1. 1
Functional connections in the active site of Alkaline Phosphatase: Interactions between
D101 and the Mg2+ ion
The Alkaline Phosphatase (AP) enzyme superfamily is a well-researched ensemble of related
enzymes that has been studied for over 100 years using various techniques. One such technique
is site-directed mutagenesis, which in combination with double mutant cycles can be used to
study the functional connection between residues in an active site. Our research focuses on
understanding the functional connection between the divalent Magnesium (Mg2+) ion and
Aspartate 101 (D101) amino acid in the AP active site, separated by more than 7 Å. Specifically,
investigating the allosteric connection between the Mg2+ and D101 and thereby the origin of
redundant function. To investigate their connection, single point mutagenesis was used to design
mutants in a hypothetical pathway between Mg2+ and D101, and study the mutants’ effects on
the redundancy between Mg2+ and D101, a reduction in redundancy suggesting mutated amino
acids to be important for redundancy. Designed mutants were expressed in Escherichia Coli
strain SM547lamdba DE3 using a pmalp2xstrepII plasmid, and purified using osmotic shock and
an amylose column (affinity resin used for MBP constructs). After verifying purity of the
enzyme and determining concentrations, the reaction rates were measured and support a model
for which the redundant function occurs through residue (D51).

2. 2
Functional connections in the active site of Alkaline Phosphatase: Interactions between
D101 and the Mg2+ ion
Introduction: the "why" section (2-3 pages)
Start with a broad picture of the problem you have chosen to study and why it is interesting.
Provide a brief review of pertinent scientific literature, describe what information is missing and
how your work addresses this gap in the literature. Previous relevant publications and patents
must be properly cited in the text of the Research Report and included in the Reference section
of your report.
Describe the specific problem to be solved, the research question to be answered, the
hypothesis(es) to be tested, or the product to be developed (if any). Provide a brief rationale for
the research and why the work is important.
Our research focuses on understanding a part of the catalytic strategy of the alkaline
phosphatase superfamily of enzymes, specifically the enzyme for which the super family is
named: Alkaline Phosphatase (AP) using site directed mutagenesis to study the impact of
specific residues on the rate of catalytic reactions. The AP superfamily is a well studied enzyme
system that has been studied for approximately 100 years throughout scientific literature. Basic
information regarding the enzyme is the mechanism, AP is a phosphomonoesterase, hydrolyzing
phosphomonoesters into a free alcohol group and phosphate group. (Figure 1) More specifically,
the active site of AP features a bimetallo Zn2+ motif that typifies members of the alkaline
phosphatase superfamily in addition to a Mg2+ ion that helps to stabilize the transferred
phosphoryl group in the transition state (Figures 2, 3, 4). Prior work has shown that both the Zn2+
ions and the nucleophile, S102, are required for measurable activity [Andrews, 2013][Plocke,
1962]. Our work adds to the wealth of information already gathered regarding the function of the
enzyme, but also using AP as a model system, increasing our knowledge of how enzymes work
in general. The potential applications of having a greater understanding of enzyme functions and

3. 3
mechanisms are countless. Basic implementations of this information are as follows: 1. Learning
how to design enzymes ourselves to achieve specific goals and functions. 2. Better understanding
drug interactions to maximize output and function, and avoid hazardous reactions between
different drugs. 3. Providing an alternative method of delivering medicine into the body,
enzymes provide an organic chemical system for the delivery of medicine. However, aside from
the numerous potential applications of enzymes, the main interest in studying the AP superfamily
is to provide a greater fundamental understanding of basic enzymatic functions and mechanisms.
Previous scientific literature has succeeded in filling in many of the gaps present in our
knowledge of AP; however, there are still countless of things that are less clear and models that
need to be more extensively tested. Prior research conducted by the Herschlag Lab at Stanford
identified functional units in the AP active site; the function of the residues in a functional unit
are dependent on the presence or absence of the other members of the same functional unit
[Sunden, Al Sadhan, 2014]. Through these studies, three energetically distinct functional units
were identified - our research attempts to dissect the molecular basis underlying the observed
energetic behavior of one of these units with energetic anti-coupling (redundancy in function).
The specific problem we aimed to solve was to elicit information about the functional
connection between a divalent Magnesium ion (Mg2+) in the active site of alkaline phosphatase
and D101 (D represents Aspartate, 101 represents where in the sequence the aspartate amino acid
is encoded). Unpublished data from the Herschlag Lab suggest that there is anti-coupling
between the Mg2+ ion and residue D101, this project aims to discover why and how these are
connected, even though the residues are separated by more than 7 Å allosteric interaction (Figure
7) [Zalatan, et al, 2008] [Bobyr, et al, 2011]. Models of potential redundant coupling between the
D101 and Mg2+ are:

