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Jul 2020

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An in vitro Coupled Assay for PEPC with Control of Bicarbonate Concentration
一种控制碳酸氢盐浓度的磷酸烯醇式丙酮酸羧化酶的体外偶联分析   

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Abstract

Phosphoenolpyruvate carboxylase (PEPC) catalyzes a critical step in carbon metabolism in plants and bacteria, the irreversible reaction between bicarbonate and phosphoenolpyruvate to produce the C4 compound oxaloacetate. This enzyme is particularly important in the context of C4 photosynthesis, where it is the initial carbon-fixing enzyme. Many studies have used kinetic approaches to characterize the properties of PEPCs from different species, different post-translational states, and after mutagenesis. Most of these studies have worked at a fixed saturating concentration of bicarbonate. Controlling the concentration of bicarbonate is difficult at low concentrations because of equilibration with atmospheric CO2. We describe here a simple, repeatable, and gas-tight assay system for PEPC that allows bicarbonate concentrations to be controlled above ca. 50 µM.


Keywords: PEPC (PEPC), C4 (C4), Bicarbonate assay (碳酸氢盐测定), Gas-controlled assay (气控化验), Malate dehydrogenase coupled assay (苹果酸脱氢酶偶联试验)

Background

The enzyme phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) catalyzes the essentially irreversible reaction between bicarbonate and phosphoenolpyruvate (PEP) to form oxaloacetate and inorganic phosphate. This reaction is a critical step in carbon metabolism in plants and bacteria, but the enzyme is most widely studied in the context of its critical role in C4 photosynthesis, where it is responsible for primary carbon fixation. In typical PEPC assays, the enzyme is coupled to malate dehydrogenase (MDH), which converts oxaloacetate to malate, consuming NADH, and leading to a decrease in absorbance at 340 nm. This reliable coupled assay has been used extensively to compare PEPCs from different species and to understand the consequences of post-translational modification on inhibitor binding and affinity for PEP (Janc et al., 1992; Duff et al., 1995; Blasing et al., 2000; Jacobs et al., 2008; Paulus et al., 2013). Studies where the concentration of bicarbonate is varied are more challenging, as background bicarbonate, arising from equilibration with atmospheric CO2, is found at concentrations above the Km for bicarbonate (often less than 100 µM), preventing accurate kinetic measurements. Measuring the rate of the PEPC reaction at low bicarbonate concentrations requires that background bicarbonate be removed as much as possible, and careful gas-tight assay procedures. Accurate kinetic measurements also require determining the concentration of residual bicarbonate, allowing calculation of the correct concentration of substrate.


We describe here a set of methods to remove most background bicarbonate, quantify the remaining amount, and reliably assay the enzyme without significant contamination from atmospheric CO2. Using these methods, we can reliably reduce background bicarbonate to less than 50 µM.


Overview: The methods described here are simple and repeatable. The major assay components, buffer and water, are sparged with nitrogen gas to reduce the background bicarbonate concentration. Assays are constructed and sealed under nitrogen to minimize contamination from atmospheric carbon dioxide. Assays are then initiated by the addition of PEPC, delivered with a gastight syringe. Background bicarbonate is determined using an endpoint assay.


Limitations: While we have found these methods suitable for measuring the properties of a wide range of PEPC enzymes (Bauwe, 1986; Phansopa et al., 2020), they are not universally applicable. For PEPC enzymes with Kmbicarbonate much below the residual background bicarbonate, the experimental design and data analysis described here are not suitable, so an alternative approach using the integrated Michaelis-Menten equation is recommended. This alternative approach requires additional controls to overcome problems associated with product inhibition, enzyme instability, and product instability leading to regeneration of CO2 in solution; these are not described here and the interested reader should consult the careful work of Bauwe (1986), and DiMario and Cousins (2019).


Also, as the product oxaloacetate is converted into malate in the assay described here, it is not possible to carry out product inhibition studies using oxaloacetate. This limitation could be resolved by detecting a substrate or the other product. An elegant alternative detection system for gaseous CO2, coupled to the substrate concentration through carbonic anhydrase, has been described using membrane-inlet mass spectrometry (DiMario and Cousins, 2019). Our attempts to develop an alternative assay to monitor the production of inorganic phosphate using the purine nucleotide phosphorylase assay (Webb, 1992) were unsuccessful, due to inhibition of PEPC by components of the coupling system (unpublished work).

Materials and Reagents

  1. Pipette tips

  2. 0.2 µm pore filter

  3. 1.5 ml mini-centrifuge tubes (Eppendorf, catalog number: 0030120159)

  4. 0.5 ml sealable UV-Cuvette (Fisher Scientific, catalog number: 10386712)

  5. Whatman membrane filters, nylon pore size 0.2 μm, diameter 47 mm (Millipore Sigma, catalog number: WHA7402004)

  6. Parafilm (Bemis, catalog number: 11772644)

  7. Two rubber septa for ST/NS 24/40 joint (Millipore Sigma, catalog number: Z553980)

  8. Two glass stoppers for ST/NS 24/40 joint (Millipore Sigma, catalog number: Z229571)

  9. 12.5 mm rubber septa for sealing cuvettes (Millipore Sigma, catalog number: Z167274)

