advanced biochemistry lab

17
An Investigation of the Importance of Amino Acids 117 and 118 in the Kinetic Regulation and Stability of E. coli Alkaline Phosphatase Hannah Barber Laboratory Partners: Meghann Kallsen Andrew Brownfield Eric Rogers University Wisconsin- La Crosse 1725 State Street Department of Chemistry and Biochemistry La Crosse, WI 54601 Wild-type E.coli alkaline phosphatase with a modified 6 histidine tail (EcAP), site-directed mutation of asparagine found at amino acid 117 to a phenylalanine in EcAP (N117F), human placental alkaline phosphatase (HuPl), Wild- type E.coli alkaline phosphatase and N117F Mutant alkaline phosphatase (APases), cell free extract (CFE), para- nitrophenylphosphate (pNPP), ethylenediaminetetraacetic acid (EDTA), tetramethylethylenediamine (TEMED), ammonium persulfate (APS), tris(hydroxymethyl)aminomethane (tris), magnesium and zinc salts (MZ salts), poly ethylene imide (PEI), site-directed mutagenesis (SDM), and sodium dodecyl sulfate (SDS)

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Page 1: Advanced Biochemistry Lab

An Investigation of the Importance of Amino Acids 117 and 118 in the

Kinetic Regulation and Stability of E. coli Alkaline Phosphatase

Hannah Barber

Laboratory Partners:

Meghann Kallsen

Andrew Brownfield

Eric Rogers

University Wisconsin- La Crosse

1725 State Street

Department of Chemistry and Biochemistry

La Crosse, WI 54601

Wild-type E.coli alkaline phosphatase with a modified 6 histidine tail (EcAP), site-directed mutation of asparagine

found at amino acid 117 to a phenylalanine in EcAP (N117F), human placental alkaline phosphatase (HuPl), Wild-

type E.coli alkaline phosphatase and N117F Mutant alkaline phosphatase (APases), cell free extract (CFE), para-

nitrophenylphosphate (pNPP), ethylenediaminetetraacetic acid (EDTA), tetramethylethylenediamine (TEMED),

ammonium persulfate (APS), tris(hydroxymethyl)aminomethane (tris), magnesium and zinc salts (MZ salts), poly

ethylene imide (PEI), site-directed mutagenesis (SDM), and sodium dodecyl sulfate (SDS)

Page 2: Advanced Biochemistry Lab

Introduction

Alkaline phosphatases share a highly conserved amino acid sequence among most of their superfamily. These

enzymes are found in almost all aspects of life as they are responsible for the important cleavage of the

phosphodiester bond [1]. There are two known regulatory sites identified on E.coli alkaline phosphatase (EcAP). The

Mg2+ regulatory site is responsible for utilization of a water molecule for the catalysis mechanism. The nucleophilic

attack of the serine-histidine-aspartate catalytic triad in the second regulatory site is responsible for the activation of

the Serine 102 residue [2]. The efficiency of this catalytic step is dependent on both the bonds formed with the Zn2+

ion, as well as the activation of the serine residue by the low barrier hydrogen bond formed between the aspartate and

histidine residues [3]. The wild-type stain of E. coli alkaline phosphatase (WT) is almost identical to the human

placental alkaline phosphatase (HuPl) in its regulatory site structure and function. It differs slightly in the amino

acids found at positions 117 and 118 (Figure 1). The differences in the amino acid sequences can be attributed the

different inhibition patterns found between these enzymes. The activity of HuPl is competitively inhibited in the

presence of phenylalanine, while the change in activity of EcAP is minimal [4]. In order to better understand the

HuPl catalytic regulatory site, the amino acid sequences were aligned and synonymous mutations were made to

EcAP in an attempt to mirror the inhibition pattern found in HuPl [5]. With these single site mutations, we expect to

see a decrease in activity in the EcAP mutants that better reflect the inhibition patterns found in HuPl. Our research

team is working with a variety of SDM mutants (N117F, N117Y, G118R, and G118Q) in hopes of better

understanding what amino acids are responsible for the inhibition patterns found in HuPl.

