
characteristic of only the
reactants are weakened
and bonds characteristic
of only product are being
formed. Free energy
profiles for a reaction in Figure 1: Energy Diagram for a Chemical Reaction, Catalyzed and Uncatalyzed [1]
the presence and
absence of a catalyst are
shown in Figure 1.
Notice that the overall free energy change for both processes is the same but
the free energy of activation for the catalyzed reaction is less than for the
noncatalyzed reaction. The maximum in the energy curve corresponds to
the activated complex.

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Characteristics:

(a) Unlike inorganic catalysts, enzymes are distinguished by a high degree of

specificity. For example the reaction of urea with water to form CO2 and
NH3, (see Equation 1)

O
|| urease
NH2-C-NH2 + H2O CO2 + 2NH3, (1)

is catalyzed by an enzyme called urease, which is absolutely specific for

H2O and urea. Though such absolute specificity is not universal among
enzymes, even those with lesser specificity act only on a limited class of
structurally related compounds.
(b)The rate of enzyme-catalyzed reactions tends to be extremely fast; in
fact, enzymes are the most efficient catalysts known.
(c) A further distinguishing characteristic of enzyme-catalyzed reactions is
that they can be subject to stimulatory or inhibitory control or both. A cell
contains probably thousands of different enzymes and though each
catalyzes one of only six basic reaction types, each prefers to react with
certain substances (substrates). The fate of a compound in a cell is
therefore determined by enzyme specificity and enzyme regulation.

Enzyme Activity: The study of enzymes and enzyme action (enzymology)

is of obvious importance to all the biological sciences. The in vivo role of an
enzyme in intermediary metabolism is often clarified considerably through
the in vitro investigation of its catalytic properties, which are reflected by the
conditions that affect the velocity of the enzyme-catalyzed reaction. In
general, the velocity or rate of an enzyme-catalyzed reaction (defined as the
rate of disappearance of substrate or appearance of product) is dependent
upon a number of factors:
1. The nature and concentration of the enzyme
2. The nature and concentration of the substrate
3. The presence of cofactors (usually inorganic ions) or coenzymes
(usually organic molecules) if needed; some enzymes must be
complexed with non-protein molecules or ions to be active
4. The concentration of modifiers, molecules which stimulate or inhibit
enzyme activity
5. Environmental parameters such as pH, temperature and ionic
strength (any factor which may disrupt protein structure or interact
with amino acid residues may affect the catalytic activity of an
enzyme).

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Figure 2: The time course of an enzymatic reaction. At time t, the velocity (rate of reaction) is equal to the slope of the tangent. [2]
The catalytic activity of an enzyme may be measured by mixing together
enzyme and substrate under carefully controlled conditions and following the
course of the reaction with time. Figure 2 shows the time course of an
enzymatic reaction. The rate of the reaction (or velocity) at any time t is
equal to the slope of a tangent to the line at t as illustrated in Figure 2.

It can be seen that the velocity decreases with time due directly to the
depletion of substrate from the reaction mixture. The goal of this type of
experiment, called an assay, is to determine the effect of a particular set of
experimental conditions on the velocity of an enzymatic reaction. The only
point during the time course of an assay at which the experimental
conditions are defined is near t = 0, the only point at which the
concentration of the substrate is exactly known. The velocity characteristic
of the original assay conditions is determined by taking the slope of the
initial portion of the assay curve. This velocity is, not surprisingly, called the
initial velocity. The effects of any parameter on the catalytic activity of an
enzyme may be determined by a series of assays of the enzyme under
conditions in which the parameter under study is varied while all other
factors that would affect activity are held constant. The effects of the
parameter would be reflected in changes in the initial velocity. For example,
the effect of pH on the action of an enzyme on a particular substrate may be
determined through a series of assays in which all conditions with respect to
enzyme and substrate are identical except the pH of the reaction mixture is
different. Enzymes are optimally active only over a very narrow range.
Therefore a plot of initial velocity versus pH might look like that in Figure 3.

