Enzyme Kinetics
Enzyme Kinetics
Enzyme Kinetics
I. INTRODUCTION
The activity of enzymes is important for the proper functioning of cells. In the context of
energy flow in living organisms, enzymes catalyze most reactions in metabolic pathways. Not
only do enzymes make most reactions possible in an intracellular environment, enzymes allow
for the control and stabilization of these reactions. It is not a stretch to say that without enzymes
life itself would be impossible.
The behavior of enzymes in response to different concentrations of the reaction chemicals
(both substrates and products) comprise the basic characteristics of each type of enzyme. This
behavior, referred to as enzyme kinetics, is responsible for much of the reaction control in
biological systems. In order to learn about enzymes and enzyme behavior, in this lab we will
examine the kinetics of the enzyme alkaline phosphatase.
O
-
H2O
substrate
alcohol
phosphate
Interestingly, the precise biological function of the enzyme is unknown. Clinical interest in
alkaline phosphatase was inspired by the observation that certain pathological conditions, such
as obstructive jaundice, rickets and other bone disorders were characterized by large increases in
the plasma concentration of the enzyme. It is particularly abundant in tissues that are involved in
the transport of nutrients. The fact that it is localized at the surface of absorptive tissues suggests
a role in the transport of nutrients across the epithelial membrane. If rats are maintained on a
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high fat diet, there is an increase in the amount of intestinal alkaline phosphatase, indicating a
role in the transport/processing of the fats (Young et al., 1981). Another hypothesized function
for intestinal alkaline phosphatase is protection from bacterial infection, whereby alkaline
phosphatase removes phosphate groups from endotoxin, a lipopolysaccharide (lipidcarbohydrate) molecule that makes up the cell wall of some types of bacteria (Poelstra et al,
1997; Kopojos et al. 2003; Verweij et al. 2004). Endotoxin induces an inflammatory response in
the host that can be fatal (septic shock), but there is no toxic response when the phosphate groups
have been removed from the endotoxin.
In this laboratory, we will be using alkaline phosphatase from bovine (cow) intestine. This
particular alkaline phosphatase has a molecular weight of 138,000 daltons (a dalton is a unit of
mass approximately equal to the mass of a hydrogen atom). It is a dimer (i.e., has two subunits),
so the molecular weight of each monomer is 138,000/2 or 69,000 daltons. The enzyme requires
++
++
4 atoms of Zn per dimer for activity. The Zn atoms are said to be cofactors for the enzyme,
meaning that they are necessary for the enzyme to be active. An enzyme with its cofactors is
called a holoenzyme; without the cofactors, it is an apoenzyme.
apoenzyme + cofactors = holoenzyme
Typically, apoenzymes are not active, so when we refer to enzymes, we usually mean
holoenzymes.
O-
H20
O-
p-Nitrophenol Phosphate
O
2 0N
OH
O-
O-
p-Nitrophenol
(yellow)
Phosphate
Substrates that change color when acted upon by an enzyme are called chromogenic substrates
and these make studying enzymes significantly easier. P-nitrophenol absorbs light at 410 nm, so
we can follow the rate of the reaction by following the increase in absorption at 410 nm by using
a spectrophotometer.
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A410
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o
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o
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o
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0
1
2
Time (minutes)
The initial linear rate of product formation is called the initial velocity, or vo. It is one of the
most important characteristics of any enzyme-catalyzed reaction. One factor that affects the
initial velocity is the substrate concentration, [S]. You will look at the relationship between vo
and [S] in lab this week.
Vmax
Velocity
(vo)
Km
Substrate Concentration
[S]
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As [S] increases, vo eventually becomes independent of [S] (why?). The velocity at which this
occurs is called Vmax, and it is the fastest that the given amount of enzyme can operate. The [S]
that yields 1/2 Vmax is another important kinetic parameter called the Michaelis-Menten constant,
designated Km. Km is important in that it indicates the [S] at which the enzyme is most effective
at altering the rate of the reaction. (Make sure that you understand why this is so.) Enzymes are
tightly regulated to maintain homeostasis, and one handy mechanism for regulation is having
your enzyme activity change based on substrate availability (Is more substrate around? - increase
the rate at which the enzyme works to compensate). In fact, it is frequently found that [S] in vivo
is near the Km for an enzyme. You can think of this as a cell exploiting the Michaelis-Menten
character of an enzyme. Furthermore, Km is important in understanding many other kinetic
parameters of enzyme activity that we will not discuss here due to National Security
Considerations. Km and Vmax are characteristics of a reaction that help characterize the enzyme
in question. (How would Km and Vmax change if you increased the amount of enzyme?)
To determine Km and Vmax, we could determine a set of v0 values at various concentrations of
S, make the graph above and read off the values. As you can imagine, this would not be very
accurate since it is difficult to obtain an accurate value for a number that is approached
asymptotically. The equation that describes the above Michaelis-Menten curve is the following:
v0 =
Vmax [ S ]
[S ] + K m
Michaelis-Menten Equation
This is called the Michaelis-Menten equation. Two algebraically inclined fellows by the
names of Lineweaver and Burke manipulated the Michaelis-Menten equation to yield the
following:
Km
1
1
=
+
v0
Vmax
Vmax [ S ]
Lineweaver-Burke Equation
(Hang in there - we are almost done.) If you plot 1/ vo vs. 1/[S], you get the following
Lineweaver-Burke plot or double-reciprocal plot:
1/v0
1/Vmax
1/[S]
-1/Km
The slope of the line is Km / Vmax, the y-intercept is 1/ Vmax and, if we extrapolate the line (i.e.,
set y = 1/v0 = 0), the x-intercept is -1/ Km. The use of the double reciprocal plot yields much
more accurate values for Km and Vmax than an interpretation of the Michaelis-Menten curve. In
this week's lab, we will determine Km and Vmax for the enzyme alkaline phosphatase.