  10. Three pieces of 1 m long 4 mm bore rubber tubing (Fisher Scientific, catalog number: 11876293)

  11. A glass funnel

  12. Two 5 ml syringes (Terumo, catalog number: Z116866)

  13. Two 120 mm, 21-gauge needles (Sterican, catalog number: 466 5643)

  14. Two 40 mm, 21-gauge small needles (BD Microlance 3, catalog number: 304432)

  15. PEPC Enzyme: Wild type or mutant PEPC purified from E. coli, stored at -80°C

  16. Phosphoenolpyruvate trisodium salt (PEP, Millipore Sigma, catalog number: P7002), store at -20°C

  17. Malate dehydrogenase enzyme (MDH, Millipore Sigma, catalog number: M2634), store at 4°C

  18. β-Nicotinamide adenine dinucleotide (NADH, Millipore Sigma, catalog number: 10107735001), store at -20°C

  19. Magnesium chloride (MgCl2, Millipore Sigma, catalog number: 63069)

  20. Potassium bicarbonate (KHCO3, Millipore Sigma, catalog number: 60339)

  21. Tricine (Millipore Sigma, catalog number: T0377)

  22. Tris(hydroxymethyl)aminomethane (Tris base) (Millipore Sigma, catalog number: 252859)

  23. Potassium chloride (KCl, Millipore Sigma, catalog number: T0377)

  24. Potassium hydroxide (KOH, Millipore Sigma, catalog number: 757551)

  25. Ultrapure deionised water

  26. Tris-HCl (see Recipes)

  27. Tricine-KOH (see Recipes)

Equipment

  1. Pipettes

  2. 1700 series gastight syringe with a cemented needle (Hamilton, catalog number: 80200)

  3. Nitrogen gas supply

  4. Nitrogen gas canister (99.998% minimum N2, BOC, catalog number: 44-W)

  5. Two 250 ml two-neck round bottom flasks (Millipore Sigma, catalog number: Z516872)

  6. Glass single bank manifold with three positions (Millipore Sigma, catalog number: Z532169)

  7. Magnetic stirrers

  8. Magnetic stirrer plate

  9. A split-beam spectrophotometer with temperature control. For example, a Cary 300 UV-Vis spectrophotometer (Agilent). A cell changer is not essential but it is convenient.

Software

  1. Cary WinUV software (Agilent, www.agilent.com)

  2. Igor Pro (Version 7.0.8.1; Wavemetrics Inc., Lake Oswego, Oregon, www.wavemetrics.com) or equivalent package capable of non-linear regression analysis.

Procedure

  1. Preparation of CO2-free assay components

    This procedure takes more than 12 h, so should be begun the day before assays are planned. The aim is to remove as much dissolved HCO3 as practically possible, by sparging the major assay components with nitrogen gas. Water and buffer make up ca. 75% of the assay solution, and are sufficiently stable to be sparged with nitrogen for extended periods. A typical degassing set-up will involve one solution of buffer and one solution of deionised water (Figure 1).



    Figure 1. A typical degassing set-up to reduce the concentration of dissolved CO2 in the assay components.


    1. Prepare buffer stock solutions (200 mM Tris-HCl pH 7.4, or Tricine-KOH pH 8.0, as described below) and obtain ultrapure water. Decant the solutions (ca. 50 ml) into separate double-neck round bottom flasks, and add a magnetic stirrer. Seal one of the necks of the round bottom flasks with a rubber septum, and seal the other with a glass stopper. Septa may be reinforced with parafilm to prevent gas leaks.

    2. Through one septum, pass a long needle, ensuring the tip of the needle is submerged in the assay component. Pass a short needle through the same septum. Create a manifold for the needle by inserting the end of the tubing into the syringe body. Use parafilm to seal the join between the syringe body and the tubing. Connect the long needle to the nitrogen supply.

    3. To begin the sparging process, turn on the gas supply indicated by bubbles emanating from the long needle. Ensure the bubbles are produced at no more than ca. one bubble per second. A faster supply will result in the loss of assay solution. Turn on the magnetic stirrer. Check all joins for any nitrogen leaks, and patch with parafilm if necessary.

    4. Extensively sparge with nitrogen in the tightly sealed container for 12 h prior to use.

    5. After a sparging period, turn off the nitrogen supply. Connect the set-up to a high-purity nitrogen cylinder. Bubble the gas for 1 h prior to the first assay at a similar rate, ca. one bubble per second.

    6. Remove the needles and seal the flask until use. As some background bicarbonate will remain after this procedure, the background concentration will need to be determined as described below in Step B4 and Procedure C. The reduced bicarbonate concentration will be stable for eight hours.


  2. Instrument set-up, reagent preparation, and quality control

    The concentration determinations described here must be carried out every day that kinetic measurements will be performed, and not just when stock solutions are prepared, defrosted, or enzymes purified. It is convenient to measure the concentrations of enzyme and substrates during the final sparge of buffer with high-purity nitrogen (1 h, as described in Step A5).

    1. Instrument set-up

      This procedure requires access to a high-quality split-beam spectrophotometer with temperature control. We have not found plate-readers suitable, due to the difficulties in preventing assays equilibrating with atmospheric CO2; if this could be solved, this procedure would be much less laborious. The instructions are given for Cary spectrophotometers, but could easily be adapted to equipment from any comparable supplier. The spectrophotometer equipment will require room for the cuvette to be seated with a gastight seal.

      Turn on the instrument at least 30 min before beginning measurements to allow the lamp to stabilize. Turn on the water bath or Peltier temperature control and set to 25°C.

    2. Preparation of coupled assay materials (malate dehydrogenase and NADH)

      Remove NADH from the freezer (-20°C) and allow it to come to room temperature before opening to prevent condensation. Prepare a NADH solution [355 mg in 25 ml for a ca. 20 mM stock in water (MiliQ)]. Determine the concentration spectrophotometrically (ϵ340 = 6220M–1cm–1). These solutions can be frozen (-20°C) in aliquots and remain stable for approximately 1 month. Do not refreeze. Malate dehydrogenase is stored at 4°C until use.

