Biology

3601 Biochemistry
Enzyme Kinetics Laboratory

Background

Enzymes, which are biological catalysts, are the machines of the cell, catalyzing chemical
reactions that are necessary to sustain metabolism. In the absence of enzymes, most
physiological chemical reactions would proceed at a rate too slow to sustain life. To
understand how enzymes contribute to physiological function, it is useful to consider the
basic thermodynamics and kinetics of chemical reactions.

Whether a chemical reaction can proceed as written depends upon the relative free energy
between the products (P) and the reactants (which we will call substrates, S). When the
free energy difference (G) between reactants and products is negative, the reaction is
called spontaneous. However, while thermodynamics can predict whether a reaction can
happen, it does not predict the rate (timeframe) at which the reaction will occur. The
actual rate of a chemical reaction depends on the free energy of the highest energy
structure on the transition between substrate and product, known as the transition state. A
thermodynamically spontaneously reaction can proceed slowly, or not at all, if the free
energy of the transition state is higher than the available thermal energy. Catalysts, such as
enzymes, work by stabilizing the transition state structure, and, as a result, lowering the
transition state free energy of a chemical reaction, allowing an otherwise slow reaction to
proceed more rapidly. To understand the mechanism by which enzymes enhance the rate
of otherwise slow chemical reactions, one needs to employ kinetics, which studies the rate
at which chemical reactions occur.

The conversion of substrate to product is given by the equation:

E + S ! E + P

[1]

The Law of Mass Action states that there is a direct relationship between the rate of a
chemical reaction and the concentration of the reactants. However, experimental
investigations of enzyme kinetics reveal that above a certain concentration of substrate,
(which differs between different enzymes) there is no further enhancement of the rate
when increasing the substrate concentration. This is explained by proposing that the
enzyme must first (reversibly) form an enzyme-substrate complex (ES), which may either
break down to enzymes and substrates, or proceed (via catalysis) to enzymes plus product.
Above a critical concentration, all enzymes are already occupied by substrate such that
there are no available free enzymes to bind additional substrate. This can be written in a
kinetic rate model as:

[2]


Michealis and Menton proposed a rate equation that described the kinetic model shown in
[2]. To derive this equation, they made three key assumptions: (1) There is an equilibrium,
or steady state, between Enzyme and substrate, and the ES complex and (2) the reaction is
observed for a short period of time after enzyme and substrate are mixed, before product
can accumulate and the reverse reaction (k-2) of E + P is significant (k-2 can be ignored).
Under these circumstances, they found that

vo = k2[Et][S]/[S] + (k2 + k1/k-1)

[3]

where:

vo is the observed rate at a given concentration of added enzyme and substrate.

It can be shown that the term k2[Et] corresponds to the maximum rate the enzyme can
convert substrate to product; accordingly, this term is called Vmax. The kinetic term (k2 +
k1/k-1) has units of concentration; this term, written as Km, corresponds to the
concentration at which the enzyme is converting substrate to product at Vmax/2. More
generally, Km is similar to a binding constant; it governs what fraction of enzyme active
sites are occupied by substrate at any given substrate concentration. The smaller the value
of Km, the less substrate required for the enzyme to function at its maximal rate. These two
terms, Vmax, and Km are the parameters that are typically used to define enzyme function.
The equation defined by Michealis and Menton equation may then be written:

vo = Vmax[S]/([S] + Km).

[4]

To find the values of Vmax and Km, it is necessary to find the initial rate of enzyme function
as a variety of substrate concentrations. Each of these plots (time, x-axis) vs. product (y-
axis) is known as a progress curve. The slope of the initial rate (P/dt) is equal to the rate
at a particular concentration of substrate. Note that the slope must be obtained only for the
initial portion of the progress curve, where the product accumulation appears to be linear.
Only in this region is the Michaelis-Menton equation valid.

From all of the progress curves, it is possible to construct a plot of substrate concentration
(x-axis) vs. the rate of the reaction (y-axis). For many enzymes, the resulting plot is a
hyperbola. The curve approaches the asymptote Vmax, and Km is the concentration where v
= Vmax/2.

In principle, these key parameters can be extracted directly from the Michaelis-Menton
plot. In practice, this is not feasible. Progress curves are rarely obtained at the high
concentration of substrate where the rate approaches Vmax. As a result, Vmax, and Km, are
not well defined on the M-M plot. However, there are superior graphical methods for
obtaining the desired information. For example, Lineweaver and Burke showed that the
inverse of both sides of the M-M could be written as:





1/vo = ((Km)/(Vmax))(1/[S]) + (1/Vmax).
[5]


This is the equation of a straight line (y = mx + b) with m = ((Km)/(Vmax)) and b = (1/Vmax).
In the linearized formulation of the equation, on a plot of 1/[S] (x-axis) vs. 1/vo (y-axis) the
x- intercept is -1/Km and the y-intercept is 1/Vmax.