v0 =
(0.375 - 0)
(0.75 - 0)
= 0.5/min
However, it is more useful to express the rate in terms of the actual amount of p-nitrophenol
formed per unit time; this allows researchers working under different experimental conditions to
compare their results. This may be given as nanomoles per minute (nmol/min) or micromoles
per minute (mol/min). The absorbance value at 410 nm can be converted to an actual
concentration using the Beer-Lambert law described in last week's lab. After a little
rearrangement:
c =
A
l
Remember that c is the concentration of the absorbing material (p-nitrophenol, in our case), A
is the absorbance measured at 410nm, and l is the length of the light path (1.0 cm for our
spectrophotometers). The extinction coefficient, , for p-nitrophenol at 410 nm is
18.5 mM-1cm-1. Therefore, using this value and the Beer-Lambert equation, you can convert an
absorbance reading into an actual amount of product formed.
Let us work through an example. Suppose that the slope of your line was 0.5/min, as described
above.
0.5 / min
Concentration = c =
(18.5 mM cm ) 1.0 cm
0.027 mM/min
27 M/min
Now things get a little messy. The units, M, are in terms of micromoles per liter, by
definition. That is what the big M means. If we have a concentration of 27 M, that means we
have 27 micromoles per liter, or 27 nanomoles/ml (make sure you understand this conversion).
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Moreover, our reaction mixture is 3.05 ml, and we want the actual total amount of product
formed in our tube, not a concentration.
So:
This then is how much p-nitrophenol was responsible for the yellow color, and represents how
much p-nitrophenol was generated by the enzyme in one minute.
In summary, youll need to follow three steps to get from your graph of absorbance vs. time to
the amount of product formed per minute:
1. Determine the slope of the straight portion of your curve (units are A410/min).
2. Convert this slope from A410/min to M/min by using the Beer-Lambert equation.
3. Convert this concentration to the total amount of product formed in the cuvette by taking
the total volume into account. Report your result in terms of nmol/min.
1. Turn on the spectrophotometer. Allow the instrument to warm up for 30 minutes before
you use it.
2. The machine should be in "Absorbance mode" i.e., the display should show some numbers
followed by an "A". If not, press 'A/T/C' to select the absorbance mode. .
3. Press
nm
or
nm
'
4. To a cuvette add 3.0 ml of Tris-HCl buffer at pH 8.0. Then add 25 l of the 100 mM stock
solution of p-nitrophenolphosphate. Cover the tube with a piece of Parafilm and invert the
tube several times to mix the solution. Insert the cuvette with the reaction mixture into the
spectrophotometer and blank the machine by pressing the '0 ABS/100%T' button. (What is
the purpose of this step?)
5. Add 25 l of the stock enzyme solution to the tube while simultaneously marking the time
(you might try to bring a watch to lab). Quickly mix the solution by inversion and put the
tube back in the sample compartment of the Spectrophotometer. Note that you start timing
when you add the enzyme solution, NOT when you finish mixing.
6. Beginning at 15 seconds, read the absorbance at 410 nm at 15 second intervals for 2 minutes.
7. Plot absorbance as a function of time on graph paper (available in lab) or on a computer in
the adjoining computer lab. Determine the period over which absorbance increases linearly
with time and calculate the initial velocity as A410/min. You can do this on the graph paper
by choosing two readings in the linear region and calculating the slope (change in absorbance
over change in time, remember?).
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8. Repeat this procedure (including the blanking step, #4!) with the same amount of enzyme at
least two more times to determine the accuracy and reproducibility of the assay method. Be
sure to invert the cuvette the same number of times for each sample you're going to be
making comparisons later which depend on consistency. You can plot all three data sets on
the same axes.
9. Once a good set of values for vo has been obtained, calculate the average initial velocity in
A410/min. Then, using the calculation given on pages 3-5 and 3-6 as a guide, convert the
initial change in absorbance per unit time to an actual rate of product formation in nmol/min.
B. Protocol for the Determination of Km and Vmax Values
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C. Report Format
Literature Cited
Kapojos J., K. Poelstra, T. Borghuis, A. Van den Berg, H. Baelde, P. Klok, and W. Bakker. 2003.
Induction of glomerular alkaline phosphatase after challenge with lipopolysaccharide.
International Journal of Experimental Pahtology 84:135-144
Poelstra, K., W. Bakker, P. Klok, M. Hardonk, and D. Meijer. 1997. A physiologic function for
alkaline phosphatase: Endotoxin detoxification. Laboratory Investigation 76:319-328.
Sakharov, I. Y., Makarova, I. E., and Ermolin, G. A. 1988. Purification and characterization of
intestinal alkaline phosphatase from harp seal. Comparative Biochemistry and Physiology, B
90B, 709-714.
Verweij W., H. Bentala, A. Huizinga-van der, A. van Loenen-Weemaes, K. Kooi, D. Meijer, and K.
Poelstra. 2004. Protection against an Escherichia coli-induced sepsis by alkaline phosphatase in
mice. Shock 22:174-179
Young, G., S. Friedman, S. Yedlin, and D. Alpers. 1981. Effect of fat feeding on Intestinal alkalinephosphatase activity in tissue and serum. American Journal of Physiology 241:G461-G468
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