    3. Preparation of phosphoenolpyruvate (PEP) stock solution.

      Remove from the freezer (-20°C) and allow to come to room temperature before opening to prevent condensation. Prepare a stock solution (15 ml of total solution with 4.3 g PEP should result in ca. 400 mM PEP solution). As PEP is supplied with an unknown amount of water of hydration, the concentration needs to be determined spectrophotometrically by an endpoint assay. Prepare assays in triplicate as described below (Procedure C) with 10 mM bicarbonate and 100 µM PEP (estimated). A ΔA340 = 0.622 is expected under these conditions; determine your stock PEP concentration using the extinction coefficient above. In this assay, it is essential that bicarbonate is in excess of PEP. This stock solution can be frozen (-20°C) and remains stable for a month.

    4. Determination of background bicarbonate (total dissolved CO2).

      The effectiveness of removing background CO2 needs to be determined with an endpoint assay. If the background bicarbonate concentration is above 50 µM, the major components will need to be sparged for a further hour with high purity nitrogen. Prepare assays in triplicate as described below (Procedure C) with no added bicarbonate and 20 mM PEP. It is essential that PEP is in excess of the background bicarbonate.


  3. Endpoint assay of PEP and background bicarbonate

    These assays for PEP and background bicarbonate are required to establish accurate substrate concentrations. The method is essentially the same as the kinetic assays, except we are interested in the total change in signal at 340 nm, not the initial rate. Concentrations of PEPC used can be adjusted to ensure that essentially full conversion to products is seen in a reasonable time frame. If you have access to a cell changer, these can be run in parallel.

    Troubleshooting tip: If your assays are not gas tight, you will see a slow linear phase in the bicarbonate assay, indicative of gas exchange with the atmosphere.

    1. Connect a glass funnel to the normal nitrogen supply to create a field of nitrogen under which to assemble the assay. Add PEP, MgCl2, NADH, KCl, MDH, and bicarbonate (if required) to the empty cuvette (Table 1). Run the nitrogen through the funnel to create a field of nitrogen, and place the cuvette with the components under the nitrogen. After removing the glass stopper, take the sparged water and buffer from the round-bottomed flasks. It is not recommended to stop the nitrogen flow through the buffer and water until all assays are assembled. Replace the glass stopper as quickly as possible to ensure the round bottom flasks are sealed after use. Pipette mix the assay components under nitrogen and seal with a septum.


      Table 1. PEPC Assay construction

      Component Final Concentration Typical volume Stock Concentration
      Buffer (Tris-HCl pH 7.4

      or Tricine-KOH pH 8.0)

      50 mM 150 µl 200 mM
      H2O (MilliQ, degassed) Adjust to give 0.6 ml total volume.
      MgCl2 20 mM 60 µl 200 mM
      NADH 150 µM 6 µl 15 mM
      MDH a 0.01 U/µl 0.6 µl 10 U/µl
      PEP b Varies 400 mM
      KHCO3c Varies x µl 6 mM
      KCl d y µl = xmax - x µl 6 mM
      PEPC e 50 nM 10 µl 3 µM


      Notes:

      1. MDH concentration varies by batch, adjust volumes accordingly.

      2. Use analytical concentrations of PEP.

      3. Adjust final concentration of bicarbonate to take into account the background. Note that this is total dissolved CO2, the true bicarbonate concentration varies with pH and I.

      4. KCl is added to prevent variation in I resulting from varying the KHCO3 and to keep a constant [K+]; x+y (µl) should be constant.

      5. Concentration of PEPC stocks vary. As 10 µl is a convenient volume to pipette accurately, it is often useful to produce a 3 µM working stock; check this concentration spectrophotometrically.


    2. Place the assembled assay in the spectrophotometer. Measure the absorbance at 340 nm for approximately 30 seconds, to determine the starting absorbance. Pause data acquisition and deliver 50 nM PEPC (final concentration) with a gas-tight Hamilton syringe (generally ca. 100 µl). Gently tip the cuvette from side to side to mix (do not invert) and return the cuvette to the spectrophotometer. Measure the absorbance for 30 min. In this time a stable endpoint will be reached. Record the absorbance of the endpoint. Determine the concentration of background bicarbonate or PEP stock from the difference in absorbance measurements.

    3. Repeat the endpoint assay three times to ensure consistent concentrations are determined. Report concentrations as mean and standard deviation.


  4. Kinetic Assays

    The goal of these assays is to determine an accurate set of initial rates, either at fixed (saturating) concentration of PEP, or varying the concentration of both PEP and bicarbonate. Reaction times and enzyme concentrations should be adjusted as needed, to ensure that data are collected in the initial rate phase of the reaction; progress to completion is not required. Assays are essentially constructed as described above. It is often useful to plot initial rates as you are collecting them; this allows ‘on-the-fly’ adjustment of the experimental design to ensure that an adequate concentration range is covered. In particular, if the lowest concentration of bicarbonate used is above the apparent Km, then modify your design to include additional points at lower concentrations.

    1. Construct the assays as before (Table 1), combining all the non-degassed components on a normal benchtop, then filling the assay with nitrogen gas, adding the sparged components, and sealing the cuvette under the field of nitrogen gas.

    2. Initiate the assay by the addition of 50 nM PEPC with a gas-tight Hamilton syringe (as before). Mix by gently shaking the cuvette from side to side (do not invert).

    3. Introduce the cuvette to the spectrophotometer and start recording. Generally, absorbance at 340 nm is measured for 15 min.