The enzyme we will study in this laboratory exercise is cellobioase, an enzyme found in
organisms, such as fungi (in particular mushrooms) that digest cellulose (from plant cell
walls) into free glucose molecules for food. Organisms that rely on cellulose as an energy
source make a number of cellulose digesting enzymes, called cellulases, which catalyze the
hydrolysis of the 1,4--D-glycosidic bond in cellulose. Each cellulase has a unique substrate
specificity, and function in the cellulose digestion process. Endocellulases digest the within
the long cellulose chains, breaking the chains into small pieces and increasing the number
of free ends. Exocellulase digest at the ends of cellulose chains, releasing the disaccharide
cellobiase, two glucose monomers connected by a (1!4) bond. This disaccharide is the
substrate from cellobioase, which catalyzes the final step in cellulose digestion, the
liberation of free glucose monomers.

Like most enzymes, the substrates and products of cellobioase are colorless. This creates a
challenge in following the breakdown of cellobiose, or the release of free glucose. To
circumvent this limitation, we will use a colorimetric substrate, p-Nitrophenyl
glucopyranoside to detect the formation of product. This substrate is a glycoside formed
from glucose and p-nitrophenol. When cleaved by the enzyme, the substrate produces a
1:1 ratio of glucose and produces p-nitrophenol. At high pH, p-nitrophenol forms the
yellow nitrophenolate anion.

The digestion reaction can be terminated by elevating the pH of the reaction; this is
accomplished by the addition of a high pH stop solution to each reaction. The amount of
nitrophenolate anion present at each time point can then be obtained by first creating a
standard curve of nitrophenolate concentration vs. absorbance. The amount of product
produced at each time, for each substrate concentration, can then be readily determined by
measuring the absorbance of the solution, and using the standard curve to determine the
amount of nitrophenolate present. From this data, it is possible to construct a M-M curve,
and a L-B plot, from which the critical enzyme kinetic parameters can be obtained.

Materials
1.5 mM p-Nitrophenyl glucopyranoside (substrate)
cellobioase enzyme
p-Nitrophenolate standards (0 nmol, 12.5 nmol, 25 nmol, 50 nmol, 100 nmol)
cuvettes
buffer solution
stop solution
15 ml conical tubes

Procedure

1. Label 8 15 ml conical tubes with the starting substrate concentrations from Table I.

2. Transfer the appropriate amount of substrate into each tube, according to Table I.

3. Transfer the appropriate amount of buffer into each tube, according to Table I.

4. Label each cuvette with the concentration, and the times, according to Table II.

5. Transfer 500 l of stop solution into each cuvette.

6. Pipette 750 l of enzyme into each substrate tube, and start the timer.

7. At the times indicated in Table II, remove 500 l from each tube and transfer it to the
appropriate cuvette that contains the stop solution.

8. Load the Simple Reads program on the spectrophotometer. Set the wavelength to
410nm, the max of the nitrophenolate anion.


9. Measure the absorption values for the standards S1-S5, and record the values in Table
III.

10. Measure the absorption values for each experiment and record the values in Table I.

Table I: Preparation of Substrate

Experiment
#

Starting
Substrate
Concentration

1
2
3
4
5
6
7
8

1.5 mM
1.0 mM
0.5 mM
.250 mM
.125 mM
.060 mM
.030 mM
.015 mM

Actual Substrate
Concentration*

Enzyme
Added
(ml)

Buffer
Added
(ml)

1.5
1.0
.50
.250
.120
.060
.030
.015

0
0.5
1.0
1.25
1.38
1.44
1.47
1.45

* calculated by taking the starting substrate concentration into the final volume of each reaction.

Table II: Absorption Data for Progress Curves**

Starting
Substrate
Concentration
1.5 mM
1.0 mM
0.50 mM
0.250 mM
.125 mM
0.60 mM
.030 mM
0.15 mM

Absorbance
t = 1
minute

Absorbance
t= 2
minutes

Absorbance
t = 4
minutes

Absorbance
t = 8
minutes

** measured at 410 nm.

Table III: p-Nitrophenolate Absorption vs. Concentration

Standard
Amount of p-
Absorbance
Nitrophenol (nmol)
(410 nm)
S1
0

S2
12.5

S3
25

S4
50

S5
100