All enzymes of a cell are unique structurally and in biological activity. They
have different substrate specificities and affinities, show different responses
to solvent condition, require different cofactors or coenzymes and may be
subject to regulation by different stimuli. However, the study of enzymes
and enzyme action is unified by a common basic reaction sequence, though
precise mechanisms may differ. Therefore, most enzyme-catalyzed reactions
show similar kinetics; that is, the reaction rates show similar dependence on

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the concentrations of the reacting species,
the enzyme and substrate(s). For example,
if a series of assays were performed on
virtually any enzyme in which the enzyme
concentration was varied while all other
conditions, including substrate
concentration, were maintained constant, a
plot of the resulting initial velocities versus
enzyme concentrations would be linear. The
rate or velocity of an enzyme-catalyzed
reaction is, therefore, directly proportional
to the concentration of enzyme or,
kinetically speaking, enzyme-catalyzed Figure 3: The pH activity profile of trypsin [1]
reactions are first order with respect to enzyme concentration. If the
reciprocal

Figure 4: The relationship of initial reaction velocity to substrate concentration in an enzyme-catalyzed reaction [1]
experiment is done in which the substrate concentration is varied while all
other conditions, including enzyme concentration, are maintained constant
quite a different situation is encountered. In this case a plot of the resulting
initial velocities versus substrate concentration is not linear but hyperbolic as
shown in Figure 4. This type of relationship has been observed to be
generally characteristic of most enzyme-catalyzed reactions. The velocity is
seen to be directly proportional to substrate concentration (kinetically a first
order relationship) at low substrate concentration and independent of
substrate concentration (kinetically a zero order relationship) at high
substrate concentration where the rate-limiting factor becomes the enzyme
concentration. (Km and Vmax will be explained later.)

Reaction Mechanism: In 1902, Henri proposed a model for enzymatic

reactions that explains this behavior and has become accepted as the basic
reaction sequence of most enzyme-catalyzed reactions. It is held that a
typical enzyme-catalyzed reaction occurs in two steps. In the first step
enzyme and substrate react reversibly to form an enzyme-substrate complex
which then in a second step decomposes to form product and free enzyme

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which then reacts with more substrate. The velocity of the reaction then is
dependent upon the rate of decomposition of the enzyme-substrate complex.
These reactions are expressed by Equations 2 and 3.
k1
E S ES
k2 (2)
k3
ES E P
and k4 (3)
where S is the substrate, P is the product, E is the enzyme, and ES is the
enzyme-substrate complex. Both reactions are considered reversible, but k4
is usually negligible compared to k1, k2, and k3. In light of this model the
diphasic nature of the plot of v0 vs. [S] becomes clear. At a constant enzyme
concentration a finite number of sites exist for substrate to bind to enzyme.
As the substrate concentration is increased a larger and larger proportion of
these sites are filled at any given instant until a point is reached at which
virtually all of the enzyme is in the ES form. Further increase in the substrate
concentration will have little effect on the velocity of the reaction. The
velocity therefore approaches a limiting value at higher concentrations of
substrate. This phenomenon is referred to as saturation. (A plot of v0 vs. [S]
at constant [E] is called a saturation curve.)

Rate Equations: In 1913 Michaelis and Menten derived an equation based

on this model, which describes the relationship of initial velocity to substrate
concentration. As refined by Briggs and Haldane in 1925, this mathematical
relationship is in wide use today and generally applicable to most enzyme
reactions. As we shall see this equation not only adequately describes the
saturation phenomenon common to all enzyme-catalyzed reactions but also
contains constants characteristic of individual enzyme-substrate interactions
that allow comparisons of kinetic properties. The evaluation of these
constants for individual enzyme-substrate reactions can be of great value in
the assessment of enzyme specificities and affinities, which in turn often
clarify the in vivo role of an enzyme reaction and yield information
concerning the active site of an enzyme. The modern treatment of enzyme
kinetics makes one primary assumption, which is that very rapidly after
enzyme and substrate are mixed a steady-state equilibrium develops in
which the rate of formation of the ES complex is balanced by the rate of its
decomposition. The rate of formation of the ES complex is proportional to
the product of the concentration of free enzyme (Ef) and the concentration of
substrate. This is expressed mathematically by Equation 4:
d [ ES ]
r forward k1 [ E f ][ S ]
dt . (4)
The formation of ES from the reverse of Equation 3 is neglected (k4 << k1, k2
and k3). Similarly we can write for the decomposition of the ES complex:

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d [ ES ]
rreverse k 2 [ ES ] k 3 [ ES ]
dt , (5)
If the steady-state assumption is valid (and it seems to be for most enzyme-
catalyzed reactions), then
r forward rreverse
(6)
k1 [ E f ][ S ] k 2 [ ES ] k 3 [ ES ]
and . (7)
The total enzyme concentration [Et] in a reaction mixture will be equal to the
sum of the concentration of the free enzyme and the ES complex:
[ Et ] [ E f ] [ ES ]
. (8)
Then
k1 [ Et ] [ ES ][ S ] k 2 [ ES ] k 3 [ ES ] . (9)
Or
k1 [ Et ][ S ]
[ ES ]
k 2 k 3 k1 [ S ] . (10)
The initial velocity, or initial rate of product formation, is therefore
k k [ E ][ S ]
v 0 k 3 [ ES ] 1 3 t
k 2 k 3 k1 [ S ] . (11)
Or, rearranging:
k 3 [ Et ][ S ]
v0
k 2 k3
[S ]
k1 . (12)
Define:
k 2 k3
Km
k1 (13)
Vmax k 3 [ E t ] .
and (14)
Then
Vmax [ S ]
v0
K m [S ] . (15)
Km is called the Michaelis-Menten constant, and Vmax is the limiting or
maximum velocity of the reaction. The Vmax of an enzyme-catalyzed reaction
is reached when all of the enzyme is in the ES form; it is the maximum rate
for the reaction, which is approached at very high substrate concentrations.

Equation 15 is known as the Michaelis-Menten equation. This equation

predicts that at low substrate concentration v0 [S] but is independent of
substrate concentration at high concentrations of substrate. A plot of v0
versus [S] will therefore be hyperbolic, as is observed experimentally.

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An interesting relationship becomes evident when v0 = Vmax. The
Michaelis-Menten equation then becomes:
Vmax V [S ]
max
2 K m [S ] , (16)
1 [S ]
2 K [S ]
m , (17)
K m [ S ] 2[ S ] , (18)
K m [S ] .
and (19)
Km therefore has units of concentration and is equal to the substrate
concentration that gives Vmax shown graphically by Figure 4.

The magnitude of Km reflects the Table 1. Km for some enzymes [1].

affinity of an enzyme for a Enzyme & Substrate Km (mM)
substrate; the lower the Km, the Catalase and
lower is the concentration of H 2 O2 25
substrate required to reach the Hexokinase and
limiting velocity and therefore the Glucose 0.15
Fructose 1.5
greater the affinity of the enzyme Chymotrypsin and
for the substrate. For example, if N- 2.5
an enzyme could react with several Benzoyltyrosinamide 12.0
substrates, a comparison of the Km N-Formyltyrosinamide 32.0
of the enzyme for each substrate N-Acetyltyrosinamide 122.0
Glycyltyrosinamide
(there is a characteristic Km for Carbonic anhydrase and
every enzyme-substrate reaction) HCO3 9.0
would determine the substrate for Glutamate dehydrogenase and
which the enzyme had the greatest Glutamate 0.12
affinity, a potentially valuable piece Alpha-ketoglutamate 2.0
of information in deciphering the in NH 4 57.0
NAD 0.025
vivo role of the enzyme in ox
0.018
metabolism. Table 1 shows some NADred
Km values for several enzyme-substrate reactions. Km is independent of
enzyme concentration but obviously dependent upon factors that influence
enzyme activity such as pH, temperature, etc.