    4. Calculate the initial rate using the instrument software (e.g., kinetic ruler in the Cary WinUV software). For each point, record the concentration of both substrates, enzyme (if varied at any point during the day), initial rate, units (e.g., µMs-1, ΔA340 min–1), and note if the trace looks in any way suspicious (e.g., non-linear, substantially lower or higher rate, or noise than expected). Rates will need to be converted into units of change in concentration with time; this can be done either immediately after the end of the assay or batchwise at the end of the day.

    5. Each concentration point should be run in triplicate. Assays should not be run in order of concentration; vary concentration of substrates between assays to prevent systematic error arising from any time-dependent decay of reagents.

    6. Repeat an endpoint assay halfway through data collection and at the end, to make sure the background bicarbonate has not increased. If it has increased, sparge the buffer solution and water for an hour with high purity nitrogen, and reperform the endpoint assay.

    7. Plot the calculated reaction velocities against bicarbonate concentration in Igor Pro (or other suitable software package), then fit using the appropriate kinetic model. Illustrative curves can be found in the literature (e.g., Figure 1 in Moody et al., 2020). As a guide, we find that a vi/[E] of ca. 40 s-1 are reasonable at saturating PEP and bicarbonate.

Data analysis

At fixed single concentrations of PEP, the data can be analyzed using the Michaelis-Menten equation (Equation 1) where vi/[ET] is the steady-state rate divided by the total enzyme concentration, kcat is the turnover number, Km is the Michaelis constant, and [S] is the substrate concentration. Estimated standard errors of parameter values are provided directly by the software and standard errors in kcat/KM can be estimated using propagation of errors; if following this procedure, it is essential to include the covariance term (Equation 3).

Alternatively, when both substrates are varied, the data can be described by Equation 2, where A and B are the two substrates.

In both cases, choose a range of substrate concentrations that span a 0.1 KM-10 KM range. Generally, 8 to 12 different substrate concentrations should be used when collecting data at a fixed concentration of PEP (i.e., analyzing using Equation 1). When both substrate concentrations are varied, then at least five concentrations of each (i.e., 25 assays in total) should be used.

The standard error in kcat/Km can be calculated from the variances in kcat and Km, and the covariance (Equation 3 [Bevington and Robinson, 1992]). Here σKm, σkcat, σkcat/Km, cov( kcat, Km ) are the standard error of Km, kcat, kcat/Km, and the covariance, respectively. Note that Igor Pro reports the variances ( i.e., σ2) in the variance-covariance matrix.

Recipes

  1. Tris-HCl, pH 7.4

    1. Dissolve 24.23 g of Tris base in ca. 900 ml of water, titrate to pH 7.53 (assuming a lab temperature of 20°C).

    2. Adjust for actual lab temperature using ΔpKa/ΔT = -0.028 and bring to a final volume of 1 L in a volumetric flask. The buffer is then passed through a 0.2 µm pore filter.

  2. Tricine-KOH, pH 8.0

    1. Dissolve 35.84 g of Tricine free acid in ca. 900 ml of water, titrate to pH 8.10 (assuming a lab temperature of 20°C.

    2. Adjust for actual lab temperature using ΔpKa/ΔT = -0.021) and bring to a final volume of 1 L in a volumetric flask. The buffer is then passed through a 0.2 µm pore filter.

Acknowledgments

NRM was funded by was supported by the Grantham Centre for Sustainable Futures, CP was funded by an ERC grant (grant number ERC-2014-STG-638333) and JDR was supported by the Biotechnology and Biological Sciences Research Council (BBSRC, UK; award number BB/M000265/1). This protocol was modified from one described in Janc et al. (1992).

Competing interests

The authors declare no conflict of interests.

References

  1. Bauwe, H. (1986). An efficient method for the determination of Km values for HCO3- of phosphoenolpyruvate carboxylase. Planta 169: 356-360.
  2. Bevington, P. R., and Robinson, D. K. (1992). Data reduction and error analysis for the physical sciences. McGraw-Hill, New York.
  3. Blasing, O. E., Westhoff, P. and Svensson, P. (2000). Evolution of C4 phosphoenolpyruvate carboxylase in Flaveria, a conserved serine residue in the carboxyl-terminal part of the enzyme is a major determinant for C4-specific characteristics. J Biol Chem 275(36): 27917-27923.
  4. DiMario, R. J. and Cousins, A. B. (2019). A single serine to alanine substitution decreases bicarbonate affinity of phosphoenolpyruvate carboxylase in C4Flaveria trinervia. J Exp Bot 70(3): 995-1004.
  5. Duff, S. M., Andreo, C. S., Pacquit, V., Lepiniec, L., Sarath, G., Condon, S. A., Vidal, J., Gadal, P. and Chollet, R. (1995). Kinetic analysis of the non-phosphorylated, in vitro phosphorylated, and phosphorylation-site-mutant (Asp8) forms of intact recombinant C4 phosphoenolpyruvate carboxylase from sorghum. Eur J Biochem 228(1): 92-95.
  6. Jacobs, B., Engelmann, S., Westhoff, P. and Gowik, U. (2008). Evolution of C4 phosphoenolpyruvate carboxylase in Flaveria: determinants for high tolerance towards the inhibitor L-malate. Plant Cell Environ 31(6): 793-803.
  7. Janc, J. W., O'Leary, M. H. and Cleland, W. W. (1992). A kinetic investigation of phosphoenolpyruvate carboxylase from Zea mays. Biochemistry 31(28): 6421-6426.
  8. Moody, N. R., Christin, P. A., and Reid, J. D. (2020). Kinetic Modifications of C4 PEPC Are Qualitatively Convergent, but Larger in Panicum Than in Flaveria. Front Plant Sci 11: 1014.
  9. Paulus, J. K., Schlieper, D. and Groth, G. (2013). Greater efficiency of photosynthetic carbon fixation due to single amino-acid substitution. Nat Commun 4(1): 1518.
  10. Phansopa, C., Dunning, L. T., Reid, J. D. and Christin, P. A. (2020). Lateral gene transfer acts as an evolutionary shortcut to efficient C4 biochemistry. Mol Biol Evol 37(11): 3094-3104.
  11. Webb, M. R. (1992). A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proc Natl Acad Sci U S A 89(11): 4884-4887.


