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BIOCHEMESTRY LABORATORY

EXERCISE A: ENZYMATIC KINETIC I

INTRODUCTION
Enzymes are natural proteins that catalyses chemistry reaction of transformation of the substrate to Product. In the next
experiment the potato acidic enzyme will be used. This enzyme is be able to hydrolyse the substrate p-
nitrophenylphosphate and it transform in p-nitrophenol. This enzyme obeys the Michaelis Menten kinetic. It is mean that
the velocity equation is the next.

𝑽𝒎𝒂𝒙[𝑺]
𝒗𝒐 = that can be represented in the Michaelis Menten plot
𝑲𝒎

- 𝒗𝒐: Initial velocity


- 𝒗𝒎𝒂𝒙: Maximal rate of velocity, it is reached when all catalytic sites on the
enzyme are saturated with substrate.
- 𝑲𝒎: Constant of Michaelis Menten, it implies that half of the active sites
on the enzymes are filled. Specific to each different enzyme.

OBJETIVE
The main objective of this experiment is determinate the Michaelis constant and maximal velocity of reaction catalysed
of this enzyme Potato acidic phosphatase. Through spectrophotometry measurement of product will be determinate the
velocity of enzymatic reaction of hydrolysis. For this is necessary prepare a standard curve for p-nitrophenol.

MATERIALS
Reagents

- 0.25 M acetate buffer pH 5.0


- 0.5 M sodium hydroxide
- 5 mM solution of p-nitrophenylphoshate in 0.25 M acetate buffer pH 5.0
- Solution of potato acidic phosphatase in 0.25 M acetate buffer pH 5.0, CE o = 0,08 mg/ml

Glassware and apparature:

- Chemical test tubes


- Volumetric flask 10 ml
- Automatic pipettes and tips
- Cuvette
- Specol
- Thermostat 370C

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PROTOCOL
1. Measurement of reaction velocity as concentration of substrate function.
- 21 dry tubes are tagged with the numbers 1, 2, 3..11 and 1’, 2’, 3’..11’.
- According to Table 1 solutions of 0.25M acetate buffer (pH=5.0) and 5.0mM p-nitrophenylphosphate are
added.

Table 1. Reagents volumes

Sample V buffer [ml] V substrate V enzyme V NaOH


nr [ml] [ml] [ml]
1 1.75 0.05 0.10 2.0
1’ 1.85 0.05 - 2.0
2 1.70 0.10 0.10 2.0
2’ 1.80 0.10 - 2.0
3 1.65 0.15 0.10 2.0
3’ 1.75 0.15 - 2.0
4 1.60 0.20 0.10 2.0
4’ 1.70 0.20 - 2.0
5 1.50 0.30 0.10 2.0
5’ 1.60 0.30 - 2.0
6 1.35 0.45 0.10 2.0
6’ 1.45 0.45 - 2.0
7 1.20 0.60 0.10 2.0
7’ 1.30 0.60 - 2.0
8 1.05 0.75 0.10 2.0
8’ 1.15 0.75 - 2.0
9 0.80 1.00 0.10 2.0
9’ 0.90 1.00 - 2.0
10 0.50 1.30 0.10 2.0
10’ 0.60 1.30 - 2.0
11 1.80 0.00 0.10 2.0

- Tubes with solutions are incubated 5 min in 37ºC


- Then phosphatase is added to tubes without (‘) according to table 1 and samples are incubated 25 min in 370
- In the meantime, start second part of exercise “Standard curve”
- All tubes are incubated during 25 min. after this is added 2ml 0.5M solution NaOH, which stops reaction
catalysed by enzyme.
- Absorbance of solution is measured using wavelength 410nm, sample 11 is used like the blank sample.

2. Standard curve

- Solution of p-nitrophenol is diluted 50-times in a volumetric flask of 10ml.


o 200 l of p-nitrophenol are added to acetate buffer to final volume 10ml
o Dissolution is mixed by flask inversion
- Solution of p-nitrophenol diluted is transferred to a other test tube and It is tagged as P (product)
- 8 dry test tubes are tagged

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- In the test tubes are adding solutions of 0.25 M acetate buffer pH = 5.0, 0.1 mM p-nitrophenol (in acetate
buffer) – standard (product) and 0.5 M NaOH in volume according to Table 2.