Every enzyme-substrate reaction has a characteristic limiting velocity Vmax,

which is dependent upon the concentration of the enzyme in the reaction
mixture and the catalytic efficiency of the enzyme with the particular
substrate involved. For an enzyme that can react with more than one
substrate, an evaluation of the limiting velocity of the enzyme reaction with
each substrate (where the enzyme concentration is identical in each
determination) can, like Km determination, yield information concerning
enzyme capabilities. The greater the Vmax for one substrate relative to
another, the greater is the difference in the reactivity of the respective ES

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complexes. Table 2 shows the effect of Table 2. Effect of substrate
substrate on relative Vmax for D-amino acid structure on Vmax for D-amino
acid oxidase (Data relative to
oxidase, an enzyme with broad specificity. D-alanine = 100) [1]
Substrate Relative Vmax
The magnitude of Vmax is indicative of the rate D-Tyrosine 297
of decomposition of the ES complex; D-Proline 231
therefore the effect of reaction conditions on D-Methionine 125
Vmax (at constant enzyme concentration) can D-Alanine 100
D-Valine 55
also yield information about the kinetics of D-Histidine 9.7
this rate-limiting step, which is the reaction Glycine 0.0
that occurs on the enzyme surface.

The determination of the constants Km and Vmax graphically from a saturation

curve requires
the estimation of an asymptotic value. Shown in Table 3 is the relationship
between the Km of an enzyme for a substrate and the substrate
concentration required to reach various degrees of saturation. As can be
seen the best estimate of Vmax is made only at high [S]/Km ratios. In many
cases, this would require very high substrate concentrations, which could be
impractical if not impossible to supply.

Table 3. Fraction of Vmax obtained when [S] is various multiples of Km

[S]/Km 1 2 3 4 5 7 10 20 50 100
v0/Vmax 0.5 0.67 0.75 0.8 0.8 0.8 0.9 0.9 0.9 0.9
0 3 8 1 5 8 9

These difficulties are overcome by the Lineweaver-Burk method of

determining Km and Vmax, which transforms the Michaelis-Menten equation
into a linear form by taking the reciprocal of Equation 15:
1 K m [S ]

v0 Vmax [ S ] , (20)
1 Km [S ]

v 0 Vmax [ S ] Vmax [ S ] , (21)
1 Km 1

and v0 Vmax [ S ] Vmax . (22)

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Equation 22 is called the Lineweaver-Burk
equation. A plot of 1/ v0 versus 1/[S] will
be linear with a slope of Km/Vmax. The
intercept of 1/ v0 ordinate equals 1/Vmax
while the intercept on the 1/[S] abscissa
equals 1/Km. A Lineweaver-Burk plot is
shown in Figure 5. Notice that in
extrapolating to the 1/v0 ordinate you are
extrapolating to infinite substrate
concentration (where 1/[S] = 0).

Other similar analysis techniques are

Eadie-Hofstee and Hanes-Woolf [5]. For Figure 5: A Lineweaver-Burk plot [1]
Eadie-Hofstee analysis, the Michaelis-
Menten equation is rearranged to Equation 23, and vo/[S] is plotted versus vo.
In an Eadie-Hofstee plot, the slope is -1/Km, and the x-intercept is Vmax.
vo Vmax v
o
[S ] Km Km . (23)
For Hanes-Woolf analysis, the Michaelis-Menten equation is rearranged to
Equation 24, and [S]/vo is plotted versus [S]. In a Hanes-Woolf plot, the x-
intercept is Km, and the slope is 1/Vmax.
[S ] [S ] K
m
vo Vmax Vmax . (24)
The Michaelis-Menten constant and Vmax can also be found through non-linear
regression.

