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Enzyme Kinetics
INTRODUCTION

Kinetics is the study of the rates of chemical reactions. For any reacting system,
thermodynamics can be used to predict whether the reaction will spontaneously occur.
The kinetics of the reaction indicate how fast the reaction actually goes. Most of the
biological reactions that occur in the cells of living organisms are greatly sped up by
protein catalysts called enzymes. Recall that catalysts dramatically increase the rate of a
reaction without affecting the equilibrium.

The objectives for this exercise on enzyme kinetics are to:

• Comprehend how enzyme kinetics relates to the chemical kinetics presented in

general chemistry courses.
• Perceive how kinetic parameters are experimentally determined.
• Learn the Michaelis–Menten equation and the meaning of KM and Vmax.

ELEMENTARY KINETICS

Our first objective for this exercise is to show how enzyme kinetics relates to the
elementary kinetics you studied in General Chemistry.

Let’s begin with a simple reaction, a unimolecular reaction that can be described as A
being converted into B.

Recall that since there is only one reactant, this is a first order reaction whose rate, or
velocity, can be described as the disappearance of the reactant over time or the
appearance of the product over time. The reaction rate is represented by the slopes of
these lines and is not constant.

For this first-order reaction, the rate is directly proportional to the concentration of reactant.
Here k is the first-order rate constant for the reaction. Although the rate constant does not
change, the velocity of the reaction begins to decrease as the concentration of the reactant
diminishes and the product accumulates. The rate of the reaction eventually slows to zero
as the product of the initial forward reaction becomes a reactant for the reverse reaction.
Equilibrium is the point where the forward and reverse reaction rates are equal, and the
overall rate of the reaction is zero.

In a simple enzyme-catalyzed reaction, the reactants are labeled S for substrates and
products are labeled P. This looks like our first example, a simple first-order reaction in
which substrate is converted to product. Similarly, the rate of an enzyme-catalyzed
reaction is either the disappearance of the substrate or the formation of the product over
time. As the reaction proceeds, the reverse reaction begins to compete with the forward
reaction until the rates are equal, and equilibrium has been reached.

The rate equation for an enzyme-catalyzed reaction with a single substrate is the same as
the rate equation for the simple nonenzymatic reaction, but only when the concentration of
substrate is so low that the enzyme has very little chance to convert it to product. Under
these unique conditions, it appears that the reaction is first order with respect to substrate.
However, this special case is much too limiting and rarely applies in biological and
experimental systems. Consequently, biochemists must use a different model to describe
the kinetics of biological reactions catalyzed by enzymes.

ENZYME KINETICS

The kinetics of an enzyme-catalyzed reaction can be studied when the concentration of

the enzyme is small compared to the concentration of the substrate. As long as the
substrate is in large excess over enzyme, altering its concentration does not change the
rate. This is quite different from the first-order reaction in the previous example, where the
rate depended on the substrate concentration. Here, the rate does not depend on the
substrate concentration, and the reaction is said to be zero order with respect to substrate.

Near the turn of the 20th century, Adrian Brown first observed this conflicting behavior of
enzyme-catalyzed reactions. It became apparent to him that the substrate must form a
complex with the enzyme. This observation marked the beginning of the study of enzyme
kinetics. Brown correctly deduced that a zero-order reaction with respect to substrate
occurred when the enzyme was saturated with substrate. Essentially, the limited number
of active sites put a ceiling on how fast the reaction could proceed. Because the enzyme
concentration is rate limiting, the rate is independent of substrate concentration. The zero-
order rate constant is referred to as the maximal velocity, or Vmax.

To account for this kinetic behavior caused by the substrate’s interaction with enzyme, the
model for an enzyme-catalyzed reaction must include an enzyme-substrate complex, or
ES complex. The reaction scheme is shown here. The rate constants k1 and k–1 are the
forward and reverse rate constants for the formation of the ES complex, and k2 is the rate
at which the ES complex converts the bound substrate into product. Notice that in order to
simplify this model, we assume that the product formation step is irreversible (that is, there
is no k–2) - the reverse reaction is zero.

Catalysts are generally not considered reactants because they facilitate reactions without
being altered. However, in an enzyme-catalyzed reaction, the enzyme does participate as
a “pseudo-reactant” because it must bind and form a complex with substrate, and the
availability of its active sites affects the overall rate of the reaction.

MICHAELIS–MENTEN EQUATION

We have seen that for an enzyme-catalyzed reaction, we can write a simple rate equation
only when the concentration of substrate is saturating or when the concentration of
enzyme is saturating. In between these two extremes, we need to use a slightly more
complex rate equation called the Michaelis–Menten equation.

The Michaelis–Menten equation is limited to the initial rate v0 so that we can ignore the
possibility of the product accumulating and undergoing the reverse reaction. The
Michaelis–Menten equation and its hyperbolic graph describe how the reaction rate varies
with substrate concentration for a one-substrate reaction. It is important to note that
despite the similarities in shape, this graph is NOT a graph of the concentration of product
forming over time.
This graph shows the reaction progress of an enzyme-catalyzed reaction. The Michaelis–
Menten model makes a steady-state assumption, meaning that the concentration of the
ES complex remains constant. This assumption is valid only when the concentration of
substrate is much greater than the total enzyme concentration. Use your mouse to
investigate the various parts of the graph.

KINETIC PARAMETERS

Vmax is the maximal velocity that can be achieved by an enzyme under the special case of
saturating substrate concentration. KM is a “lumped” rate constant incorporating all the rate
constants for ES and P formation: k1, k–1, and k2. It is equal to the substrate concentration
that gives 1/2-saturation of the enzyme. It is therefore also equal to the substrate
concentration at one-half the Vmax.