Materials and Methods Preparation of Solutions

Solutions of 1 M tris, 3 M NaCl, and 1 M imidazole were prepared individually from their corresponding salts. Both

the tris and the imidazole solutions were brought to a pH of 8.0. A solvent solution, Buffer A, was prepared at a

concentrations of 10 mM tris and 300 mM NaCl. A Buffer B solution was prepared at concentrations of 10 mM tris,

300 mM NaCl, and 400 mM imidazole. Dialysis buffer was prepared at concentrations 10 mM tris, 50 mM NaCl, and

1 mM EDTA.

Cellular Lysis

The volume of APase was approximated and twice the volume was added in Buffer A. The solution was vortexed

until homogenous and placed on ice. Four rounds of sonication were performed using a Branson Sonicator with

microtip attachments at a setting of five. The samples were placed on ice between each round. PEI was added to the

sonicated cells to adjust the solution to a concentration of 0.4% PEI and mixed by inversion. The samples were then

Page 3: Advanced Biochemistry Lab

centrifuged at 13,000 rpm for 10 minutes. The supernatant was recovered and the final volume recorded. A 200 !L

aliquot was removed for control quantification.

Protein Concentration

The protein concentration was read using a NanoDrop 2000 at an absorbance reading of 280 nM. The extinction

coefficient was 33140 M-1cm-1 for both APases and the molecular weights for WT and N117F were 49773.1 g/mol

and 49740.6 g/mol respectively.

EcAP Activity Assay

The activity was measured for both the CFE and the purified sample using a SPECTRA MAX 190 microplate reader.

The test tubes eluted in the last peak were tested individually for activity. Each well contained 0.1 M tris, 1X MZ

salts, and a final enzyme concentration of 10 nM. A concentration of 0.5 mM pNPP was added prior to reading. Test

tubes with significant activity were pooled and placed in dialysis buffer for 48 hours. The dialysis solution was

exchanged twice during this time.

Amicon

Ideal protein concentrations were stored in aliquots ranging from 50 !M to 250 !M. If the concentration was below

50 !M, the solution was amiconed to filter out excess buffer and salt. If the concentration exceeded 250 !M the

solution was diluted in Buffer A.

Protein Purification

His-tagged EcAP was purified from the CFE sample using Ni2+-NTA chromatography. A program created in

BioLogic LP was set-up to linearly increase the concentration of Buffer B from 6% to 100%. The solution was

collected in test tubes based on the order of the elution time. The test tubes corresponding to the final peak were

tested for activity.

Michaelis-Menten Kinetics

Michaelis-Menten data was collected varying the concentration of pNPP from 5 !M to 500 !M. Both WT and N117F

enzymes were diluted to a final concentration of 20 nM in solution containing 0.1 M tris, 1X MZ salts, and varying

concentrations of pNPP. The activity was then multiplied through by the enzyme factor, 0.036709, to obtain the

corrected velocity.

Page 4: Advanced Biochemistry Lab

SDS Page

Sample purity was confirmed by 12% SDS-PAGE analysis. 20 µL of 0.5 mg/ml protein containing 1X loading dye

was loaded alongside Ladder #7780S. The gel was run at 200V until the dye front ran off the gel.

Temperature Testing

As a preliminary measure, temperature testing was preformed using the same concentrations found in the Arrhenius

plate set-up to ensure the temperature chosen for the construction of the Arrhenius plot did not denature the enzymes

(See Arrhenius Data Collection section).

Arrhenius Data Collection

From the temperature testing results, five temperatures were chosen with as much variance as possible. Plate set-up

was prepared the same across each temperature. Each plate contained 20 nM of enzyme, 0.1 M tris, 1X MZ salts, and

pNPP concentrations ranging from 5 !M-500 !M. A total of three plates were run at all five temperatures for each

enzyme.

Results

In order to analyze the data collected, the purification of the APases needed to be confirmed. The percent

yield was 51% for the WT EcAP with a fold purification of 2.85 (Table 1). N117F had a 32% yield with a fold

purification of 2.40 (Table 1). In addition, the SDS-page gel displayed in Figure 2 shows a single band present at the

approximate molecular weight of N117F (47,773 g/mol), which further confirmed the purification process. Since the

specific activity of the N117F mutant was a 4-fold lower than WT, the mutant was incubated at room temperature in

the presence of 0.1 M tris and 1X MZ salts. It was observed that the increase in activity was directly related to the

amount of time the enzyme was allowed to incubate in this solution.

