4: Kinetic Characteristics of Acid Phosphatase

I. Introduction

Phosphatases are enzymes that catalyze the hydrolysis of phosphate monoesters with consequent release of inorganic phosphate and are widely distributed in nature.

The seeds of plants are a particularly rich source of typical acid phosphatases. The amount of phosphatase activity in seeds usually increases sharply upon germination and then falls as the seedling develops. The precise physiological function of this phosphatase is not known, but it is probably employed in releasing phosphate from organically bound storage forms, such as inositol hexaphosphate (phytic acid), for use in the metabolism of the germinating seedling.

In this experiment, acid phosphatase from wheat germ is used to develop skills in assaying and studying general kinetic properties of enzymes. The assay to be used takes advantage of the broad specificity of the phosphatase by using the artificial substrate, p-nitro-phenyl phosphate (pNPP). The degree of hydrolysis of the substrate is determined by photometric measurement of the p-nitrophenol liberated in the reaction. In alkaline solution the p-nitrophenolate ion absorbs light strongly in the region of 405 nm.

Since you are able to use a spectrophotometer and standard cells with a 1.0 cm light path , you can use the extinction coefficient for p-nitrophnol in 0.02M NaOH ( e = 18.8x10³ M^-1cm^-1 at 405 nm) to calculate the mmoles of product formed from your absorbance readings.

II. Materials and Methods

A. Materials:

1. 0.06 M Disodium p-nitrophenyl phosphate (pH 4.8), (pNPP, keep cold)

2. 0.5 M Sodium hydroxide

3. 0.05 U/ml Acid phosphatase in 0.1% bovine serum albumin.

4. 0.025 M Sodium phosphate (pH 4.8)

5. 0.05 M Citrate buffer (pH 4.8)

B. Equipment:

1. Test tubes

2. Pipette and tips

3. Water bath

4. Test tube rack

5. Spectrophotometer

C. Procedure:

A. Determination of the Michaelis Constant:

Because the assay for phosphatase is a fixed time assay, the stock enzyme solution is diluted to yield a constant rate of product formation throughout the assay period. This is particularly important in this experiment because phosphatase is subject to product inhibition.

1. Prepare a series of tubes containing 50 ml of 3, 6, 12, 30 and 60 mM pNPP.

2. Add 200 ml of 0.05 M citrate buffer (pH 4.8) to each tube to yield a final volume of 250 ml.

3. Incubate the substrate tubes and the tube of enzyme for 5 minutes in the 37^oC water bath.

4. At time intervals add 0.25 ml of enzyme to substrate tube after 30 sec to another. Mix and immediately remove to water bath.

5. After 10 min add 0.5 ml of 0.5 M sodium hydroxide and 2 ml H₂O to each tube. This stops the enzyme-catalyzed reaction and makes the pH alkaline for measurement of p-nitrophenol.

6. Measure the absorbance at 405 nm of these tubes against the corresponding “Zero-time” tube as a blank.

B. Inhibition by Inorganic Phosphate:

1. Repeat the experiment for determination of the Michaelis constant, with the modification that 0.05 ml of 0.025 M sodium phosphate (pH 4.8) is added to each tube and the volume of buffer added to each tube is adjusted so that the final volume (before adding enzyme) is 0.25 ml. (Note: This experiment must be done with the same dilution of acid phosphatase and on the same day as the determination of the Michaelis constant. why?).

方法:

1. 準備並標記實驗用玻璃試管(雙重複): 共21支。

Blank +Ⅰ(S only:5*2) + (S+I:5*2)

2. 分別將50ml各濃度pNPP (3, 6, 12, 30, 60 mM) 加到組別Ⅰ及Ⅱ。

3. 組別Ⅰ:加200ml 0.05M citrate buffer；

組別Ⅱ:加150ml 0.05M citrate buffer 和50ml 0.025M Na₃PO₄ (inhibitor)，此時總體積皆為250ml。

4. 將組別Ⅰ, II的試管以及酵素放到37℃水浴槽靜置5分鐘，使溫度皆達37℃。

※步驟5和6皆在水浴槽旁操作。

5. 在T=0 min時, 加250ml酵素到試管→震盪→放回37℃水浴槽。間隔30秒加250ml酵素至下一管，重覆步驟5至所有試管皆加入酵素。

6. 在T=10 min時，加500ml 0.5M NaOH 到試管終止反應→震盪→放回試管架。間隔30秒重覆步驟6至所有試管皆終止反應。

7. 將所有試管帶回座位，每管加入2ml d.H₂O→震盪使混合均勻。

8. Blank: 250ml citrate buffer + 250ml 酵素 + 500ml 0.5M NaOH + 2ml d.H₂O = 3ml (total)。

9. 讀OD₄₀₅ nm。

III. Results

1. Calculate the m moles of p-nitrophenol liberated by the enzyme at each substrate concentration and time in the presence and absence of inhibitor.

利用Lambert-Beer’s Law: A/ε= [pNP]_{3ml solution；}ε=18.8*10^3M-1CM^-1 ，分別計算兩組在各個受質濃度時pNP之莫耳數。

2. Calculate the reaction velocity (m moles of p-nitrophenol formed/ml of reaction mixture/10 min ) at each substrate concentration.

分別計算兩組在各個受質濃度時pNP的V_o值。

3. Prepare a Lineweaver-Burk plot of your data, express pNPP concentration in moles/liter. Determine the V_max and the Michaelis constant for pNPP, expressing each in appropriate units.

分別將V_o與受質濃度取倒數，作Lineweaver-Burk圖 (S only及S+I 兩條線畫在同一個圖)，由直線關係式分別計算出在 S only及S+I時的V_max及K_m值。

4. Which type of inhibition do you observe?

依據你的作圖結果，判別sodium phosphate為哪一類型的抑制劑?並說明此抑制劑的特徵。