4. 4
1. Mg2+ and D101 are connected through D51 (tested via mutating D51 to glycine or
asparagine). Mg2+ and Zn2+ both interact with the shared metal ligand D51 (Aspartate
51). The zinc ion in turn interacts with the nucleophile, S102 (serine 102), which sits
next to D101 (Figures 2, 3, 4). Thereby the residues would be connected through D51.
2. Mg2+ and D101 are connected through the Mg2+ water. Mg2+ and D101 are connected
through the water molecule that is bound to the Mg2+ ion. This either occurs through
positioning the substrate relative to the amide backbone that sits next to D101 or by
positioning the S102 nucleophile that sits next to D101. (Figures 2, 3)
Nevertheless, there may also be other ways to connect Mg2+ and D101 that are currently not
understood. In this report we explore the validity of the first model. To test the first model we
used site directed mutagenesis and constructed a cubic double mutants cycle. No specific
research has attempted to determine the functional connection between the Mg2+ ion and the
D101 amino acid, this paper fills in the relevant gaps in the literature. Thus, we hypothesize that
removing any of the residues connecting D101 and Mg2+ (in the case of model 1, this residue is
D51) will result in a decrease in redundancy between the two.
The methods and procedures we followed in this project were similar to the standard
procedures that are used throughout the literature [Zalatan et al., 2008]. The overall procedure
we performed was as follows: First, create gene mutants using PCR (site directed mutagenesis).
Second, express the genes and enzymes in Escherichia Coli (E. Coli). Third, perform osmotic
shock on the cells to lyse them and extract the enzyme. Fourth, wash and elute the enzyme for
purification. Fifth, run gel electrophoresis to verify presence and purity of AP enzyme. Sixth,
perform buffer exchange to concentrate the purified enzymes and measure the enzyme
concentration Seventh, run kinetic assays to measure the activity of the enzymes. Eighth,

5. 5
perform analysis of data using Michaelis-Menten Kinetics methods to analyze the results.
(Figures 5, 6) Each of the processes is described in further detail as follows:
First, the gene mutants were created using polymerase chain reaction. The genes in the
pmalp2xstrepII plasmid construct were mutated to produce the desired gene sequence for the
altered AP active site residues. Then, standard PCR procedure was used to accumulate more of
the plasmid and the gene of interest. [Zalatan et al., 2008]
Second, the plasmids were grown in E. Coli and enzyme expression was induced using
IPTG (isopropyl-beta-D-thiogalactopyranoside), which induces expression of the cloned genes
under control of the lac operon. The plasmids contain a carbenicillin resistance gene that allows
the cells with plasmids to grow and reproduce in rich media containing carbenicillin. The
plasmids were taken up in E. Coli, SM547lamdbaDE3, a knockout strain of E. coli that does not
normally produce any Alkaline Phosphatase. This is beneficial in that we are able to only
produce the AP enzymes that have undergone the desired site-directed mutagenesis, and
minimizes the risk for contamination. 2 liters of rich media was prepared (20 g tryptone, 5 g
yeast extract, 5 g NaCl, 2 g glucose) per mutant, then autoclaved for 20 minutes. The E. Coli
cells were then inoculated (30 mL luria broth, 30 µL carbenicillin, cells), and incubated at 37 C
overnight while shaking. The next day, the inoculated E. Coli cells were added to the rich media,
and left to grow for about 2-3 hours while shaking until the OD600 reached 0.6 to 0.8. Once the
desired OD was reached, 0.3 mM (600 µL) IPTG was added to the 2 L culture to induce protein
production, and growth continued overnight at 30 C while shaking.
Third, lyse the E.Coli cells by performing osmotic shock to extract the mutated AP
enzyme. The osmotic shock procedure is carried out as follows. Harvest the cultured cells by
pelleting them; centrifuge for 20 minutes at 4000 rpm at 4°C, discard supernatant and save pellet.