The mutant purification results were compiled for N117F, N117Y, G118Q, and G118R. It was concluded

after multiple attempts at purification of mutant N117Y, that purification of N117Y through this process was

inconclusive. The relative changes in kcat and Km values were noted as a reference for data collection (Figure 3). The

velocity of the reaction for the three mutants and WT EcAP [Purified by Andrew Brownfield] were tested at varying

temperatures for Arrhenius data collection.

The results from the Arrhenius plot seemed to correlate with what was predicted despite the addition of

vanadate to each plate. Arrhenius data collection is typically run without an inhibitor, but the competitive nature of

the regulatory site kept the Vmax value relatively constant with the addition of the10 nM vanadate, which is a known

inhibitor for both WT and mutant enzymes [Reference Meghann Kallsen: Inhibitor Testing]. Since the enzyme

concentration was kept at 20 nM, the resulting kcat values at 25°C did not change (relative to error) compared to the

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kcat values calculated after protein purification for WT, G118R, and G118Q (Table 4). The difference in the kcat value

for N117F did fall outside the standard deviation. It is possible that since the kcat values were obtained at different

times, the error in these values could be attributed to amount of time N117F was allowed in incubate in the 10% MZ

salt solution which would have a significant effect due to its low activity. The data however was averaged for each

mutant in hopes of obtaining the general trend displayed by each mutant.

The Arrhenius plot showed a similar transition state energy for WT, G118R, and G118Q, however the

transition state energy appeared to drastically increase for G118R (Figure 4, Table 4). Due to inconsistent data

collection for G118R across the increasing temperature range, the experiment would need to be re-run in order to

draw any accurate conclusions. Another error in experimental set-up was found in the incubation time allowed

among replicate data sets. The rate of the reaction was directly related to the temperature, so it was crucial to keep the

incubation time of each plate constant. The large degree of error found in the Arrhenius data can be attributed to this

inconsistency. It took approximately 2 minutes to read each plate and since all three replicate experiments were set

up in the same plate, the third plate was allowed an incubation time approximately four minutes longer than the first

plate. This showed a significant increase in the rate of the reaction (Figure 5). The effect the incubation time had on

the reaction rate became increasingly more evident at higher temperatures. Since this trend was found consistently

across all enzymes, the average was taken in order to compare the relative effect of temperature on the transition

state. In order to form a more complete understanding of the exact kinetics of these enzymes, data would need to be

collected in which the allowed incubation time was monitored and kept consistent.

Discussion

The data collected from the purification of N117F confirmed the fact that a mutation of the asparagine found

at position 117 to a phenylalanine greatly reduced the enzymes ability to catalyze the reaction. The specific activity

appeared to be reduced by a 4-fold in N117F. Although the decrease in specific activity is significant, the relative

accuracy of these activities can only be approximated due to differences in experimental set-up. Since the activity of

N117F is significantly lowered by the mutation, it became increasingly more sensitive to certain chemicals used in

the procedure. In particular it was found that the dialysis buffer contained EDTA, which is a known metal chelate.

The EDTA present in the dialysis buffer was predicted to have stripped the APase of the zinc and magnesium ions

essential for the binding and catalysis of the substrate. The WT enzyme was somehow able to overcome this setback

by either forming stronger bonds to the metal ions or the enzyme was more efficient at re-binding the metal ions that

were lost during its brief interaction with the MZ salts before the plate was read. Regardless of the mechanism used

by WT, N117F lacked the ability to maintain the active site ions after being exposed to EDTA. An incubation of

N117F in a 10% MZ salt solution was necessary in order to visualize activity. This increased the velocity of N117F,

but since WT was not incubated in a similar solution a direct comparison cannot be made. However, this discrepancy

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was caught early enough that a laboratory partner was able to preform the purification of the WT sample in a similar

manner.

The mutants purified by each member of research team were brought together for analysis of the purification

result. The process was successful in purifying mutants, N117F, G118R, G118Q, and WT. Attempt at purification of

N117Y was performed multiple times and for an unknown reason was not able to be purified following the same

technique and was disregarded for the remainder of the lab. Kinetic experiments were run on each of the three

mutants and WT in hopes of better understanding the effect these mutations had on the rate of the reaction. Circular

Dichroism Spectroscopy and Dynamic Light Scattering techniques were also run in hopes of uncovering the changes

in size or structural stability that may have arose from these mutations.

