Alkaline Phosphate Activity:
The Removal and Reintroduction of
Zinc and Magnesium
Jesse Caballero, TJ Corley,
Sherilyn Mumme, Joe Quiroz
Introduction
Introduction: Alkaline Phosphatase
- The purposed active site structure dependent on 2 Zinc and 1 Magnesium
- Indigo= Serine, Cyan= Histidine, White= Aspartic acid
- Purple= Zn, Green= Mg, Orange= PO4, Red= H20
Theory
• Alkaline Phosphatase (E.C. 3.1.3.1) from Escherichia coli exists within the
periplasmic region
– subject to elevated environmental constituents; therefore, retains structure under
varying conditions. Proven to have increased resistance to: degredation,
inactivation, denaturation, and intrinsically has a greater rate of activity.
• Alkaline Phosphatase, obtained from swine kidney, was inhibited by EDTA
and cyanide due to the chelating removal of metal from active site
• H. Csopak et al. showed that in absence of EDTA, only two Zn2+ were
required for full enzyme activity. Furthermore, saturating AP in EDTA
removed Zinc and Magnesium rendering inactive.
– Also reintroduce Co2+ producing activity within alkaline phosphatase but with a new
degree of specificity.
• Alkaline phosphatase’s structural stability due to periplasmic location
produced a protein that remained structurally unaltered when metal ions
were removed; under specific conditions
Hypothesis
• Removal of active site metal ions would render alkaline phosphatase
inactive; however, reactivation is obtained when metal ions are
reintroduced into deactivated alkaline phosphatase.
• Since H. Csopak et al. achieved reactivation when experimenting
with similar transition metals, it is possible to reactivate alkaline
phosphatase with optimal metal ions to achieve the same rate of
activity as unaltered AP
• Stec et al. purposed a three metal ion active site within AP
– This claim was enhanced by Vallee et al. which indicated magnesium was
ignored by previous experimentation, but aided in activity
• Wilson et al presented the capability of alkaline phosphatase to have
four zinc-binding sites, which are not equivalent, but enhance
Csopak et al.’s claim of only 2 zinc requirement for activity.
Secondary Experiment
– Structural stabilizing role of Magnesium within Alkaline
phosphatase increased rate of activity; however, is not directly
involved in general base catalysis
• Herschlag et al. contradicted previous claims that third metal
ion site, magnesium, provides general base catalysis.
– Their results indicate the third metal ion stabilizes the transferred
phosphoryl group within the transition state.
– Removal of Mg2+ effected both phosphate and sulfate monoester
hydrolysis reactions; however, does not significantly effect phosphate
diester hydrolysis
– Magnesium does not mediate general base catalysis
• Observing the activity of Alkaline phosphatase after
reactivation in varying metal concentrations will give insight to
metal triad active site functionality
Overview of methods
Major Steps
• Changing of the original Alkaline Phosphatase
buffer using dialysis
• Chelating of the Alkaline Phosphatase using
EDTA and dialysis to remove the EDTA and
metals
• Addition of Zinc and/or Magnesium for
samples
• Activity assays of samples
Experimental Design
• Alkaline phosphatase was chelated over night
by saturation with EDTA
• In activation of AP was confirmed with activity
assay.
• Stoichiometric amounts of metals were added
to 50 µL of inactivated enzyme for 24 hours at
room temperature
• Enzyme assay was performed after over night
incubation
Methods - Dialysis
Dialysis exchanges the salts and other small
molecules across a filtration membrane.
Protein is eluted into a new buffer of
workable sets of parameters for activity
assays
Ethylenediaminetetraacetic acid
(EDTA)
• EDTA is a excellent
chelating molecule for
metals such as
Magnesium and Zinc
• The negative charges on
the Oxygen stabilize the
positively charged metal
Cofactors
• Metals provide Alkaline phosphatase with
chemical properties that help carry out the
hydrolysis of phosphate on a variety of
substrates.
• In the wild type of Alkaline Phosphatase Zinc
and Magnesium are found in stoichiometric
ratios of 2 Zn and 1 Mg per molecule of
enzyme
Activity Assay
• The enzyme of interest is a
phosphatase so a substrate paranitrophenol phosphate (PNPP) is
particularly useful.