6. 6
Resuspend the pelleted cells in 400 mL sucrose solution (30mM Tris pH 8.0, 20% sucrose, 1mM
EDTA), gently shake at room temperature for 10 minutes. Transfer product to 400 mL centrifuge
bottles, balance each out within plus or minus 1 gram of each other. Pellet cells in centrifuge at
9000 RPM at 4°C for 10 minutes. In the cold room (4°C), remove supernatant in each bottle,
resuspend pellets in 400 mL ice cold water, and shake gently on a shaker for 10 minutes. Pellet
cells twice in centrifuge for 20 minutes, 9000 RPM at 4°C. Place liquid from all bottles
containing the same mutant enzyme into a large beaker. Bring solution to 10mM Tris pH 7.4,
200 mM NaCl, 10µM ZnCl2 by adding 8 mL Tris pH 7.4, 1 M, 32 mL NaCl 5 M, 160 µL ZnCl2
50 mM. Filter with 500 mL filter units to remove insoluble material, such as cell debris.
Fourth, purify the enzyme over amylose affinity resin, wash and elute the enzyme; the
enzyme will be isolated in the resin through the MBP tag on the AP enzyme. Clean a running
tube and a elution column with 0.5 M NaOH and Tris pH 8. Prepare a 10 mL amylose column,
letting it settle for 5 minutes in 4°C cold room. Set regulator to flow dilute protein solution over
column at 4°C overnight with a speed of flow around one drop per two seconds. After the
protein solution has dripped through, save the flow through in freezer for later tests through gel
electrophoresis. Wash the amylose column with 15 mL column buffer; save the flow through in
separate tubes with appropriate labels (perform the wash 3 times). Elute the protein with 10 mL
elution buffer; store in a similar manner to the wash flow throughs (repeat elutions 4 times).
Check the elution flow throughs (fractions) for the presence of enzymes by using Bradford
solution (Bradford solution should turn blue to indicate the presence of a protein, but the strength
of the blue color should diminish with each progressing fraction). Resuspend amylose column in
30 mL column buffer and store in fridge.

7. 7
Fifth, run gel electrophoresis to verify presence and purity of AP enzyme using an SDS-
PAGE gel. Assemble gel caster, test with water to ensure no leakage. Prepare running gel: 1.9
mL H2O, 1.8 mL Acrylic amide, 1.3 mL Tris 1.5 M pH 8.8, 100 µL 10% SDS. Add 100µL
persulfate (APS) and 10 µL TEMED to running gel to begin polymerisation process. Mix by
pipetting up and down, then immediately add running gel to gel caster apparatus. Add 100 µL
isopropanol (butanol) into the gel caster, above the running gel. Wait 10-15 minutes for the
running gel to set. Discard isopropanol from the gel caster. Prepare stacking gel: 0.5 mL H2O,
0.1 mL acrylic amide, 0.1 mL Tris buffer 1M pH 6.8. Add 10µL persulfate (APS) and 10µL
TEMED to stacking gel to begin coagulating process. Add stacking gel to the caster, placing the
well molds inside the liquid gel, let sit fr 10-15 minutes. Meanwhile, heat samples for 5 minutes
after adding 5 µL dye (15%SDS, 50% glycerol, 25% β-mercaptoethanol, 0.01% bromophenol
blue) into 10 µL of sample (each sample should be in a separate tube; samples should include
flow through, washes, and fractions). After moving the completed gel to the gel box, add non-
hazardous buffer to completely cover the base, rear, and bottom of the gel. Add ladder to the first
well, samples to all other wells, run gel at 130 V until the samples level out, then run at 170 until
the first band of ladder runs off of the gel. Remove gel from gel box, stain with dye (10% acetic
acid, 40% methanol, 0.1% coomasie blue in water) for 30 minutes, immerse in 10% acetic acid
overnight to de-stain, then strands should be visible. Compare strand lengths of proteins to the
ladder to confirm presence of the proper enzyme.
Sixth, perform buffer exchange to concentrate the purified enzymes. Based on
observations and readings from the gel, select the fractions with the most enzyme (typically the
darkest band on the gel contain the most enzyme). Place contents from the fractions into a filter
tube; store in a bucket of ice throughout the process. Balance out filter tube with a tube of water