An Arrhenius plot was constructed in hopes of better understanding the changes in the transition state energy

that may have arose between the WT and the mutants. The Arrhenius plot is typically run without inhibitor, but since

competitive inhibition does not change the Vmax value the addition of 10 nM vanadate did not cause a change in the

kcat value. This was confirmed in comparing the kcat values obtained following protein purification to the kcat values

extracted from the Arrhenius plot at 25°C. The Arrhenius plot showed similar activation energies among WT,

G118Q, and N117F, but a drastic jump in activation energy seemed to appear in G118R. The standard deviation in

the linear fit of G118R was large and initially the point at 25°C seemed to be an outlier. Upon comparison of the kcat

values found earlier in the semester, the value at for G118R at 25°C remained relatively consistent. It would then be

suspected that the values for 30°C and 35°C were in fact the outliers, but with no data to support this assumption both

points remained. However, if further testing of G118R proved that the 30°C and 35°C points were an inaccurate

representation of the kcat value at these temperatures, the linear fit of G118R would most likely result in a transition

state energy similar to WT. N117F was also thought to have an outlying point at 35°C and was this point was initially

deleted. The results of the Circular Dichroism Spectroscopy experiment predicted a melting temperature of 35°C and

if this was correct, a decrease in the kcat would be expected due to the denaturation of the enzyme [Personal

communication Andrew Brownfield]. The increase in activity of N117F again at 40°C and 45°C, however remained

unexplained. This increase in activity at temperatures of 40°C and 45°C could be a result of residual enzyme leftover

in the wells that was activated at higher temperatures or it could be due to the nature degradation of pNPP. These

hypotheses would require further tests that would include running a control plate without added enzyme in an attempt

to recover an activity similar to the one displayed for N117F at temperatures of 40°C and 45°C. Also it would be

beneficial to run each experiment again to ensure the above values are in fact trends and not due a universal error in

plate set-up.

A significant decrease in activity based on these mutations can be seen in the column fraction table and

although the Arrhenius plot appears to correlate with these findings, it is impossible to know the accuracy of these

results without running further tests. The competitive inhibition pattern found in WT appears to still competitively

affect each of the mutants as the kcat did not significantly change with the addition of the vanadate. The results of the

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Van’t hoff analysis would have be key in validating the effects of inhibitor binding, but unfortunately the huge

standard deviations caused analysis to be inconclusive [Personal Communication Meghann Kallsen]. The Km values

in the column fraction table also remain constant between enzymes, indicating that the substrate binding is not

affected by these mutations. The decrease in turnover rate could stray away from kinetics altogether and cause a

decrease in the structural stability of the enzymes that is caused by their unique mutations. It is impossible to form

any conclusions based on the data obtained. General trends may be pulled from this data and compared to future

results, but due to the large error in standard deviations these conclusions say little on their own.

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!

!

Figure 1: Pymol images of the A) the asparagine (blue) located at amino acid 117 in EcAP regulatory catalytic site, B) the phenylalanine (blue) located at amino acid 107 in the HuPl regulatory catalytic site displaying the interaction distances formed with the incoming phenylalanine molecule (yellow) and C) the overlay of the regulatory sites for EcAP and HuPl highlighting (blue) the similarities in positioning of N117 in EcAP and F107 in HuPl. [Image created by Andrew Brownfield]

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Table 1: WT and N117F EcAP Column Fraction Table Sample Volume Activity Total

Activity Protein Conc. Total

Protein Specific Activity

% Yield Fold Purification

mL U/mL U mg/mL mg U/mg WT CFE 2.4 210 ± 8 510 31.0 ± 0.5 74 6.9 100% 1 WT Purified N117F CFE N117F Purified

1.4 1.7 2.0

180 ±!20 56 ± 30 15 ± 7

260 96 30

9.47 ±!0.04 24.11±!0.08 2.689 ± 0.005

13 41 5.4

20 2.3 5.6

51% 100% 32%

2.9 1 2.4

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Table 2: Column Fraction Table for WT and Mutant EcAP used in Arrhenius Data Collection Sample Volume Activity Total

Activity Protein Conc. Total

Protein Specific Activity

% Yield Fold Purification

mL U/mL U mg/mL mg U/mg WT* 4.5 270 ± 20 1200 3.41 ± 0.02 15 38 80% 2.3 G118R G118Q N117F

1.5 1.35 2.0

90 ±!20 140 ± 50 15 ± 2

100 180 30

1.5 ±!0.2 5.98±!0.04 2.689 ± 0.005

2.2 8.1 5.4

60 17 5.6

40% 14% 32%

20 0.83 2.4

*Wild-type enzyme purified by Andrew Brownfield

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*Wild-type enzyme purified by Andrew Brownfield

!!