Table 2. Reagents volumes

V NaOH [ml]
Sample nr V buffer [ml] V p-nitrophenol
[ml]
1 1.70 0.20 2.0
2 1.50 0.40 2.0
3 1.30 0.60 2.0
4 1.10 0.80 2.0
5 0.90 1.00 2.0
6 0.70 1.20 2.0
7 0.50 1.40 2.0
8 1.90 0.00 2.0

- Absorbance of each tube is measured in the spectrophotometer using wavelength 410nm, sample 8 is used like
the blank sample.
- The results are noted in the notebook and collected in the part of results.

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CALCULATIONS AND DIS CUSSION OF RESULTS
1. Calculate concentration of p-nitrophenol after dilution – used standard in standard curve experiment 𝐶𝑝𝑘 [M].

Sample V p-NPH (mL) [p-NPH] (µM)


1 0.2 5.1
2 0.4 10.3
3 0.6 15.4
4 0.8 20.5
5 1 25.6
6 1.2 30.8
7 1.4 35.9
Calculations performed:

0,1 𝑚𝑚𝑜𝑙 1 1000 𝑚𝐿 1000 µ𝑚𝑜𝑙


0,20 𝑚𝐿 𝑜𝑓 𝑝𝑁𝑃𝐻 × × × × = 5,1 µ𝑀
1000 𝑚𝑙 3,9 𝑚𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 1𝐿 1𝑚𝑚𝑜𝑙

2. Prepare standard curve plot. And find out linear equation for this.

μmol
Sample Abs Calibration curve of p-nitrophenol
p-NPH
Blank 0 0 0.7
1 0.02 0.1 0.6
Absorbance

2 0.04 0.196 0.5


3 0.06 0.289 0.4
0.3
4 0.08 0.376 y = 4.6143x + 0.007
0.2
5 0.1 0.47 R² = 0.9997
0.1
6 0.12 0.56 0
7 0.14 0.649 0 0.05 0.1 0.15
p-nitrophenol (μmol)

The equation is:

Y = 4,6143x + 0,007

3. Calculate weight of phosphatase in reaction mixture 𝑚𝐸 [mg]. Volume of reaction mixture Vr = 1.90 ml.

The weight of phosphatase can be calculated knowing that their concentration in solution is 0,08 mg/mL.

Calculations performed:

0,08 𝑚𝑔 𝑜𝑓 𝐸𝑛𝑧𝑦𝑚𝑒
0,1 𝑚𝐿 𝑜𝑓 𝐸𝑛𝑧𝑦𝑚𝑒 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 × = 0,008 𝑚𝑔 𝑜𝑓 𝐸𝑛𝑧𝑦𝑚𝑒
1 𝑚𝐿 𝑜𝑓 𝐸𝑛𝑧𝑦𝑚𝑒 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

The concentration of the phosphatase in the reaction mixture can be obtained If we divide this amount into the final
volume of the reaction mixture, which is 1,9 mL.

Calculations performed:

0,008 𝑚𝑔 𝑜𝑓 𝐸𝑛𝑧𝑦𝑚𝑒
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑝ℎ𝑜𝑠𝑓𝑎𝑡𝑎𝑠𝑒 𝑖𝑛 𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑚𝑖𝑥𝑡𝑢𝑟𝑒 = = 4,2 ∙ 10−3 𝑚𝑔/𝑚𝐿
1,9 𝑚𝐿 𝑜𝑓 𝑅𝑀𝑖𝑥

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4. Calculate concentration of substrate (p-nitrophenylphosphate, p-NPH-P) ([S] calculated in μM) in reaction mixture
and inverse 1[𝑆].

Sample mL of p-NPH-P [p-NPH-P] (µM) 1/[p-NPH-P]


solution (L/µmol)
1 0.05 131.6 0.0076
2 0.1 263.2 0.0038
3 0.15 394.7 0.0025
4 0.2 526.3 0.0019
5 0.3 789.5 0.0013
6 0.45 1184.2 0.0008
7 0.6 1578.9 0.0006
8 0.75 1973.7 0.0005
9 1 2631.6 0.0004
10 1.3 3421.1 0.0003
Calculations performed:

5 𝑚𝑚𝑜𝑙 𝑜𝑓 𝑝𝑁𝑃𝐻𝑃 1000 µ𝑚𝑜𝑙 1 1000 𝑚𝐿


[𝑆] = 0,05 𝑚𝐿 𝑜𝑓 𝑠𝑜𝑙 𝑝𝑁𝑃𝐻𝑃 × × × × = 131, 6 µ𝑀
1000 𝑚𝑙 𝑜𝑓 𝑠𝑜𝑙 𝑝𝑁𝑃𝐻𝑃 1 𝑚𝑚𝑜𝑙 1,9 𝑚𝐿 1𝐿

Once you have the [p-NPH-P] you just have to make the inverse:

1 1 𝐿
= = 0,0076
[𝑆] 131,6 µ𝑚𝑜𝑙

5. Calculate absorbance ΔA410 /kor/:for amount of p-nitrophenol raised in enzymatic reaction. Using standard curve
from exercise A calculate amount of p-nitrophenol raised in enzymatic reaction (𝑛𝑝𝑡 calculated in µmol).

