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LSC-10034

The effect of temperature, pH, substrate concentration and inhibitors on the enzyme activity.

Student number: 14003336

Word count: 3 804

Student number: 14003336

Introduction

Aims:

1. To determine optimum wavelength.

2. To produce calibration graph.

3. To investigate the effect of the following factors on the enzyme activity:

pH,

substrate concentration,

inhibitor,

temperature.

Background information

Enzymes are biocatalysts that speed up the chemical reaction.

The reaction is (Watson, 2014):

up the chemical reaction. The reaction is (Watson, 2014): Rate of reaction is the amount of
up the chemical reaction. The reaction is (Watson, 2014): Rate of reaction is the amount of

Rate of reaction is the amount of product produced per unit of time. In this experiment it is mol/min.

The rate of the enzyme reaction depends on various factors:

pH,

substrate concentration,

inhibitor,

temperature.

When the substrate concentration is low, molecules react slowly and there are fewer collisions which result in low rate of reaction. However, when the concentration of substrate increases, the solution becomes more saturated, the rate increases. At the beginning substrate reacts very fast, but to a lesser and lesser and finally it reaches the maximum rate (

(Hames, Hooper, 2009). This happens because there is not enough enzyme to convert all the

it reaches the maximum rate ( (Hames, Hooper, 2009). This happens because there is not enough

Student number: 14003336

substrate at once, enzyme is being damaged, a reversible reaction starts, or pH deviates from its optimum value. It can be solved by the addition of enzyme (Berg, Tymoczko, Stryer, 2012). The relationship between the substrate concentration and velocity can be presented as a Michaelis-Menten equation.

velocity can be presented as a Michaelis-Menten equation. In order to calculate the rate of reaction,

In order to calculate the rate of reaction, the following formula is used (Watson, 2014):

of reaction, the following formula is used (Watson, 2014): where is called the Michaelies constant and
of reaction, the following formula is used (Watson, 2014): where is called the Michaelies constant and

where is called the Michaelies constant and is the substrate concentration at which the rare

of reaction is equal to half maximum velocity. The does not change with substrate

concentration. enzyme bonding.

It is affinity indicator. Low value means high affinity and weak substrate-

indicator. Low value means high affinity and weak substrate- The Michaelis-Menten graph has many limitations, one

The Michaelis-Menten graph has many limitations, one of them are inaccurate values due to

hyperbolic form.

equation. The example of the graph (Watson, 2014):

can be calculated more accurately using the Lineweaver-Burk

form. equation. The example of the graph (Watson, 2014): can be calculated more accurately using the

and

form. equation. The example of the graph (Watson, 2014): can be calculated more accurately using the

Student number: 14003336

Student number: 14003336 Temperature and pH also affect enzymatic reactions. In both cases with the increase

Temperature and pH also affect enzymatic reactions. In both cases with the increase of pH/ temperature, there is an increase in the rate of reaction, until it reaches peak value and starts to denature. In case of temperature, enzyme get too much energy and the side chains move, destroying the tertiary structure and changing the active site. Non-covalent interactions are broken. The enzyme cannot work(Martini, Nath, Bartholomew, 2012). With pH, even small change in pH can result in big difference in the rate of reaction. It happens because, pH deviation causes changes in charges carried by ionisable side chains of amino acids. This changes the intermolecular forces, tertiary structure is changed and protein denatures (Hames, Hooper, 2009).

There are different types of inhibitors. They lower enzyme activity. One of them is competitive inhibitor, it is reversible inhibitor which competes with substrate in order to bind to enzymes. When it wins, the enzyme-inhibitor complex cannot react and the rate of reaction decreases or falls to zero. With the increase of inhibitor, there is an increase in slope in the Lineweaver-Burk equation and y-intercept remains unchanged. Competitive inhibitors do not affect value. The increase in substrate concentration can overcome the inhibitor (Berg,

do not affect value. The increase in substrate concentration can overcome the inhibitor (Berg, Tymoczko, Stryer,

Tymoczko, Stryer, 2012).