8. 8
and centrifuge for 20 minutes at 3500 RPM. Afterwards, save the flow through that remains after
centrifuging. Make 250 mL storage buffer (10 mM Tris pH 7, 50 mM NaCl, 200 uM ZnCl2) in a
graduated cylinder (include 1 mM Mg2+ depending on whether enzyme contains the E322Y
amino acid or not). Fill the filter with buffer, balance again with a tube of water, and centrifuge
for 20 minutes at 3500 RPM. Save the flow through again and repeat centrifuge process with
storage buffer. Remove the filter and collect the enzyme that is found in the bottom. Measure
concentration of the enzyme using a spectrophotometer, record for later calculations.
Seventh, run kinetic assays to measure the activity of the enzymes. Wash cuvettes in
cuvette wash with distilled water and acetone to avoid any possible contamination with
previously tested enzymes. Select the appropriate enzyme, substrates, and buffer. Fill all 8
cuvettes with 64 μL buffer, 32.5 μL of substrate in order of varying concentration levels (will
vary over 128-fold concentration range), and 32.5 μL enzyme in the first 7 cuvettes (sterile water
in the 8th). Mix until the solution is inside each cuvette is clear, without any cloudiness or
fogginess or bubbles. Set up spectrophotometer, making sure the water heater is set to 25 C
(room temperature) and the visible light is turned on. Place the cuvettes in a spectrophotometer,
which takes absorbance readings every 10-60 seconds, until a sufficient amount of the reaction
rate has been recorded (10% of reaction). After the run is completed remove cuvettes and record
all of the data measurements for later calculations.
Eighth, perform analysis using Michaelis Menten Kinetics methods to analyze the results
using the help of a downloadable software named KaleidaGraph [KaleidaGraphTM]. The
explanation for Michaelis Menten Kinetics calculations are provided in the results section.
Our mentor assisted us in identifying the necessary gene mutation and interacting with
the 3rd party company that verified the gene sequences following site directed mutagenesis. She