Sample Abs Δabs μmol p-NPH


BLANK 0 0 0
1 0.369
0.367 0.078
1’ 0.002
2 0.585
0.580 0.124
2’ 0.005
3 0.692
0.685 0.147
3’ 0.007
4 0.716
0.701 0.151
4’ 0.015
5 0.856
0.842 0.182
5’ 0.014
6 0.905
0.885 0.191
6’ 0.020
7 1.003
0.977 0.211
7’ 0.026
8 1.092
1.059 0.229
8’ 0.033
9 1.129
1.072 0.232
9’ 0.057
10 1.138
1.084 0.235
10’ 0.054
Calculations performed:

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∆𝐴𝑏𝑠 = 𝐴𝑏𝑠 𝑠𝑎𝑚𝑝𝑙𝑒 𝑋 − 𝐴𝑏𝑠 𝑠𝑎𝑚𝑝𝑙𝑒 𝑋′

∆𝐴𝑏𝑠 = 0,369 − 0,002 = 0,367

The calibration curve performed with µmol of p-nitrophenol in “x” axis and absorbance in “y” axis is y = 4,6143x + 0,007

We have to isolate the “x” and substitute the “y” for the value of absorbance:

𝑦 − 0,007
𝑥=
4,6143

0,367 − 0,007
𝑥= = 0,078
4,6143

So, in sample 1, as the ΔAbsorbance is 0,367, the amount of p-nitrophenol is 0,078 µmol.

6. Calculate velocity of p-nitrophenol raising in enzymatic reaction 𝑣𝑐. Reaction time t = 25 min.

Vc
Sample
(µmol/min)
1 0.0031
2 0.0050
3 0.0059
4 0.0060
5 0.0073
6 0.0077
7 0.0085
8 0.0092
9 0.0093
10 0.0094
To calculate the velocity of the reaction we have to divide the amount of p-nitrophenol obtained into the time of
reaction, which is 25 minutes.

𝑛𝑝𝑡 (µ𝑚𝑜𝑙)
𝑣𝑐 =
𝑡 (𝑚𝑖𝑛)

0,078 µ𝑚𝑜𝑙
𝑣𝑐 = = 0,0031
25 𝑚𝑖𝑛

7. Calculate amount of product raised in 1 minute for 1 mg of enzyme – velocity 𝒗𝒔𝒑 and inverse.

Sample 𝒗𝒔𝒑 (µmol/ mgE·min) 1/𝒗𝒔𝒑 (mgE· min/µmol)


1 0.389 2.571
2 0.622 1.607
3 0.737 1.356
4 0.755 1.325
5 0.909 1.100
6 0.956 1.046
7 1.057 0.946
8 1.147 0.872
9 1.161 0.861
10 1.174 0.852

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We can calculate the 𝑣𝑠𝑝 by divide the 𝑣𝑐 value calculated previously into the mg of enzyme calculated in exercise 3:

𝑣𝑐 (µ𝑚𝑜𝑙/𝑚𝑖𝑛)
𝑣𝑠𝑝 =
𝑚𝐸 (𝑚𝑔)

0,0031 𝑣𝑐 (µ𝑚𝑜𝑙/𝑚𝑖𝑛)
𝑣𝑠𝑝 = =
0.008 𝑚𝐸 (𝑚𝑔)

8. Results note in the form of Table 3.

Sample Δabs [S] μM 1/[S] Np Vc 𝒗𝒔𝒑 (µmol/ 1/𝒗𝒔𝒑 (mgE·


(L/μmol) (µmol) (µmol/min) mgE·min) min/µmol)
Blank 0 0 0 0 0 0 0
1 0.367 131.6 0.0076 0.078 0.0031 0.389 2.571
2 0.580 263.2 0.0038 0.124 0.0050 0.622 1.607
3 0.685 394.7 0.0025 0.147 0.0059 0.737 1.356
4 0.701 526.3 0.0019 0.151 0.0060 0.755 1.325
5 0.842 789.5 0.0013 0.182 0.0073 0.909 1.100
6 0.885 1184.2 0.0008 0.191 0.0077 0.956 1.046
7 0.977 1578.9 0.0006 0.211 0.0085 1.057 0.946
8 1.059 1973.7 0.0005 0.229 0.0092 1.147 0.872
9 1.072 2631.6 0.0004 0.232 0.0093 1.161 0.861
10 1.084 3421.1 0.0003 0.235 0.0094 1.174 0.852

9. Using data from Table 3 prepare plots:

a) [S] in “x” axis against Vsp in “y” axis.