• The conjugated Pi-system of PNPP
changes when the phosphate is
hydrolyzed by AP at a wave length
of 410nm
• The absorption of substrate is
correlated to the activity of Enzyme
Extensive Materials and methods
Materials
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108 mM Alkaline Phosphatase
Tris/HCl
Tris/ClSorval Centrifuge
Centrifuge dialysis tube and 40 kDa membrane
EDTA
MgCl2 • 6H2O
ZnCl2 • 6H2O
Cary 50 Spectrophotometer
Changing of the original Alkaline
Phosphatase buffer:
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75 µL of the stock Alkaline Phosphatase was added to 2425 µL of buffer in a centrifuge
dialysis tube with a 40 kDa membrane. 2.5 mL of water was used on the opposite side of
the centrifuge. The solution was run in a centrifuge for 30 minutes at 8,000 rpm.
The supernatant was kept and the remaining solution was discarded. The supernatant
was less than 10 µL.
1 mL of Tris buffer was added to the centrifuge dialysis tube with the supernatant and
centrifuged for 10 minutes at 3,000 rpm.
Again, the supernatant was kept and the remaining solution was measured and then
discarded. 1 mL of Tris buffer was added to the 428 µL of the supernatant and the
solution was centrifuged for 10 minutes at 3,000 rpm.
260 µL of the supernatant (which included the Tris buffer and the Alkaline Phosphatase)
was collected after the dialysis. To get the supernatant off of the membrane it was
centrifuged for 5 minutes at 3,000 rpm with the membrane upside down.
1 mL of Tris buffer at pH 7.4 was added to the supernatant in order to dilute and store
the solution in 4°C.
An activity assay was done on the resulting protein solution.
Chelating of the Alkaline Phosphatase
and removal of EDTA:
• 1 mL of 0.1 M EDTA was added to 0.5 mL of the Tris/AP solutions. This mixture
was allowed to come to equilibrium over 20 hours at room temperature.
• A dialysis was again carried out in order to remove the EDTA and the chelated
metals. The solution was centrifuged for 30 minutes at 8,000 rpm. 1.41 mL of
solution was removed.
• 1.5 mL of Tris buffer was added to the 90 µL of the denatured enzyme and the
original Tris buffer. The centrifugation was carried out for 10 minutes at 3,000
rpm. 0.79 mL of the AP solution was recovered after the dialysis.
• 1 mL of Tris buffer was added and the solution was centrifuged for a final time
for 10 minutes at 3,000 rpm.
• To get the supernatant off of the membrane it was centrifuged for 5 minutes at
3,000 rpm with the membrane upside down. 0.91 mL of the denatured Alkaline
Phosphatase in Tris buffer was collected and used to run the samples.
• An activity assay was done on the solution containing the chelated protein.
Addition of Zinc and/or Magnesium:
• The Zinc and Magnesium were added according to
stoichiometric ratios. 0.203 g of MgCl2 was added to
10 mL of Tris buffer and 0.136 g of ZnCl2 were
added to 10 mL of Tris buffer.
• Then for each of the enzyme solutions, 1.8 µL of the
metal solutions were added to the denatured
enzyme/Tris buffer solution until a volume of 50 µL
is reached.
• These samples were incubated at room
temperature for 20 hours.
Samples
• The different mixtures were
– 1 Zn2+ : 0 Mg2+ : 1 AP
– 2 Zn2+ : 0 Mg2+ : 1 AP
– 0 Zn2+ : 1 Mg2+ : 1 AP
– 1 Zn2+ : 1 Mg2+ : 1 AP
– 2 Zn2+ : 1 Mg2+ : 1 AP
– 1 Zn2+ : 2 Mg2+ : 1 AP
• All ratios are according to stoichiometry
All of the activity assays were
conducted in the following manner:
• 0.5 mL of 1 µM PNPP was added to 0.45 mL of the
Tris buffer at pH 7.4. This solution was used as the
blank.
• 50 µL of the enzyme solution was added and the
enzyme kinetics at 410 nm was taken and analyzed
with a molar absorptivity of 17500 M-1 cm-1. The
change in absorption was collected for each of the
different samples.
• Each of the samples differed in the concentration of
the metals (Zn2+ or Mg2+). 15 different mixtures
were measured.
Results
Results
Enzyme activity
Activity 1 (µM/min)
Activity 2 (µM/min)
Activity 3 (µM/min)
1 Zn
18.977
84.95*
55.67
2 Zn
36.67
68.78*
60.8
1 Mg
0.572
1.00
n/a
1 Zn 1 Mg
23.81
29.62
n/a
2 Zn 1 Mg
64.85
71.71
n/a
1 Zn 2 Mg
44.23
13.84
60.43
Discussion
Dialysis
• A Dialysis was run initially to remove the
contaminating metals, Mg2+ and Zn2+
• To inactive AP, the chelation of Mg2+ and Zn2+
was necessary. This was accomplished by
saturating the enzyme with EDTA.