9. 9
also performed the PCR procedure. We completed the remaining methods of the procedure (2-8)
with initial guidance and assistance from our mentor.
To find our results, we analyzed the data gathered from the kinetic assays of the AP
mutants. We collected data on the initial reaction rates until 10% completion of the reaction.
Once the data was on the computer, plots and graphs were created using Kaleidagraph
[KaleidaGraphTM]. We plotted the absorbance at 400 nm (the output from the spectrophotometer)
against time in second for each of the 8 different cuvettes’ data sets. (Figure 5) The resulting rate
of absorbance/time was plotted against the substrate concentrations used in the assays, forming a
Michaelis Menten curve (Figure 6). (The Michaelis Menten equation is (m1*m0)/(M2+m0),
where m1= vmax, m0=substrate concentration, M2=KM) [#4, Le, 2010] Reading the Michaelis
Menten curve to determine the Vmax value, which had the units of absorbance/seconds, the Vmax
value was converted to a kcat (units: 1/seconds) by dividing the Vmax value (m1 in Kaleidagraph)
with the enzyme concentration used in the experiment and the extinction coefficient for the
product, 16652 /(M cm). (We divide by the extinction coefficient to convert absorbance to
concentration, utilizing Beer’s law: Absorbance = extinction coefficient * b * concentration,
where b = 1 cm, the width of the cuvette) [Le et al., 2010]. The KM is the m2 value on
Kaleidagraph, and corresponds to the substrate concentration where the activity of the enzyme is
half of the Vmax value. Then, the kcat/KM (/M s) value can be calculated [Le et al., 2010], which is
used to compare the various mutants as a measure of reaction speed.
The purpose of our experiment was to find how D101 and Mg2+ interact with each other.
Previous research by the Herschlag Lab at Stanford University [Sunden et al., 2014] found that
D101 and Mg2+ do in fact interact, we carried out a series of experiments after mutating D51 to

10. 10
asparagine or glycine, and created several other mutants as comparisons. The following is a list
of all the mutants we expressed, purified, and ran through kinetic assays:
● Mutants created in unpublished data: [Sunden et al., 2014]
○ D153A/R166S/K328A
○ D101A/D153A/R166S/K328A
○ D153A/R166S/E322Y/K328A
○ D101A/D153A/R166S/E322Y/K328A
● Mutants created for D51G Cubic Double Mutant Cycle (Figure 15):
○ D51G/D153A/R166S/K328A
○ D51G/D101A/D153A/R166S/K328A
○ D51G/D101A/R166S/E322Y/K328A
○ D51G/D101A/D153A/R166S/E322Y/K328A
After gathering data on all the mutants and calculating the kcat/KM values as explained above, we
compared the double mutant cycles between 4 related mutants. To create the double mutant
cycles, we took the 4 mutants and took the ratio between the kcat/KM values. (Figure 8) The
kcat/KM value of the mutant at the beginning of the arrow was always divided by the kcat/KM
value of the mutant at the tip of the arrow. The proportions gave us 4 values, a, b, c, d (Figure 8).
The possible outcomes are that a = c, which results in no coupling (i.e. the kcat/KM values do not
increase/decrease in the presence or absence of the other residue in question) , a > c, where there
is coupling kcat/KM (i.e. there is energetic coupling, meaning that the ratio of kcat/KM is greater in
the presence of the other residue in question as opposed to without it), or a < c, where there is
redundancy (i.e. the two residues are energetically anti-coupled, the ratio of kcat/KM is higher
when the residue in question is absent as opposed to present). After analyzing the double mutant
cycles to determine the coupling of the two residues, 6 double mutant cycles could be combined
to form a cubic double mutant cycle since there are 3 variable residues. Below are several data
sets for double mutant cycles, demonstrating how we tested for the redundant coupling between
D101 and Mg2+.

11. 11
Data Tables 1 & 2: [Sunden et al., 2014]
Mutant kcat/KM (/M s)
D153A/R166S/K328A 3600
D101A/D153A/R166S/K328A 5500
D153A/R166S/E322Y/K328A 4.3
D101A/D153A/R166S/E322Y/K328A 0.17
Mutants Ratio of kcat/KM
(/M s) values
D153A/R166S/K328A to D101A/D153A/R166S/K328A 0.65
D153A/R166S/K328A to D153A/R166S/E322Y/K328A 840
D101A/D153A/R166S/K328A to D101A/D153A/R166S/E322Y/K328A 32000
D153A/R166S/E322Y/K328A to D101A/D153A/R166S/E322Y/K328A 25
The triple mutant background D153A/R166S/K328A was used for the mutants in Tables 1 & 2.
Removing D101 (mutating it to alanine) from the active site with magnesium does not have a
significant effect on catalysis, as indicated by the ratio of 0.65. However, if we remove the
magnesium by mutating the magnesium ligand E322 to a tyrosine (E322Y) [Zalatan et al., 2008]
and look at the effect of mutating D101 in that background, there is a 25 fold redundant effect.
Since 0.65 < 25, the double mutant cycle suggests redundancy between D101 and the Mg2+
(represented by E332 residue) as mentioned before. (Figures 9, 10)
Data Tables 3 & 4: [Sunden et al., 2014]
Mutant kcat/KM (/M s)
D153A/R166S/E322Y/K328A 4.3
D51G/D101A/R166S/E322Y/K328A 2.2
D101A/D153A/R166S/E322Y/K328A 0.17

