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The University of Lethbridge Chemistry 2740 Laboratory
Department of Chemistry & Biochemistry Experiment 1
KINETICS OF WHEAT GERM ACID PHOSPHATASE
Phosphatase enzymes catalyze the hydrolysis of phosphate esters to produce
inorganic phosphate,
R-O-P + H2O ––––––> R-OH + HPO42-
Phosphatase enzymes involved in synthetic pathways are very substrate specific (eg:
glucose-6-phosphatase, fructose-1,6-diphosphatase and glycerol-2,3-diphospha-
tase). Acid phosphatase is a more nonspecific enzyme and will cleave many
different phosphate esters. The exact biochemical role of acid phosphatase is
somewhat obscure but is thought to be mainly a digestive enzyme.
In this experiment, the objectives will be to examine the kinetics and inhibition of
wheat germ acid phosphatase. Para-nitrophenyl phosphate (PNPP) is a suitable
substrate. Hydrolysis of PNPP produces para-nitrophenol and inorganic phosphate.
The addition of excess alkali used to quench the reaction also quantitatively
converts the p-nitrophenol to its anion which has a λmax at 405 nm of the visible
spectrum. Assuming a path length of 1.0 cm and a molar absorptivity constant of
18.8 x 103 liter mole-1cm-1 the concentration can be calculated. The kinetics of the
reaction is best represented by the Michaelis-Menten Equation.
V max [S]
ν = (1)
Km + [S]
Where ν is for the initial rate, Km the Michaelis-Menten constant, and V max is the
maximum velocity. The constants Km and V max are the kinetic parameters often
used to characterize an enzyme. The Michaelis-Menten equation based on model (2)
is only applicable under the following conditions: when the initial rates are
measured; when the substrate [S] is much, much greater than the total enzyme
concentration [E]; and when a steady state exists for the enzyme-substrate complex
[ES].
k1 k3
[E] + [S] [ES] [E] + [P] (2)
k2 k4
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Chemistry 2740 Laboratory
Experiment 1
Assuming the formation of the ES complex from product is negligible, the Michaelis-
Menten constant (Km) is then equal to
k2 + k3
Km = (3)
k1
The equation is derived by setting the rate of formation of the ES complex equal to
the rate of is breakdown
k1[E][S] = (k2 + k3)[ES] (4)
then
[E][S] = Km[ES]. (5)
Manipulating equation (5) together with the conservation of the enzyme
[E]t = [E] + [ES] (6)
we can derive the following equation to solve for the ratio [ES]/[E]t
([E]t - [ES][S])
= Km (7)
[ES]
which rearranges to
[ES] [S]
= (8)
[E]t Km + [S]
When all of the enzyme is in the form of the enzyme-substrate complex, Vmax is
obtained and is equal to
V max = k3([E] + [ES]). (9)
If conditions are such that the enzyme is not saturated and we assume the
formation of the product and enzyme from the enzyme-substrate complex is the slow
step in the reaction, then the velocity v is
v = k3[ES]. (10)
Dividing equation (10) by (9) gives
v [ES]
= (11)
V max [E] + [ES]
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Chemistry 2740 Laboratory
Experiment 1
Finally substituting equation (11) into (8) gives the Michaelis-Menten equation (1).
The two quantities V max and Km are, respectively, the maximum velocity that would
be achieved at saturated substrate levels and the relative affinity of the enzyme for
the substrate. The lower the magnitude of the Michaelis-Menten constant (Km) the
higher the affinity the enzyme has for the substrate.
The action of most enzymes is inhibited by a variety of substances. The inhibition
may be irreversible leading to the permanent inactivation of an enzyme. The
affinity of the inhibitor for the enzyme is expressed quantitatively through the Ki or
dissociation constant for the enzyme inhibitor complex. The derivation of the
equation and the calculation of Ki are not required for the objective of this
experiment.
The three types of inhibition that can be distinguished are competitive,
noncompetitive, and uncompetitive. Lineweaver-Burke plots (Figure 1) are often
employed to distinguish between the three types of reversible inhibition.
Figure 1: Lineweaver-Burke Plot of Phosphatase
0.15
1 0.10
!
0.05
–0.25 0.25 0.50 0.75 1.0
1
[S]
No Inhibitor
Competitive Inhibitor
Noncompetitive Inhibitor
Uncompetitive Inhibitor
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Chemistry 2740 Laboratory
Experiment 1
A more complete derivation of the Michaelis-Menten equation, the Lineweaver-
Burke equation and enzyme inhibition information can be obtained from any
number of introductory biochemistry text books.
Apparatus
Pharmacia Biotech Novaspec II spectrophotometer; pH meter and calibration
buffers, ice bucket; Eppendorf pipettes; vortex mixer and timer.
Reagents
p-nitrophenyl phosphate (PNPP), wheat germ acid phosphatase, BSA, potassium
hydroxide, sodium acetate, 0.1 M MgCl2 and unknown inhibitor solution.
Waste Disposal
A 4 liter bottle for the collection of wastes is supplied with the experimental set up.
All excess stock reagents, unused reaction solutions and solutions from the reaction
vessel should be disposed of in this bottle. The glassware can then be given a single
small rinse into the waste container before being cleaned further in the sink.