Mikaelis-Menten Kinetics
2.5
Vsp (µmol/ mg enz * min)

1.5

0.5

0
0 200 400 600 800 1000 1200 1400
[S] μM

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b) 1/[S] in “x” axis against 1/Vsp in “y” axis.

Lineweaver-Burk y = 231.24x + 0.7967


R² = 0.9924
0,003

0,003
1/Vsp (mg enz*min/μmol)

0,002

0,002

0,001

-1/Km 0,001
1/Vmax
0,000
00,000 00,000 00,000 00,000 00,000 00,000 00,000 00,000 00,000
-0,001
1/[S] (L/μmol)

10. Using the curve calculate Michaelis Menten constant [μM] for tested enzyme and maximal celocity performed
enzymatic reactions:

The equation of Mikaelis-Menten is the following one:

𝑉𝑚𝑎𝑥 · [𝑆]
𝑉=
𝐾𝑀 + [𝑆]

By inverting the equation, you can obtain the Lineweaver-Burk equation.

1 𝐾𝑀 1 1
= · +
𝑉 𝑉𝑚𝑎𝑥 [𝑆] 𝑉𝑚𝑎𝑥

If we compare this equation with the structure of a line y = mx + n, the slope corresponds to Km/Vmax and the ordered
in origin is 1/Vmax.

With the graphical representation of the 1/[S] in the “x” axis and 1/V in the “y” axis we can obtain the equation of a line
and obtain the Km and Vmax from this one.

𝑦 = 231,24𝑥 + 0,7967

1
0,7967 =
𝑉𝑚𝑎𝑥

𝑽𝒎𝒂𝒙 = 𝟏, 𝟐𝟓 µ𝐦𝐨𝐥/ 𝒎𝒈𝑬 · 𝐦𝐢𝐧

𝐾𝑀
231,24 =
𝑉𝑚𝑎𝑥

231,24 · 𝑉𝑚𝑎𝑥 = 𝐾𝑀

𝐾𝑀 = 1,25 · 231,24

𝑲𝑴 = 𝟐𝟗𝟎, 𝟐 µ𝑴

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11. Summarize and discuss results.

With regard to the data of the concentration of p-nitrophenol calculated from the calibration curve we can say that is
quite accurate if we consider that the calibration curve has a regression coefficient very close to 1 (0,9999) which means
that all the points fit very good as a line.

When the [S] is been graphically represented against the velocity it can be appreciated a hyperbolic-like curve,
characteristic of Mikaelis-Menten enzymatic kinetics. The 4th value maybe should be higher to adjust better to the
hyperbolic curve, but it can be clearly appreciating the fast increase of the beginning of the curve and then a stabilization
when the enzyme it is supposed to be saturated, working at a Vmax velocity.

Thanks to the linearization of Lineweaver-Burk, the values of Vmax and Km can be easily obtained. The regression
coefficient of this line its also very close to 1 (0,9924) which means that the adjust to a line shape its quite good.

Talking about the concrete values of Km and Vmax that we obtained:

It’s been searched that values in the range between 10-8-10-6 M are considered that has a high affinity substrate-enzyme,
and a Km over 10-2 M are considered very low affinity1. The value that we have obtained its Km = 290,2 µM. If we convert
this value from µM to M it is 2,902 · 10-4 M, so their affinity is in the middle of the extreme values, neither too much
affinity not to low2.

The Vmax obtained show to us that 1 mg of enzyme its able to convert 1,25 μmol of subtract into product in one minute,
which is also quite good turnover number for an enzyme.

REFERENCES
1. Enzyme Kinetics. Available at:
https://www.chem.wisc.edu/deptfiles/genchem/netorial/modules/biomolecules/modules/enzymes/enzyme4.
htm. (Accessed: 24th November 2018)

2. Km values. Available at:


https://employees.csbsju.edu/hjakubowski/classes/ch331/transkinetics/kmkcatvalues.htm. (Accessed: 24th
November 2018)

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