12. 12
D51G/D101A/D153A/R166S/E322Y/K328A 0.12
Mutants Ratio of kcat/KM
(/M s) values
D153A/R166S/E322Y/K328A to D51G/D101A/R166S/E322Y/K328A 2.0
D153A/R166S/E322Y/K328A to D101A/D153A/R166S/E322Y/K328A 25
D51G/D101A/R166S/E322Y/K328A to
D51G/D101A/D153A/R166S/E322Y/K328A
18
D101A/D153A/R166S/E322Y/K328A to
D101A/D153A/R166S/E322Y/K328A
1.4
Next we mutated, D51 to glycine and D101 to alanine in the E322Y background. The effect of
mutating D51 to glycine was small, within 2 fold of the E322Y mutant. While the effect of
mutating D101 is 25-fold as described before. The effect of mutating each of the residues was
roughly the same no matter whether or not other residue was present. Since 2.0 is roughly equal
to 1.4, this suggests that D51 and D101 are not coupled in the absence of magnesium. This does
however not exclude coupling between them if magnesium is present, but it suggests that D51
does not help the catalysis if magnesium is absent. (Figures 11, 12)
Data Tables 5 & 6 [Sunden et al., 2014]
Mutant kcat/KM (/M s)
D101A/D153A/R166S/K328A 5500
D51G/D101A/D153A/R166S/K328A 3.5
D101A/D153A/R166S/E322Y/K328A 0.17
D51G/D101A/D153A/R166S/E322Y/K328A 0.12
Mutants Ratio of kcat/KM
(/M s) values

13. 13
D101A/D153A/R166S/K328A to D51G/D101A/D153A/R166S/K328A 1300
D101A/D153A/R166S/K328A to D101A/D153A/R166S/E322Y/K328A 3200
D51G/D101A/D153A/R166S/K328A to
D51G/D101A/D153A/R166S/E322Y/K328A
29
D101A/D153A/R166S/E322Y/K328A to
D51G/D101A/D153A/R166S/E322Y/K328A
1.4
Then, we mutated D101 and looked at the interaction between D51 and the magnesium ion in
that background. In this case, mutating D51 to a glycine had a huge effect, decreasing activity
1300 fold, However, when mutating D51 to glycine in the E322Y mutant, activity did not
decrease further. That means that magnesium and D51 are 1,000 fold coupled (1300 >1.4). Thus
suggesting that D51 and magnesium are interacting with each other in the D101 background.
(Figures 13, 14)
Since we have 3 variable residues, D51, D101, and E322 (Mg2+ ligand), a cubic double
mutant cycle can be constructed to summarize the data. (Figure 15) The 6 double mutant cycles
and final cubic double mutant cycle suggest that D51 and E322Y are coupled (energetically
coupled), more so if D101 is gone, which matches the prediction of our model because Mg2+ is
more significant in the D101A background. Further, D51 and D101 are not coupled in the
presence of E322Y, but are anti-coupled in the WT background (WT background is TM
background: D153A/R166S/K328A). Finally, D101 and E322Y are anti-coupled in the WT
background, but are anti-coupled the absence of D51.
Thus, the data shows that the redundant coupling between D101 and Mg2+ is gone in the D51
glycine mutational background, which suggests that the observed redundancy between Mg2+ and
D101 goes through D51, supporting model 1 as described above.