In preparing reaction solutions, only remove as much reagent from the stock
container as is necessary to make the reaction mixtures.
Procedure
Note: All glassware MUST be washed with phosphate free soap.
Measure approximately 75 mL of a previously prepared 1.0 M sodium acetate
buffer, and adjust to pH 5.7 using the conjugate acid. Use a portion of this buffer to
prepare 50.00 mL of 5mM sodium acetate buffer [to be used later] and keep the rest
for use elsewhere. Weigh accurately 0.2785 g of p-nitrophenyl phosphate (PNPP)
into a clean and dry 25 mL Erlenmeyer flask. Dissolve the substrate in a 15 mL
aliquot of double distilled water. Weigh accurately 4.5 mg of wheat germ acid
phosphatase into a 10 mL volumetric flask previously charged with 10 mg of bovine
serum albumin (BSA). Dissolve the enzyme and BSA in 5 mM sodium acetate
buffer [pH 5.7] and dilute to the calibration mark with 5 mM sodium acetate buffer.
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Chemistry 2740 Laboratory
Experiment 1
Obtain 5 clean and dry 5-mL volumetric flasks and label these from “B” to “F”, then
label 7 clean and dry small beakers “A” to “G”. Prepare dilutions of the substrate as
outlined in the following table and transfer the contents into the labeled beakers.
Table 1: Dilution series from 0.05 M stock PNPP substrate
Volumetric 0.05 M PNPP double distilled Diluted PNPP Beakers
flask (mL) water (mL) (M)
--- --- ~5.00 0.0 A
B 0.10 4.90 0.0010 B
C 0.25 4.75 0.0025 C
D 0.50 4.50 0.0050 D
E 1.00 4.00 0.0100 E
F 2.50 2.50 0.0250 F
--- ~5.00 --- 0.0500 G
Obtain a total of 21 clean and dry 16 x 150 mm reaction tubes. The experiment
must be performed three times and the data averaged. Into the reaction tubes add
500 µL of 1.0 M sodium acetate, 500 µL of 0.1 M MgCl2, 500 µL of p-nitrophenyl
phosphate (from beakers A to G) and 3.30 mL of double distilled water.
Equilibrate these solutions to 37°C in the water bath for approximately 5 minutes.
Initiate the reaction in the first test tube with the addition of a 200-µL aliquot of the
wheat germ acid phosphatase, mix with the vortex and start the timer. Initiate the
reaction the same way in each of the remaining test tubes at 0.5-minute intervals. If
the appropriate reagents have been added according to the above protocol there
should be a total volume of 5.00 mL in each reaction tube. Incubate the reaction in
each test tube at 37°C for exactly 5.00 minutes. After 5.00 minutes has passed for a
test tube, the reaction is then quenched with the addition of 2.5 mL of 0.5 M KOH
and carefully mixed with the vortex (note that the final volume is now 7.5 mL). The
quenched solution should be set aside undisturbed, to allow the cloudy precipitate to
form and settle to the bottom of the reaction tube. The assay is repeated in
triplicate for each of the different substrate concentrations (“A” to “G”).
Set the Nova spectrophotometer to a λmax of 405 nm. Calibrate to zero absorbance
with the appropriate blank for each run (which should be clear and colourless) and
measure the absorbance for each of the assay tubes. The precipitate at the bottom
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Chemistry 2740 Laboratory
Experiment 1
of the reaction tube must be avoided at all costs as the particulate matter will
interfere with absorbance readings. The supernatant can be removed carefully for
analysis with the use of clean pasteur pipettes to fill the cuvettes.
It would be wise to charge and equilibrate all 21 reaction tubes before starting the
first assay with the addition of the phosphatase enzyme. As there are 21 assays
initiated at 0.5-minute intervals, each requiring 5.00 minutes of incubation before
quenching, this experiment will require good cooperation and communication
between partners in order to complete the exercise in the allotted time.
Before leaving the laboratory, please enter names, date, and experimental
data into the computer.
Calculations and Report
Determine the micromoles of p-nitrophenol produced per reaction mixture and
express the phosphatase activity (or velocity, υ) in units of micromoles of
product.min–1.mg protein–1. As the Michaelis-Menten plot results in a rectangular
hyperbola, the resulting estimates of Km and V max are not very precise. Better
values for Km and V max are derived by taking the double reciprocal form of the
Michaelis-Menten equation (1) to give a linear equation called the Lineweaver-
Burke equation (12):
1 1 Km 1
= + (12)
v V max V max [S]
A Lineweaver-Burke plot of the dependent variable 1/v versus the independent
variable 1/[S] will result in an intercept of 1/Vmax and a slope of Km/V max.
A statistically better methodology is the Eadie-Hofstee plot, obtained by dividing
both sides of the Michaelis-Menten equation (1) by the substrate concentration [S].
Rearrangement gives a linear equation called the Eadie-Hofstee equation (13):
v
v = –Km [S] + Vmax.
(13)
A graph of v vs. (v/[S]) should yield a straight line with a y-intercept equal to V max
and a slope of –Km.
Determine Km and Vmax values using both methods (Lineweaver-Burke and
Eadie-Hofstee) to describe the wheat germ acid phosphatase enzyme.
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