Student number: 14003336

Data collection and presentation

Preliminary investigations

To determine optimum wavelength needed to determine the absorbance of product (PNP), to both control (1 ml of distilled water) and sample (0.1 ml of 1mM PNP and 0.9 ml of distilled water) tubes 2ml of diluting solution was added. The solution were mixed and next the reaction was stopped by the addition of 3 ml of NaOH. Absorbance measurement was taken using spectrophotometer at wavelength range of 340-500nm.

using spectrophotometer at wavelength range of 340-500nm. In order to determine the optimum wavelength, data from

In order to determine the optimum wavelength, data from Table 1 were presented on graph.

wavelength range of 340-500nm. In order to determine the optimum wavelength, data from Table 1 were

Student number: 14003336

From the Graph 1, it can be determined that the wavelength

From the Graph 1, it can be determined that the wavelength absorbance and therefore is an

absorbance and therefore is an optimum wavelength .

absorbance and therefore is an optimum wavelength . gives the peak Absorbance depends on the concentration

gives the peak

Absorbance depends on the concentration of the solution and the distance which the light has to pass. The more saturated solution, the bigger absorbance value (Watson, 2014). Therefore according to the Beer-Lambert Law, only concentrations that give the absorbance value below 1.000 will be used in the next part of the experiment. To narrow PNP concentration range, absorbance measurement of solutions containing different amounts of 1nM PNP were taken. The following results were obtained.

Table 2 Absorbance value obtained at different 1mM PNP volume using spectrophotometer.

Test tube number

Amount of reagent (ml)

Absorbance

1mM PNP

Distilled water

1

0.000

1.000

0.000

2

0.010

0.990

0.026

3

0.050

0.950

0.172

4

0.100

0.900

0.745

5

0.250

0.750

1.111

6

0.500

0.500

1.770

7

0.750

0.250

2.664

8

0.900

0.100

3.053

9

1.000

0.000

2.276

It was observed that after 0.1M NaOH was added to each tube, colour change was observed. First test tube was colourless, 2 nd had a slightly yellow colour which became more saturated with the increase of PNP volume in the test tube. The last test tube (number 9) had a dark yellow colour.

To determine the concentration that gives the absorbance 1.000, the volume of 1mM PNP were converted into the concentration of PNP. Dilution equation was used (Watson,2014):

of PNP. Dilution equation was used (Watson,2014): where C is the concentration and V is the

where C is the concentration and V is the volume. Therefore to calculate the concentration of PNP in the test tube number 1, the following equation is used:

the volume. Therefore to calculate the concentration of PNP in the test tube number 1, the
the volume. Therefore to calculate the concentration of PNP in the test tube number 1, the

Student number: 14003336

Table 3 Concentration of 1 mM PNP calculated for different 1mM PNP volumes.

Test tube number

Amount of reagent (ml)

Absorbance

Concentration

of

PNP

1mM

Distilled

(mM)

PNP

water

1

0.000

1.000

0.000

0.000

2

0.010

0.990

0.026

0.010

3

0.050

0.950

0.172

0.050

4

0.100

0.900

0.745

0.100

5

0.250

0.750

1.111

0.250

6

0.500

0.500

1.770

0.500

7

0.750

0.250

2.664

0.750

8

0.900

0.100

3.053

0.900

9

1.000

0.000

2.276

1.000

3.053 0.900 9 1.000 0.000 2.276 1.000 From the Graph 3, it can be determined that

From the Graph 3, it can be determined that for the PNP concentration equal to 0.260mM, the absorbance is equal to 1.000. This means that the concentration range of 1mM PNP, for which it is directly proportional to absorbance is 0.00-0.25ml of PNP. Hence for these values absorbance readings were obtained.

Student number: 14003336

Table 4 The absorbance value at different volume of 1mM PNP.

Test

tube

Volume of reagent (ml)

Absorbance

number

1mM PNP

Distilled water

1

0.000

1.000

0.000

2

0.030

0.970

0.094

3

0.060

0.940

0.214

4

0.090

0.910

0.302

5

0.120

0.880

0.391

6

0.150

0.850

0.474

7

0.180

0.820

0.589

8

0.210

0.790

0.713

9

0.250

0.750

0.793

To calculate the amount of PNP, the following equation is used (Watson, 2014):

of PNP, the following equation is used (Watson, 2014): where N is the amount of PNP,

where N is the amount of PNP, C is the concentration of PNP and V is the volume of PNP.

To calculate the amount of PNP in test tube number 3:

PNP. To calculate the amount of PNP in test tube number 3: Table 5 The amount
PNP. To calculate the amount of PNP in test tube number 3: Table 5 The amount

Table 5 The amount of PNP ( moles) at different volume of PNP reagent (ml).

Test

tube

Volume of reagent (ml)

Absorbance

Amount

of

number

1mM

Distilled water

PNP( moles)

PNP

1

0.000

1.000

0.000

0.000

2

0.030

0.970

0.094

0.030

3

0.060

0.940

0.214

0.060

4

0.090

0.910

0.302

0.090

5

0.120

0.880

0.391

0.120

6

0.150

0.850

0.474

0.150

7

0.180

0.820

0.589

0.180

8

0.210

0.790

0.713

0.210

9

0.250

0.750

0.793

0.250

Finally, the calibration graph was produced.