In virtually any cell extract or protein preparation, individual enzymes will be

present in such trace amounts that the determination of the concentration of
an enzyme by a direct chemical or physical test is next to impossible. As a
result, the amount of an enzyme present is determined in terms of its
catalytic activity. As we have seen, the concentration of an enzyme in a
reaction mixture is directly proportional to the limiting velocity of the
enzyme-catalyzed reaction.

Relative measurements of the enzyme concentration are therefore made by

assaying under identical conditions as near saturating substrate levels as
possible. If a sufficient degree of saturation cannot be achieved
experimentally, limiting velocities may be determined by assaying individual
enzyme solutions at different substrate concentrations and obtaining Vmax
from a Lineweaver-Burk plot. The catalytic activity of an enzyme is normally
expressed in enzyme units. A unit of enzyme activity is defined as that
amount of enzyme causing the transformation of one micromole (10 6 mole)
of substrate per minute at 25C under optimal conditions of measurement.
In the isolation of an enzyme, it is often desirable, if not necessary, to

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determine the degree of purity achieved by each purification step. A
measure of the purity of an enzyme preparation is the specific activity, which
may be defined as the number of enzyme units per milligram of protein. As
undesired proteins are selectively removed at each purification step, the
specific activity increases. When purity is achieved the specific activity
reaches a maximal, constant value.

The enzyme -galactosidase of E. coli K12 is an inducible enzyme. Normally

present in only trace amounts, large-scale synthesis of this enzyme occurs
only when its substrate is present in significant quantities in the organisms
environment. For example, when E. coli K12 is grown with glucose as the
major carbon source, the intracellular levels of -galactosidase will be very
low. However, when this organism is grown with a -galactoside, such as
lactose, as the principle carbon source, the intracellular concentration of -
galactosidase will be quite high. In this experiment you will investigate the
reaction kinetics of a -galactosidase-catalyzed reaction.

Experimental Considerations: The compound o-nitrophenyl--

galactopyranoside (ONPG) can act as a substrate (reactant) for -
galactosidase (catalyst or enzyme). The products of the reaction are one
molecule of galactose and one molecule of o-nitrophenol (ONP) per molecule
of ONPG hydrolyzed. When the reaction mixture is buffered to a slightly
alkaline pH (7.7), the ONP once formed instantly ionizes, producing a highly
colored species that absorbs strongly at 420 nm. -galactosidase can
therefore be conveniently assayed under these conditions by a continuous or
kinetic assay following the production of product (ONP) with time, by
spectrophotometric measurements. The initial velocities derived directly
from this spectrophotometric assay will have units of OD/unit time, change
in optical density or absorbance per unit time (usually minutes). More
meaningful units such as moles substrate hydrolyzed per minute per ml are
generally desired. The optical density or absorbance of the assay solution at
any time will be directly proportional to the concentration of product, which
in this case is numerically equal to the concentration of substrate
hydrolyzed. Therefore, to convert units of OD/min to units of moles
substrate hydrolyzed/ml-min the absorptivity of ONP must first be
determined. This may be accomplished very easily by allowing an assay in
which the initial concentration of ONPG is known to go to completion and
measuring the final OD or absorbance. At completion all of the ONPG will
have been hydrolyzed, therefore the final concentration of ONP equals the
initial concentration of ONPG. From this data the absorptivity of ONP may be
calculated from Beers law as shown in Equation 25,
ODc

Ci L , (25)
where

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= absorptivity of ONP
ODc = the optical density at completion
Ci = initial concentration of ONPG = the concentration of ONP at
completion
(in moles/ml), and
L = path length (1 cm).

Once the absorptivity of ONP has been calculated, OD may be interpreted as

moles product formed/ml (or moles substrate hydrolyzed/ml) with
Equation 26,
OD
C
L , (26)
which is a transformation of the Beers law equation.