简介

[摘要] 磷酸烯醇式丙酮酸羧化酶 (PEPC) 催化植物和细菌碳代谢的关键步骤,即碳酸氢盐和磷酸烯醇式丙酮酸之间的不可逆反应,生成 C 4化合物草酰乙酸。这种酶在 C 4光合作用的背景下特别重要,它是最初的固碳酶。许多研究使用动力学方法来表征来自不同物种、不同翻译后状态和诱变后的 PEPC 的特性。大多数这些研究都是在固定的饱和碳酸氢盐浓度下进行的。由于与大气 CO 2平衡,在低浓度下难以控制碳酸氢盐的浓度. 我们在此描述了一种简单、可重复且气密的 PEPC 检测系统,该系统允许将碳酸氢盐浓度控制在大约 10倍以上。50 µM。

[背景]磷酸烯醇式丙酮酸羧化酶 (PEPC; EC 4.1.1.31) 催化碳酸氢盐和磷酸烯醇式丙酮酸 (PEP) 之间基本上不可逆的反应,形成草酰乙酸和无机磷酸盐。该反应是植物和细菌碳代谢的关键步骤,但该酶在 C 4光合作用中的关键作用方面得到了最广泛的研究,在 C 4光合作用中它负责初级碳固定。在典型的 PEPC 检测中,该酶与苹果酸脱氢酶 (MDH) 偶联,后者将草酰乙酸转化为苹果酸,消耗 NADH,并导致 340 nm 处的吸光度降低。这种可靠的偶联试验已被广泛用于比较来自不同物种的 PEPC,并了解翻译后修饰对抑制剂结合和 PEP 亲和力的影响(Janc等人,1992;Duff等人,1995;Blasing等人,1995 年)。, 2000; Jacobs et al. , 2008; Paulus et al. , 2013)。碳酸氢盐浓度变化的研究更具挑战性,因为与大气 CO 2平衡产生的背景碳酸氢盐浓度高于碳酸氢盐的K m (通常小于 100 µM),从而妨碍了准确的动力学测量。在低碳酸氢盐浓度下测量 PEPC 反应的速率需要尽可能多地去除背景碳酸氢盐,以及仔细的气密分析程序。准确的动力学测量还需要确定残余碳酸氢盐的浓度,从而计算出正确的底物浓度。
我们在这里描述了一组方法来去除大多数背景碳酸氢盐,量化剩余量,并可靠地检测酶,而不会受到大气 CO 2 的显着污染。使用这些方法,我们可以可靠地将背景碳酸氢盐降低到 50 µM 以下。
概述:此处描述的方法简单且可重复。主要测定成分,缓冲液和水,用氮气鼓泡以降低背景碳酸氢盐浓度。检测在氮气下构建和密封,以最大限度地减少大气二氧化碳的污染。然后通过添加 PEPC 启动检测,使用气密注射器输送。使用终点测定法确定背景碳酸氢盐。
局限性:虽然我们发现这些方法适用于测量各种 PEPC 酶的特性(Bauwe,1986 年;Phansopa等人,2020 年),但它们并不普遍适用。对于K m碳酸氢盐远低于残留背景碳酸氢盐的PEPC 酶,此处描述的实验设计和数据分析不适合,因此建议使用集成 Michaelis-Menten 方程的替代方法。这种替代方法需要额外的控制来克服与产物抑制、酶不稳定性和导致溶液中 CO 2再生的产物不稳定性相关的问题;这些在此不作描述,感兴趣的读者应查阅 Bauwe (1986)和 DiMario 和 Cousins (2019)的细致工作。
此外,由于产物草酰乙酸在此处描述的测定中转化为苹果酸,因此不可能使用草酰乙酸进行产物抑制研究。这种限制可以通过检测底物或其他产品来解决。已经使用膜入口质谱法描述了一种优雅的气态 CO 2替代检测系统,通过碳酸酐酶与底物浓度耦合(DiMario 和 Cousins,2019)。我们尝试使用嘌呤核苷酸磷酸化酶测定法(Webb,1992)开发一种替代测定法来监测无机磷酸盐的产生,但没有成功,因为偶联系统的组分抑制了 PEPC(未发表的工作)。