• A second dialysis was required to remove
EDTA. This yields a solution containing only the
apoenzyme.
Activity Assay
• PNP- was the substrate used because AP is a
phosphatase.
• PNP- provides absorption at 410 nm due to its
conjugated Pi system
• Initially Velocity, V0, was calculated by taking the
change of the concentration of the AP at 410 nm and
dividing by the change in time. The time interval was
the first 10 seconds.
• The concentration of PNPP which was added completely
saturated the enzyme, thus V0 is the equivalent to Vmax.
This provides consistency for measuring activity
Activity Assay
• The activity of AP after saturation of EDTA
indicated successful chelation
• The activity of the chelated enzyme was 0.59
µM/min. This indicates that there was some
enzyme left in the solution that was completed
chelated; however, compared to the original
activity (73 µM/min) this was significantly
decreased.
Brownian motion of metal ion solutions
• Activation observed in
varying solutions showed
a potential ratio
• Zn1 was less than Zn2
because due to higher
concentrations a greater
amount of metal was
able to be reintroduced
into the AP
Enzyme
activity
Activity 1
(µM/min)
Activity 2
(µM/min)
Activity 3
(µM/min)
1 Zn
18.977
84.95
55.67
2 Zn
36.67
68.78
60.8
1 Mg
0.572
1.00
n/a
1 Zn 1 Mg
23.81
29.62
n/a
2 Zn 1 Mg
64.85
71.71
n/a
1 Zn 2 Mg
44.23
13.84
60.43
Brownian motion of metal ion solutions
• Mg showed slight
activation; however, since
this is known to not be
compatible with enzyme
activity, the activity is
compared to postdeactivation AP
– The rate of activity of
“deactivated” AP was
comparative to Mg solution;
therefore, EDTA was not
successful at fully
deactivating the enzyme
and Mg was not able to
increase activity alone
Enzyme
activity
Activity 1
(µM/min)
Activity 2
(µM/min)
Activity 3
(µM/min)
1 Zn
18.977
84.95
55.67
2 Zn
36.67
68.78
60.8
1 Mg
0.572
1.00
n/a
1 Zn 1 Mg
23.81
29.62
n/a
2 Zn 1 Mg
64.85
71.71
n/a
1 Zn 2 Mg
44.23
13.84
60.43
Brownian motion of metal ion solutions
• Ratio between Zn1Mg1
and Zn2Mg1 in
comparison to Zn1 and
Zn2
– Increased enzyme
activity; however,
magnesium is known to
not produce activity
alone. Therefore,
magnesium's enzyme
functionality is aiding, not
inducing activity
Enzyme
activity
Activity 1
(µM/min)
Activity 2
(µM/min)
Activity 3
(µM/min)
1 Zn
18.977
84.95
55.67
2 Zn
36.67
68.78
60.8
1 Mg
0.572
1.00
n/a
1 Zn 1 Mg
23.81
29.62
n/a
2 Zn 1 Mg
64.85
71.71
n/a
1 Zn 2 Mg
44.23
13.84
60.43
Mechanism: The Three Metal Triad
• In the free enzyme, three water molecules fill the
active site and the Ser102 hydroxyl group
participates in a hydrogen bond with the Mg coordinated hydroxide ion.
HO-(Ser102)
(Mg2+)-OH
Mechanism: The Three Metal Triad
• Upon binding of the phosphomonoester, which forms a
Michaelis enzyme-substrate complex, the Ser102
becomes fully deprotonated for nucleophilic attack and
there is an associated transfer of this proton to the Mgcoordinated hydroxide group to form a Mg-coordinated
water molecule.
• Coordination of Zn2 stabilizes the deprotonated Ser102.
(Zn2+)
-O-(Ser102)
(Mg2+)-OH2
31
Mechanism: The Three Metal Triad
• In the first in-line displacement, the negatively charged
Oxygen of Ser102 attacks the phosphorus center of the
substrate in the enzyme-substrate complex to form a
covalent serine-phosphate intermediate
• Zn1 participates in this step by coordinating the bridging
oxygen atom of the substrate and facilitating the departure of
the alcohol leaving group.