Example:
To illustrate the mechanics of these calculations lets consider some
hypothetical data. Suppose that in a particular spectrophotometric assay for
-galactosidase, the concentration of substrate (ONPG) was 10 moles/ml
and the initial velocity was determined to be 0.05 OD/min. And lets say
that when this assay was allowed to go to completion, the final OD was 2.0.
The problem is to convert the velocity units of OD/min to units of moles
substrate hydrolyzed/ml-min. The first step is to calculate the absorptivity of
ONP, the product, using Equation 25 as shown below:
[ONPG ]initial [ONP ] final 10
mole/ml.
Therefore
2
0 .2
(1 cm)(10 moles / ml ) ml/molecm.
Using Equation 26
OD / min
C / min
L ,
or
0.05 OD / min
C / min
(0.2 ml / moles cm)(1 cm) .
Therefore the initial velocity equals 0.25 mole product formed/ml-min or
0.25 moles substrate hydrolyzed/mlmin.

Procedure:
Preparation of solutions:
80 mM Phosphate Buffer (this is normally prepared by the teaching
assistant):
Add 10.09 g Na2HPO4 (dibasic sodium phosphate) and 1.074 g NaH2PO4

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(monobasic sodium phosphate) to a 1-L volumetric flask and add distilled
water to the 1-L mark.

1.50 mM ONPG Solution:

The ONPG is stored in the freezer chest in the SW corner of the lab. Let the
ONPG warm up to room temperature before opening the container. Add 45.2
mg ONPG to a 100 ml volumetric flask and dilute with 80 mM Phosphate
Buffer to the 100 ml mark.

Enzyme Solution:
The enzyme is also stored in the freezer. Let the enzyme warm up to room
temperature before opening the container. Accurately weight about 50 mg
of enzyme (approximately the amount of enzyme that fits on the tip of a
spatula) and dilute it with 50 ml (accurately measured) of the 80 mM
Phosphate Buffer.

Experiment:
1. Set the spectrophotometer to 420 nm and turn it on.
2. Prepare a set of solutions by mixing the chemicals as suggested in Table
4. Use pipettes. Make a set of cuvettes for each temperature in your
study.

Table 4. Cuvette solution volumes

Cuvette Number 0 1 2 3 4 5 6 7 8 9
Volume of 80 mM
phosphate buffer 3.0 2.8 2.6 2.4 2.2 2.0 1.5 1.0 0.0
Replicate
(pH 7.7), ml
of 1-8
Volume of 1.50 mM
0.0 0.2 0.4 0.6 0.8 1.0 1.5 2.0 3.0
ONPG, ml

3. Place one set of filled cuvettes in the water bath set to the elevated
temperature for your literature values. Also divide the diluted enzyme
and heat half of it. Keep the other set at room temperature. Record the
temperatures.
4. Using the enzyme at the same temperature as cuvette 0, add 1.0 ml of
the enzyme solution to the cuvette. Zero the spectrophotometer using
this cuvette.
5. Add 1.0 ml of same-temperature enzyme to one of cuvettes 1 - 9. Start
the stopwatch as soon as you add the enzyme. Mix the cuvette by
inverting and immediately insert it in the spectrophotometer chamber.
6. Measure the absorbance at an interval of 15 seconds for about 5 to 6
minutes. You must start making readings IMMEDIATELY! Do not discard
the solutions. Return the solutions to the water bath or counter, as
appropriate.
7. Check the zero of the spectrophotometer again using cuvette 0.
8. Repeat Steps 5 through 7 for the rest of the cuvettes of that temperature.
9. Repeat Steps 4 through 8 for the cuvettes at the other temperature.

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10. Allow all cuvettes from each set to stand at their appropriate
temperatures for ~1 hour (when the reaction is assumed to be complete).
Measure the absorbance of each assay, if it is still within the range of the
instrument.
11. Clean up. Dispose of solutions down the drain with plenty of water.
Throw cuvettes and pipet tips in the trash. Put away dry glassware. Wash
glassware and hang it up to dry. Turn off the spectrophotometer and
water bath.