关键字:PEPC, C4, 碳酸氢盐测定, 气控化验, 苹果酸脱氢酶偶联试验

材料和试剂

1.     移液器吸头

2.     0.2 µm 孔径过滤器

3.     1.5 ml 微型离心管(Eppendorf,目录号:0030120159

4.     0.5 ml 可密封 UV-CuvetteFisher Scientific,目录号:10386712

5.     Whatman 膜过滤器,尼龙孔径 0.2 μm,直径 47 mmMillipore Sigma,目录号:WHA7402004

6.     封口膜(Bemis,目录号:11772644

7.     ST/NS 24/40 接头的两个橡胶隔垫(Millipore Sigma,目录号:Z553980

8.     ST/NS 24/40 接头的两个玻璃塞(Millipore Sigma,目录号:Z229571

9.     用于密封比色皿的 12.5 mm 橡胶隔垫(Millipore Sigma,目录号:Z167274

  1. 三件 1 m 4 mm 内径橡胶管(Fisher Scientific,目录号:11876293
  2. 一个玻璃漏斗
  3. 两个 5 毫升注射器(Terumo,目录号:Z116866
  4. 两个 120 毫米,21 号针(Sterican,目录号:466 5643
  5. 两个 40 毫米,21 号小针(BD Microlance 3,目录号:304432
  6. PEPC 酶:从大肠杆菌中纯化的野生型或突变型 PEPC ,储存于 -80°C
  7. 磷酸烯醇式丙酮酸三钠盐(PEPMillipore Sigma,目录号:P7002),储存在-20°C
  8. 苹果酸脱氢酶(MDHMillipore Sigma,目录号:M2634),储存在4°C
  9. β-烟酰胺腺嘌呤二核苷酸(NADHMillipore Sigma,目录号: 10107735001), 储存于 -20°C
  10. 氯化镁(MgCl Millipore Sigma,目录号:63069
  11. 碳酸氢钾(KHCO Millipore Sigma,目录号:60339
  12. TricineMillipore Sigma,目录号:T0377
  13. 三(羟甲基)氨基甲烷(Tris 碱)(Millipore Sigma,目录号:252859
  14. 氯化钾(KClMillipore Sigma,目录号:T0377
  15. 氢氧化钾(KOHMillipore Sigma,目录号:757551
  16. 超纯去离子水
  17. Tris-HCl(见配方)
  18. Tricine-KOH(见食谱)

设备

1.     移液器

2.     1700 系列带胶结针头的气密注射器(Hamilton,目录号:80200

3.     氮气供应

4.     氮气罐(最低 99.998% N BOC,目录号:44-W

5.     两个 250 ml 双颈圆底烧瓶(Millipore Sigma,目录号:Z516872

6.     具有三个位置的玻璃单排歧管(Millipore Sigma,目录号:Z532169

7.     磁力搅拌器

8.     磁力搅拌板

9.     带温度控制的分束分光光度计。例如,Cary 300 紫外-可见分光光度计(安捷伦)。换细胞器不是必需的,但它很方便。

软件

1.     Cary WinUV 软件(安捷伦,www.agilent.com

2.     Igor Pro7.0.8.1 版;Wavemetrics Inc.,俄勒冈州奥斯威戈湖,www.wavemetrics.com)或能够进行非线性回归分析的等效软件包。

 

程序

A.    CO 2测定组分的制备

此过程需要超过 12 小时,因此应在计划分析的前一天开始。目的是通过用氮气鼓泡主要测定组分来尽可能多地去除溶解的HCO 。水和缓冲液组成约。75% 的测定溶液,并且足够稳定,可以长时间用氮气鼓泡。典型的脱气装置包括一种缓冲溶液和一种去离子水溶液()。

 

 

一种典型的脱气装置,用于降低测定组分中溶解的 CO 2浓度。

 

1.     制备缓冲液储备溶液(200 mM Tris-HCl pH 7.4,或 Tricine-KOH pH 8.0,如下所述)并获得超纯水。将溶液(50 毫升)倒入单独的双颈圆底烧瓶中,并添加磁力搅拌器。用橡胶隔膜密封圆底烧瓶的一个颈部,并用玻璃塞密封另一个。隔垫可以用封口膜加固以防止气体泄漏。

2.     通过一个隔垫,穿过一根长针,确保针尖浸没在化验组件中。将一根短针穿过同一个隔垫。通过将管子的末端插入注射器主体,为针头创建一个歧管。使用封口膜密封注射器主体和管道之间的连接。将长针连接到氮气源。

3.     要开始喷射过程,请打开由长针发出的气泡指示的气体供应。确保气泡的产生不超过大约。每秒一个气泡。更快的供应将导致测定溶液的损失。打开磁力搅拌器。检查所有连接处是否有氮气泄漏,如有必要,用封口膜修补。

4.     使用前在密闭容器中用氮气大量喷射 12 小时。

5.     喷射期后,关闭氮气供应。将装置连接到高纯度氮气瓶。在第一次测定之前,以类似的速率将气体鼓泡 1 小时,大约。每秒一个气泡。

6.     取出针头并密封烧瓶直至使用。由于此过程后将保留一些背景碳酸氢盐,因此需要按照以下步骤 B4和过程 C 中的描述确定背景浓度。降低的碳酸氢盐浓度将稳定 8 小时。

B.    仪器设置、试剂制备和质量控制

此处描述的浓度测定必须在进行动力学测量的每一天进行,而不仅仅是在制备、解冻或纯化酶时。在用高纯度氮气(1 小时,如步骤 A5 中所述)最后喷射缓冲液期间,可以方便地测量酶和底物的浓度。

1.     仪器设置

此过程需要使用具有温度控制的高质量分束分光光度计。我们还没有找到合适的读板器,因为很难防止与大气 CO 2平衡的分析;如果能解决这个问题,这个过程就不会那么费力了。这些说明是针对 Cary分光光度计给出的,但可以很容易地适用于任何可比供应商的设备。分光光度计设备需要空间让比色皿安装气密密封。

在开始测量前至少 30 分钟打开仪器,使灯稳定。打开水浴或 Peltier 温度控制并设置为 25°C

2.     偶联测定材料的制备(苹果酸脱氢酶和 NADH

从冰箱 (-20°C) 中取出 NADH 并在打开前使其达到室温以防止冷凝。制备 NADH 溶液 [355 mg in 25 ml for a ca. 20 mM 水溶液 (MiliQ)]。用分光光度法确定浓度ε 340 = 6220 – cm – )。这些溶液可以分装冷冻 (-20°C) 并保持稳定约 1 个月。不要重新冷冻。苹果酸脱氢酶在 4°C 下储存直至使用。