(Zn2+)
OR
O
P
O-(Ser102)
(Zn2+)
32
Mechanism: The Three Metal Triad
•
In the second in-line displacement step, a nucleophilic hydroxide ion
coordinated to Zn1 attacks the phosphorus atom, hydrolyzing the covalent
serine-phosphate intermediate to form the non-covalent enzyme-phosphate
product complex and regenerate the nucleophilic Ser102.
•
Zn1 lowers the pKa of the coordinated water molecule to effectively form the
nucleophilic hydroxide ion
Pi
-O-(Ser102)
33
Mechanism: The Three Metal Triad
• The Mg-coordinated water molecule acts as a general
acid to reprotonate the oxygen of Ser102. Protonation of
Ser102 may facilitate departure of the phosphate product
from the non-covalent complex.
Product
HO-(Ser102)
(Mg2+)--OH
34
Mechanism: The Three Metal Triad
• Alternatively, the Mg-coordinated water molecule may
directly protonate the phosphate group for its release.
The release of phosphate from the complex to give the
free enzyme may be facilitated also by the increased
mobility of the Arg166 side-chain.
35
-Mechanism: Three Metal Triad
Stec et al. 1309
36
Conclusion
Conclusion
• Co-factors were successfully removed from Alkaline
phosphatase shown by inactivity post EDTA
• Addition of Co-factors were successfully reintroduced
and activity was partially restored.
• It was found that Mg alone did not restore AP activity,
but did help increase activity in the presence of Zn.
• Zn alone can restore activity but is more effective in
the presence of Mg.
Application
The Importance of Zinc in biological
systems
• Phosphorylation is important in process in biological
systems. As seen by the data from this experiment,
the concentration of Zinc in the environment of the
Alkaline Phosphatase is essential for activity.
• Numerous experiments have been done on AP. As
Garen and Levinthal state, AP can be studied in a
number of conditions. Zinc concentration is another
condition that can be monitored.
Phospholipase C (PLC)
•
This three metal-ion mechanism proposed for alkaline phosphatase can be
related directly to a whole class of enzyme mechanisms involving three metal
ions, such as that observed in phospholipase C
•
Enzymes which cleave phospholipids just before the phosphate group
•
Synonymous with the human forms of this enzyme, which play an important
role in eukaryotic cell physiology: signal transduction pathways.
http://en.wikipedia.org/wiki/File:Phospholi
pases2.png
41
Further Experiments
• Complete inactivation of enzyme was not achieved. For future
studies observations at complete enzyme inactivation would result
in more concrete results.
• The role of pH in the enzyme catalysis is of particular interest for
further investigation. Because alkaline phosphatase requires a higher
pH for optimal activity the removal of metals at different pH and re
addition could provide interesting results on the mechanism of AP.
• When Zinc is reintroduced to AP, oxygen my be deprotonated which
would lower the pH of the environment and affect the protein
activity because of the acidic interactions in other areas.
• Proteins stability is dependent on structure. Magnesium provides
structural stability, so addition of cofactors at different temperatures
could result in interesting results.
• Different metal ions could be analyzed for its affect on the metal
binding sites. For example, the affect of Co2+
References
References
• Coleman, J. (1992) “Structure and Mechanism of Alkaline
Phosphatase” Annu. Rev. Biophys. Biomol. Struct.
• Garen, A., and Levinthal, C. (1959) “A Fine-Structure Genetic and
Chemical Study of the Enzyme Alkaline Phosphatase of E. coli.”
Biochimica et Biophysica Acta.
• Ninfa, A., Ballou, D., and Benore, M. (2010) Basic Procedures in the
Biochemistry Laboratory, in Fundamental Laboratory Approaches for
Biochemistry and Biotechnology. 2nd Ed.
• Stec, B., Holts, K., and Kantrowitz, E. (2000) “A Revised Mechaniem
for the Alkaline Phosphatase Reaction Involving Three Metal Ions.” J.
Mol. Biol.
• Zalatan, J., Fenn, T., and Herschlag, D. (2008) “Comparative
Enzymology in the Alkaline Phosphatase Superfamily to Determine
the Catalytic Role of an Active-Site Metal Ion.” J. Mol. Biol.
References
• Hough, E., Hansen, L. K., Birknes, B., Jynge, K., Hansen, S.,
Hordvik, A., Little, C., Dodson, E. J. & Derewenda, Z. (1989).
High-resolution (1.5 AÊ ) crys- tal structure of phospholipase C
from Bacillus cereus. Nature, 38, 357-360.
• http://en.wikipedia.org/wiki/Phospholipase_C
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