14. 14
Figure 1: Alkaline Phosphatase Enzyme
Structure [Bobyr et al., 2011]
Figure 2: Alkaline Phosphatase Active Site
[Zalatan et al., 2008]
Figure 3: Alkaline Phosphatase Active Site
Close-Up [Bobyr et al., 2011]
Figure 4: Alkaline Phosphatase Active Site 3-
Dimensional Close-Up [Bobyr et al., 2011]
Figure 5: Absorbance vs. Time Graph from
Kaleidagraph Software [KaleidaGraphTM]
Figure 6: Michaelis Menten Curve from
Kaleidagraph Software [KaleidaGraphTM]

15. 15
Figure 7: Active site of Alkaline Phosphatase,
Mg2+ and D101 highlighted [Zalatan et al.,
2008]
Figure 8: Example of Double Mutant Cycle in
TM Background [Sunden, Al Sadhan, 2014]
Figure 9: TM Background Double Mutant
Cycle [Sunden et al., 2014]
Figure 10: TM Background Double Mutant
Cycle Active Site Modifications [Sunden et
al., 2014]

16. 16
Figure 11: E322Y/TM Background Double
Mutant Cycle
Figure 12: E322Y/TM Double Mutant Cycle
Active Site Modifications [Sunden et al.,
2014]
Figure 13: D101A/TM Background Double
Mutant Cycle
Figure 14: D101A/TM Background Double
Mutant Cycle Active Site Modifications
[Sunden et al., 2014]

17. 17
Figure 15: D51G Cubic Double Mutant Cycle

18. 18
The results support the model proposed in the introduction: Mg2+ and D101 are
redundantly coupled and connected through D51. Based on the data, since we have 3 variable
residues, D51, D101, and E322 (Mg2+ ligand), a cubic double mutant cycle was constructed to
summarize the data for D51G mutants. The 6 double mutant cycles and final cubic double
mutant cycle suggest that D51 and E322Y are energetically coupled, more so if D101 is gone,
which matches the prediction of our model because Mg2+ is more significant in the D101A
background. Further, D51 and D101 are not coupled (i.e. redundantly coupled/energetically
coupled) in the presence of E322Y, but are anti-coupled in the WT background. Finally, D101
and E322Y are anti-coupled in the WT background, but are not connected in the presence of the
D51 mutation.
All three of those results from the double mutant cycles described in Tables 1-6 and
Figures 9-15 support the model that D101 and the Mg2+ ion are redundantly coupled through
D51. In the absence of one of the three residues, the kcat/KM value for the mutant decreases,
indicating a slower reaction rate; however, in the presence of all of them, the kcat/KM values not
only increase, but the ratios of the kcat/KM values in those scenarios versus when the residue is
lacking is more extreme.
Thus, the implications of our findings further the initial findings in the unpublished data
from the Herschlag Lab at Stanford [Sunden et al., 2014]. In other words, not only does there
exist anti-coupling between the Mg2+ ion and D101 residue even though the residues are
separated by more than a 7 Å allosteric interaction, but this interaction appears to occurs through
the D51 residue. This finding is significant in that it verifies that there is redundant coupling
between two seemingly unrelated parts of the active site, which would not typically be expected
to interact due to the 7 Å gap; further, there is a third, unrelated residue involved in this linkage.