3.     磷酸烯醇式丙酮酸 (PEP) 原液的制备。

从冰箱 (-20°C) 中取出并在打开前使其达到室温以防止冷凝。准备储备溶液(15 ml 总溶液和 4.3 g PEP 应该导致大约. 400 mM PEP 溶液)由于 PEP 含有未知量的水合水,因此需要通过终点分析法通过分光光度法确定浓度。使用 10 mM 碳酸氢盐和 100 µM PEP(估计值),如下所述(程序 C)准备一式三份的测定。在这些条件下,预计Δ A 340 = 0.622 ;使用上述消光系数确定您的库存 PEP 浓度。在该测定中,碳酸氢盐必须超过 PEP。该储备溶液可以冷冻 (-20°C) 并保持稳定一个月。

4.     测定背景碳酸氢盐(总溶解 CO )。

需要通过终点分析来确定去除背景 CO 2的有效性。如果背景碳酸氢盐浓度高于 50 µM,则需要用高纯度氮气将主要成分再鼓泡一小时。如下所述(程序 C),在不添加碳酸氢盐和 20 mM PEP 的情况下准备一式三份的测定。PEP 必须超过背景碳酸氢盐。

 

C.    PEP 和背景碳酸氢盐的终点测定

需要对 PEP 和背景碳酸氢盐进行这些检测,以建立准确的底物浓度。该方法与动力学分析基本相同,除了我们感兴趣的是 340 nm 处信号的总变化,而不是初始速率。可以调整所用 PEPC 的浓度,以确保在合理的时间范围内看到基本上完全转化为产品。如果您可以使用细胞更换器,它们可以并行运行。

故障排除提示:如果您的检测不是气密的,您将在碳酸氢盐检测中看到缓慢的线性阶段,表明与大气进行了气体交换。

1.     将玻璃漏斗连接到正常的氮气供应,以创建一个氮气场,在该场下组装化验。将 PEPMgCl NADHKClMDH 和碳酸氢盐(如果需要)添加到空试管中(表 1)。将氮气通过漏斗以创建一个氮气场,并将装有组件的比色皿放在氮气下。取下玻璃塞后,从圆底烧瓶中取出喷过的水和缓冲液。在组装所有检测之前,不建议停止通过缓冲液和水的氮气流。尽快更换玻璃塞,以确保圆底烧瓶在使用后密封。移液器在氮气下混合测定成分并用隔膜密封。

 

1. PEPC 检测构建

笔记:

a.     MDH 浓度因批次而异,相应地调整体积。

b.     使用分析浓度的 PEP

c.     调整碳酸氢盐的最终浓度以考虑背景。请注意,这是总溶解 CO ,真实碳酸氢盐浓度随 pH 值和 I 变化。

d.     添加 KCl 是为了防止因改变 KHCO 3而导致 I 的变化并保持恒定的 [K+]x+y (µl) 应该是常数。

e.     PEPC库存的浓度各不相同。由于 10 µl 是准确移液的方便体积,因此生产 3 µM 工作储备液通常很有用;用分光光度法检查该浓度。

 

2.     将组装好的检测放在分光光度计中。测量 340 nm 处的吸光度约 30 秒,以确定起始吸光度。暂停数据采集并使用气密 Hamilton 注射器(通常100 µl)提供 50 nM PEPC(最终浓度)。轻轻地将比色皿从一侧倾斜到另一侧以混合(不要倒置)并将比色皿放回分光光度计。测量吸光度 30 分钟。此时将达到一个稳定的终点。记录终点的吸光度。根据吸光度测量值的差异确定背景碳酸氢盐或 PEP 库存的浓度。   

3.     重复终点测定 3 次以确保确定的浓度一致。将浓度报告为平均值和标准偏差。

D.    动力学分析

这些测定的目标是确定一组准确的初始速率,无论是在固定(饱和)浓度的 PEP 下,还是在改变 PEP 和碳酸氢盐的浓度下。应根据需要调整反应时间和酶浓度,以确保在反应的初始速率阶段收集数据;不需要完成进度。测定基本上如上所述构建。在收集初始利率时绘制它们通常很有用;这允许对实验设计进行即时调整,以确保覆盖足够的浓度范围。特别是,如果 最低c所用碳酸氢盐的浓度高于表观,然后修改您的设计以包括较低浓度的其他点。

1.     像以前一样构建化验(表 1),将所有非脱气成分组合在一个正常的台式机上,然后用氮气填充化验,添加喷射的成分,并在氮气场下密封比色皿。

2.     通过添加 50 nM PEPC 与气密的汉密尔顿注射器 (如前) 来启动检测。通过轻轻摇晃比色皿来混合(不要倒置)。

3.     将比色皿引入分光光度计并开始记录。通常,在 340 nm 处测量吸光度 15 分钟。

4.     使用仪器软件(例如Cary WinUV 软件中的动力学标尺)计算初始速率。对于每个点,记录两种底物的浓度、酶(如果在一天中的任何时间点变化)、初始速率、单位(例如μMs -1 ΔA 340分钟)——),并注意轨迹是否看起来可疑(例如,非线性,实质上 低于或高于预期的速率或噪音)。速率需要转换成浓度随时间变化的单位;这可以在测定结束后立即进行,也可以在一天结束时分批进行。