! Figure 2: 12% acrylamide SDS Page gel displaying approximate molecular weights of purified WT and mutant EcAP against a 7780S ladder.

Ladder G118Q WT N117F Ladder (N117Y) G118R WT*

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A)!!

!

B)!!

!

Figure 3: A) Normalized and B) Specific velocity plots displaying average enzymatic velocity of WT EcAP (20 nM) vs. [pNPP] (0.5 !"-50 !") and mutant EcAP (20 nM) vs. [pNPP] (5!!"-500!!") [Both WT and mutant enzymes were ran in a solution containing 0.1 M tris, 1X MZ salts, and varying concentrations of pNPP.]

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Table 3: Kinetic Parameters of WT and Mutant EcAP Sample Vmax Km kcat kcat/Km !M/min !" sec-1 M-1sec-1 WT* 3.0 ± 0.1 14 ± 1 2.5 ± 0.3 3.6 x 105 G118R 1.6 ± 0.2 12 ± 1 1.3 ± 0.2 1.1 x 105 G118Q 1.9 ± 0.2 12 ± 4 1.6 ± 0.2 1.4 x 105 N117F 0.65 ± 0.08 13 ± 4 0.56 ± 0.03 4.4 x 104 *Wild-type enzyme purified by Andrew Brownfield

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!

! Figure 4: Arrhenius plot of WT and mutant enzymes ran at 20 nM in solutions containing 0.1 M tris, 1X MZ salts, 10 nM NaVO4, and varying concentrations of pNPP.

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Figure 5: Arrhenius plot displaying the effect of incubation time on the rate of reaction for G118Q. Each plate contains solutions at 20 nM G118Q, 0.1 M tris, 1X MZ salts, 10 nM NaVO4, and varying concentrations of pNPP. Incubation times varied from no incubation time in plate 1 to a 4-minute incubation for plate 3 at each temperature.

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Table 4: Transition State Energies and kcat Values from Arrhenius Plot at 25°C for WT and Mutant EcAP Sample Reference kcat (No Inhibitor)* kcat (10 nM NaVO4) △H∓ sec-1 sec-1 kJ/mol WT* 2.5 ± 0.3 2.7 ± 0.1 18 ± 7 G118R 1.3 ± 0.2 1.6 ± 0.8 70 ± 10 G118Q 1.6 ± 0.2 1.44 ± 0.03 19 ± 3 N117F 0.56 ± 0.03 0.38 ± 0.04 11 ± 3 *Wild-type enzyme purified by Andrew Brownfield *Reference kcat calculated after protein purification (as seen in Table 3) !!!!!!!!!!!!!!!!!!!!!!

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References

1. Galperin, M.Y., Jedrzejas, M.J. (2001) Conserved Core Structure and Active Site Residues in Alkaline

Phosphatase Superfamily Enzymes. Protein.45, 318-324

2. Kim, E.E., Wyckoff, H.W. (1991) Reaction mechanism of alkaline phosphatase based on crystal structures.

Two-metal ion catalysis. J. Mol. Biol. 218, 449-464.

3. Chaidaroglou A, Brezinski JD, Middleton SA, Kantrowitz ER. (1988) Function of arginine in the active site

of Escherichia coli alkaline phosphatase. Biochemistry 27:8338-8343.

4. Zhang, L., Buchet, R., & Azzar, G. (2004). Phosphate Binding in the Active Site of Alkaline Phosphatase

and the Interactions of 2-Nitrosoacetophenone with Alkaline Phosphatase-Induced Small Structural

Changes. Biophysical Journal, 86(6), 3873–3881.

5. Le Du, M.H., Stigbrand, T., Taussig, M.J., Menez, A., and Stura, E.A. (2001). Crystal Structure of Alkaline

Phosphatase from Human Placenta at 1.8 Å Resolution. J. Biol. Chem. 276, 9158-9165.

!