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Advanced Biochemistry Lab

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  • 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)

  • 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

  • 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.

  • SDS Page

    Sample purity was confirmed by 12% SDS-PAGE analysis. 20 L 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 25C did not change (relative to error) compared to the

  • 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

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