5.     每个浓度点应重复运行三次。测定不应按浓度顺序进行;不同测定之间的底物浓度,以防止试剂随时间衰减而引起的系统误差。

6.     在数据收集的中途和最后重复终点分析,以确保背景碳酸氢盐没有增加。如果它增加了,用高纯氮气将缓冲溶液和水喷射一小时,然后重新进行终点分析。

7.     Igor Pro(或其他合适的软件包)中根据碳酸氢盐浓度绘制计算的反应速度,然后使用适当的动力学模型进行拟合。可以在文献中找到说明性曲线(例如Moody等人2020 年的图)。作为指导,我们发现/[E] of ca40 s -1在使 PEP 和碳酸氢盐饱和时是合理的。

数据分析

 

在固定的单一 PEP 浓度下,可以使用 Michaelis-Menten 方程(方程 1)分析数据,其中/[E ]是稳态速率除以总酶浓度,cat是周转数,m是米氏常数,[S] 是底物浓度。参数值的估计标准误差由软件直接提供,cat M 标准误差可以使用误差传播来估计;如果遵循此程序,则必须包括协方差项(等式 3)。

 

或者,当两种底物都发生变化时,数据可以用公式 2 描述,其中 A B 是两种底物。

 

在这两种情况下,选择跨越 0.1 -10 M范围的底物浓度范围。通常,在固定浓度的 PEP 下收集数据时,应使用 8 12 种不同的底物浓度(,使用公式 1 进行分析)。当两种底物浓度不同时,应分别使用至少 5 个浓度(总共 25 次测定)。

cat m的标准误差可以从catm协方差(等式[Bevington and Robinson, 1992])。这里σ Km σ kcat σ kcat/Km , cov( cat Km ) 分别是cat cat m和协方差的标准误。注意,伊戈尔临报告的方差(σ 2中的方差-协方差矩阵)。

 

   

 

食谱

 

1.     Tris-HClpH 7.4

a.     24.23 Tris 碱溶解在 900 ml 水,滴定至 pH 7.53(假设实验室温度为 20)。

b.     调整实际实验室温度使用 Δp /ΔT =-0.028 并在容量瓶中使最终体积为 1 L。然后将缓冲液通过 0.2 µm 孔过滤器。

2.     Tricine-KOHpH 8.0

a.     35.84 Tricine 游离酸溶解在35.84 900 毫升水,滴定至 pH 8.10(假设实验室温度为 20°C

b.     使用 Δp /ΔT = -0.021)调整实际实验室温度,并在容量瓶中使最终体积达到 1 L。然后将缓冲液通过 0.2 µm 孔过滤器。

 

致谢

 

NRM 由格兰瑟姆可持续未来中心资助,CP ERC 资助(资助号 ERC-2014-STG-638333)资助,JDR 由生物技术和生物科学研究委员会(BBSRC,英国;奖)资助编号 BB/M000265/1)。该协议是从 Janc人中描述的协议中修改而来的(1992)

 

利益争夺

 

作者声明没有利益冲突。

 

参考

 

1.     Bauwe, H. (1986)一种测磷酸烯醇式丙酮酸羧化HCO3 m的有效方植物169356-360

2.     Bevington, PR Robinson, DK (1992)物理科学的数据简化和误差分析。纽约麦格劳希尔。

3.      Blasing, OE, Westhoff, P. Svensson, P. (2000)Flaveria4磷酸烯醇式丙酮酸羧化酶进化酶的羧基末端部分中的保守丝氨酸残基是4特异性特的主要决定因J Biol Chem 275(36)27917-27923

4.     DiMario, RJ Cousins, AB (2019)单个丝氨酸到丙氨酸的取代降低了Flaveria trinervia中磷酸烯醇式丙酮酸羧化酶的碳酸氢盐亲和力J Exp Bot 70(3)995-1004              

5.     Duff, SM, Andreo, CS, Pacquit, V., Lepiniec, L., Sarath, G., Condon, SA, Vidal, J., Gadal, P. Chollet, R. (1995)来自高粱的完整重组4磷酸烯醇丙酮酸羧化酶的非磷酸化磷酸化和磷酸化位点突变 (Asp8) 形式动力学分Eur J Biochem 228(1): 92-95

6.     Jacobs, B.Engelmann, S.Westhoff, P. Gowik, U. (2008)Flaveria C4 磷酸烯醇丙酮酸羧化酶的进:对抑制剂 L-苹果酸的高耐受性的决定因素植物细胞环境31(6): 793-803

7.     Janc, JW, O'Leary, MH Cleland, WW (1992)玉米磷酸烯醇式丙酮酸羧化酶的动力学研究。生物化学3128):6421-6426

8.     Moody, NR, Christin, PA Reid, JD (2020)PEPC 动力学修定性收敛,但Panicum Flaveria Front Plant Sci 111014

9.     Paulus, JKSchlieper, D. Groth, G.2013 年)。由于单一氨基酸取代,光合碳固定效率更高。国家通讯社41):1518

10.  Phansopa, C.Dunning, LTReid, JD Christin, PA2020 年)。横向基因转移是高效4生物化学的进化捷径Mol Biol Evol 37(11): 3094-3104

11.  韦伯先生 (1992)无机磷酸盐的连续分光光度法测定和测量生物系统中磷酸盐的释放动力学。Proc Natl Acad Sci USA 89(11): 4884-4887

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引用:Moody, N. R., Phansopal, C. and Reid, J. D. (2021). An in vitro Coupled Assay for PEPC with Control of Bicarbonate Concentration . Bio-protocol 11(24): e4264. DOI: 10.21769/BioProtoc.4264.
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