For a reaction that follows the Michaelis–Menten kinetic model, the KM is inversely related
to the proportion of total enzyme (Et) which is in complex with substrate. In other words,
the higher the KM, the lower the fraction of enzyme with a bound substrate. Although
loosely related, the KM is not the same as the enzyme’s affinity for substrate, for it also
takes in to account the decomposition of the ES complex to product. The most useful
definition for KM is that it is a constant that describes the dependence of v0 on S, and
dictates the steepness of the shape of the Michaelis–Menten curve.

Experiment with the sliders to change Vmax and KM. Note how the shape of the line
changes with KM.

A KINETIC EXPERIMENT

To help you understand the hyperbolic relationship between the substrate concentration
and the rate of an enzyme-catalyzed reaction which is described by the Michaelis–Menten
equation, we will now demonstrate a kinetic experiment.

To experimentally determine the relationship between substrate concentration and initial

velocity, a biochemist sets up a series of test tubes for the reaction. Each tube contains a
constant amount of enzyme. But the amount of the substrate placed in each tube varies.
For easy analysis, the biochemist often chooses a substrate that yields a product that has
a different color or different fluorescence. In order to measure the initial velocity, the
reaction is only run for a short length of time. This is done so that only a small percentage
of substrate is converted into product, and therefore there will be no kinetic contribution
from the reverse reaction of product formation, and k–2 can still be ignored.

The amount of product formed over time is monitored. The amount of product formed
divided by the brief time of incubation is the slope of the early linear phase of the reaction.
This rate of product formation is the initial velocity of the reaction. Note that the tubes
containing the most substrate yield the highest initial velocities.

A plot of initial velocity versus substrate concentration yields a hyperbolic curve consistent
with the Michaelis–Menten model of enzyme activity. The important kinetic parameters
Vmax and KM can be estimated from this graph. The Vmax is the point where the enzyme is
fully saturated and cannot achieve a higher initial velocity Vmax and is therefore the upper
limit for v0. The KM is the substrate concentration that corresponds to 1/2 of the Vmax. More
precise values for Vmax and KM can be obtained by using a curve-fitting program.
Remember that the Michaelis–Menten equation is a mathematical expression for a
hyperbola.

VIRTUAL ENZYME KINETICS

Now imagine that you are a biochemist attempting to characterize the kinetic behavior of a
new mutant of the motor protein kinesin You will need to set up reaction tubes and collect
data to determine the values for Vmax and KM. You will be monitoring the ATPase activity of
the kinesin by quantifying the conversion of radiolabeled ATP to ADP.

Plan your experiment by indicating the proper order of the steps.

First, prepare six 1 milliliter reaction samples. Use the micropipettor to add samples drop-
wise to the tubes. Be careful: sometimes the order of addition matters. Be sure to leave
room in the tube for substrate, and do not add substrate to any tube until all samples are
ready. When all the samples are prepared, add substrate to each tube and start the timer.

Now that you are ready to start the reactions, add substrate and start the timer. After the
desired incubation time, quench the reactions with detergent to inactivate the enzyme.

You now need to determine the amount of product formed in each reaction tube. You will
use thin layer chromatography to separate the ADP product from the leftover ATP
substrate and quantify the radiolabeled ADP by phosphoimaging and densitometry. The
values obtained are now added to the table.

From the experimental data, calculate the initial velocities and enter the values into the last
row of the table.

Now use your data to construct a Michaelis–Menten plot. First, select the proper axis
labels, then add your x and y data to plot a curve.

While you could roughly estimate Vmax and KM by eye or achieve more precise values by
fitting the data computationally, it is instructive to do the curve fitting by hand. Use the
sliders for Vmax and KM to create a curve that is computed from the Michaelis–Menten
equation and that fits your data. Note how changing each kinetic parameter affects the
shape of the curve. Manipulate the parameters until you are satisfied with the fit, and then
press "Accept fit."

From your data you have obtained values for Vmax and KM. You can now use these kinetic
parameters to compare the ATPase activities of your kinesin mutant and wild-type kinesin,
in order to determine the effect of the mutation. This information, coupled with the location
of the mutation in the three-dimensional structure of kinesin, could lead to new insights into
the mechanism of this motor protein.

LINEWEAVER-BURK PLOTS

While computers can easily determine kinetic parameters from a hyperbolic Michaelis–
Menten plot, these graphs are unwieldy for visually estimating values for Vmax and KM. For
this purpose, the Michaelis–Menten equation can be rearranged to a linear equation with
the form y = mx + b. The resulting plot is called a Lineweaver-Burk or double-reciprocal
plot.
KM and Vmax can be readily obtained from the x and y intercepts of a Lineweaver-Burk plot.
The y-intercept is 1/Vmax and the x-intercept is –1/KM. Try varying the Vmax and KM on the
following graphs.

CONCLUSION

Chemical kinetics and enzyme kinetics are related, but because of the complex of enzyme
with bound substrate, enzyme kinetics only correlates well with chemical kinetics in the
extreme cases with very limited substrate or vast excess of substrate.

Michaelis–Menten enzyme kinetics describes the behavior of enzymes over a wide range
of substrate concentrations. Two important kinetic parameters are Vmax and KM.

Vmax is the maximal velocity of an enzyme-catalyzed reaction at saturating substrate

concentration. KM is loosely related to substrate affinity, but a more correct description is
that it determines the shape of the Michaelis–Menten hyperbolic curve.

Kinetic experiments can quantify how the rate of an enzyme-catalyzed reaction changes
with the concentration of substrate. Data obtained can be plotted and fit to the Michaelis–
Menten equation or rearranged into a double-reciprocal plot. From these graphs, the
kinetic parameters KM and Vmax can be obtained.