Prelab Report Calculations:

Find literature values of the Michealis-Menten constant and the maximum
velocity of our enzyme, which is derived from Aspergillus oryzae. At what
temperature(s) can you find data?

Many of the compounds in this experiment are not in the Chemical Reactivity
Worksheet database. For those that are not in the database, check the SDS
for reactivity hazards and report.

Do Suggested Calculations 1 6 and 8 below for the data in Table 5. Error

analysis is not required for the prelab report.

Table 5. Optical densities for ONPG decomposition

by -galactosidase

Time,
Cuvette 4 Cuvette 7
Min:sec
Cuvette 1
0:15 0.014 0.048 0.064
0:30 0.024 0.094 0.127
0:45 0.035 0.139 0.190
1:00 0.041 0.190 0.246
1:15 0.052 0.225 0.305
1:30 0.061 0.270 0.357
1:45 0.070 0.320 0.408
2:00 0.078 0.355 0.467
2:15 0.085 0.397 0.518
2:30 0.093 0.438 0.573
2:45 0.102 0.480 0.623
3:00 0.109 0.516 0.679
3:15 0.115 0.555 0.728
3:30 0.126 0.597 0.774
3:45 0.135 0.631 0.834
4:00 0.139 0.667 0.876
4:15 0.145 0.709 0.915
4:30 0.151 0.744 0.960
4:45 0.160 0.778 1.015
5:00 0.164 0.809 1.061
2+ (off-
60:00
0.425 1.734 scale)

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Prelab Quiz: You will describe the procedure to the TA, without using a
handout.

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Suggested Calculations and Report Requirements:
1. Calculate the absorptivity for each cuvette using equation (25), where
the OD is the absorbance measured in step 10 of the procedure above.
2. Convert all OD units to concentration units using equation (26) and the
absorptivity calculated above for the two sets of experiments. Should you
use a different value for each cuvette, or an average for each set, or an
average overall?
3. Calculate the initial velocities (v0) by plotting these concentrations versus
time and finding the slope of the curve at t = 0.
4. Make the Lineweaver-Burke graph by plotting 1/v0 vs. 1/[S] for each set,
where [S] is the substrate (ONPG) concentration. This plot is a straight
line and is represented by equation (22). Therefore calculate Vmax and Km
by finding the slope and the intercept of the line.
5. Plot v0 vs. [S] and estimate Vmax and Km using a non-linear least squares
fitting of the data to equation (15). [Create cells that contain guesses for
Vmax and Km. Calculate, for each experimental data point, the right-hand
side of equation (15) based on these guesses. Calculate the square of the
difference between the right-hand and left-hand sides of equation (15) for
each experimental data point. Add all of the squares of the difference up.
Use Solver to minimize the sum of the squares by changing the guesses
for Vmax and Km.]
6. Similarly to #4, analyze your data at both temperatures with the Eadie-
Hofstee and Hanes-Woolf methods.
7. Estimate errors for all of your Vmax and Km except those found by non-
linear least squares regression.
8. Make a plot at each temperature for v0 vs. [S] that has your experimental
data and four theoretical models. Compare your results (by all methods)
for room temperature and for your other temperature, and explain the
difference.

References and Further Reading:

(1) Lehninger, A. L., Biochemistry. Worth Publishers, Inc., N.Y. (1970).
Chapter 8.

(2) Price, N.C., R.A. Dwek, R.G. Ratcliffe, and M.R. Wormald, Principles and
Problems in Physical Chemistry for Biochemists, 3rd Ed. Oxford, Oxford
(2001). Chapters 9 and 11.

(3)Bohinski, R.C., Modern Concepts in Biochemistry. Allyn and Bacon, Boston

(1973). Chapter 5.

(4) Morris, J. G., A Biologists Physical Chemistry. Addison-Wesley, London

(1968). Chapter 11.

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(5) McGilvery, R. W., Biochemistry. W. B. Saunders, Philadelphia (1970).
Chapters 6 and 7.

Enzyme Kinetics Page 16