19. 19
It opens a relevant gap in the literature with regards to why there is such an interaction within the
Alkaline Phosphatase active site, and whether these sorts of unlikely interactions have been
disregarded but are actually more prevalent than expected. Further, the broader implication for
the study of Alkaline Phosphatase is why there would be an interaction of this type in the first
place, does it provide the enzyme with a functional advantage in catalyzing the reaction, or is this
redundancy can merely be a coincidence that is a side effect of the various other functions of
D101 and Mg2+.
Ultimately, our results strongly suggest that the redundant coupling pathway between the
D101 and Mg2+ residues occurs through the D51 residue, even though it appears as if they should
not be able to form an interaction with each other. The D51 residue, which is situated next to the
Zn2+ bimetallo site, opening the way for broader questions of why this is possible. Additionally,
it moves the discussion towards why the unlikely connection occurs through D51 as opposed to
the number of other residues present in the active site, questions that literature in the field has not
yet explored.
The lab’s main focus is in the fundamental understanding of biochemistry. Using
different testing methods, we were able to find how D101 and Mg2+ are connected. To
accomplish this, we had to find a way of comparing different paths and identifying which one is
most likely the way it travels. Though we were able to find that they are redundantly coupled in
the presence of D51 but not in it’s absence, we were not able to rule out a couple other paths to
show how D101 and Mg2+ are connected. One route is through the Mg2+ water molecule which
was proposed as the other possible model; tests still need to be done to confirm/debunk this
alternative model. Other pathways might also exist that are not as apparent, leaving room for
hundreds of unaccounted for possibilities of the related function between D101 and Mg2+.

20. 20
However, our research was able to narrow down that our hypothesized model is the most likely
way the two residues interact in the AP active site.
The conclusions we reached through our research is supported not only in the report but
also in conjunction with the literature [Zalatan et al., 2008] [Bobyr et al., 2011]. Unpublished
data from the Herschlag Lab [Sunden et al., 2014] confirmed that the D101 and Mg2+ residues
interact in several different backgrounds; however, these studies did not indicate how or why
they interact. Our experiments and results have supported these findings and added to them by
providing reasonably strong evidence of the redundant coupling between D101 and Mg2+
through D51. Further, the data is self-supporting in that the cubic double mutant cycles created
to explain the relationship between D101 and Mg2+ supports the connection in each of the 6
double mutant cycles. The cubic double mutant cycle compares the kcat/KM values for the D51G
mutants in the TM Background and provided convincing evidence to support our model of
redundant coupling of D101 and Mg2+ through D51.
There are more experiments we could do to further verify our findings. The most logical
experiment to perform would be similar tests in a D51N (Asparagine) background, which is a
more conservative structural mutation than glycine, to further verify our findings. Next, we
could express more mutants to explore the effects of different residue combinations on the
reaction rates of the AP mutant, specifically, with the S102 residue. The S102 residue is of
importance because of it located adjacent to the Zn2+ bimetallo site, which is located in the
middle of the proposed pathway between D101 and Mg2+ (Figure 3). Furthermore, x-ray
crystallography tests need to be completed to confirm the positioning and determine whether
any mutations that we expressed changed the shape or conformity of the active site of the AP
mutant in consistency with our models. If there was one thing we had to change in the way we

21. 21
performed the work, it would be the organization of our procedure. What we ended up doing
was performing many of the preliminary steps in our method first (i.e. purified many enzyme
mutants) and leaving kinetic assays and Michaelis Menten data analysis until later on in the
process. This was not the best strategy because if we discovered later in the process that we did
not have enough enzyme, or that our enzyme did not catalyze the reactions when put in the
kinetic assays, then we had to go restart the process for creating the mutant, which is always
easier done closer to the initial time we expressed and purified the mutant.
There are several questions that still remain unanswered after our experiment. First of
all, our test provides compelling evidence for a redundant coupling through D51, but more tests
can be done to support the conclusion we arrived at. Second, now that we know that there is
redundant coupling through D51 and have a model for how the Mg2+ and D101 are connected,
the next logical question is why they are connected. Third, what is the molecular basis for the
redundant coupling? There are several potential models, including: 1. Nucleophile S102
positioning (less well positioned nucleophile results in less catalysis), 2. Backbone amide that
can position substrate, or 3. Zn2+ positioning (the one above S102 that also interacts with a non-
bridging oxygen on the substrate). Now that we have determined that Mg2+ and D101 are
connected through D51, the Herschlag Lab will continue to study these models and uncover the
molecular basis for the redundant coupling.

22. 22
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