Student number: 14003336

Student number: 14003336 Graph 3 shows that increase of PNP amount ( mol) is directly proportional

Graph 3 shows that increase of PNP amount ( mol) is directly proportional to increase in absorbance value. It is supported by high value of

in absorbance value. It is supported by high value of Effect of substrate on enzyme activity

Effect of substrate on enzyme activity

To understand the influence of substrate on enzyme activity, in this part of experiment, different concentrations of substrate (PNPP) were used. Two sets of controlled and experimental tubes were prepared. In both, the amount of citrate buffer (pH=4.8) and 10mM PNPP were the same. The only difference was that each control tube had 1ml more of distilled water. It was to equal the mass in experimental and control tubes, because 1.0 ml of enzyme solution was added to each experimental tube.

Student number: 14003336

Table 6 The absorbance values at different amount of substrate (10mM PNPP).

Test

Amount of reagent (ml)

 

Absorbance

tube

10mM

Distilled water

Citrate

buffer

PNPP

pH=4.8

1

0.010

0.990

 

1.000

0.087

3

0.015

0.985

 

1.000

0.120

5

0.025

0.975

 

1.000

0.135

7

0.050

0.950

 

1.000

0.179

9

0.100

0.900

 

1.000

0.234

11

0.200

0.800

 

1.000

0.293

13

0.500

0.500

 

1.000

0.329

15

1.000

0.000

 

1.000

0.364

In the test tubes 1-7, there is a higher increase in absorbance than in test tubes 11-15.

To calculate PNPP concentration ([PNPP]), dilution equation is used (Watson,2014):

([PNPP]), dilution equation is used (Watson,2014): where C is the concentration and V is the volume.

where C is the concentration and V is the volume. Therefore to calculate the concentration of PNP in the test tube number 3, the following equation is used:

in the test tube number 3, the following equation is used: Table 7 Concentration of PNPP
in the test tube number 3, the following equation is used: Table 7 Concentration of PNPP

Table 7 Concentration of PNPP at different 10mM PNPP volumes.

Test

Amount of reagent (ml)

 

Absorbance

[PNPP]

tube

10mM

Distilled water

citrate

buffer

(mM)

PNPP

pH=4.8

1

0.010

0.990

 

1.000

0.087

0.033

3

0.015

0.985

 

1.000

0.120

0.05

5

0.025

0.975

 

1.000

0.135

0.083

7

0.050

0.950

 

1.000

0.179

0.167

9

0.100

0.900

 

1.000

0.234

0.333

11

0.200

0.800

 

1.000

0.293

0.667

13

0.500

0.500

 

1.000

0.329

1.667

15

1.000

0.000

 

1.000

0.364

3.333

To calculate the amount of PNP produced, the calibration graph (Graph 4) is used. The absorbance value for each tube is divided by the line gradient ( . To calculate the

tube is divided by the line gradient ( . To calculate the rate of reaction, the

rate of reaction, the amount of PNP is divided by time of reaction (15 minutes).

Student number: 14003336

Table 8 The amount of PNP produced and the rate of reaction in each tube.

Test

Amount of reagent (ml)

   

rate

tube

10mM

Distilled

citrate

buffer

PNP ( mole)

( mole/min)

PNPP

water

pH=4.8

1

0.010

0.990

1.000

0.281

0.019

3

0.015

0.985

1.000

0.388

0.026

5

0.025

0.975

1.000

0.437

0.029

7

0.050

0.950

1.000

0.579

0.039

9

0.100

0.900

1.000

0.757

0.05

11

0.200

0.800

1.000

0.948

0.063

13

0.500

0.500

1.000

1.064

0.071

15

1.000

0.000

1.000

1.177

0.078

0.071 15 1.000 0.000 1.000 1.177 0.078 From the Graph 4, it can be observed that

From the Graph 4, it can be observed that at the beginning with the increase of substrate, there is a sharp increase in the rate of reaction. However, with time the increase is slower and finally at some PNPP concentration the rate of reaction does not increase anymore and the maximum

Student number: 14003336

rate (

Menten equation.

Student number: 14003336 rate ( Menten equation. is reached. The values of and can be determined

is reached. The values of

and14003336 rate ( Menten equation. is reached. The values of can be determined from the Michaelis-

rate ( Menten equation. is reached. The values of and can be determined from the Michaelis-

can be determined from the Michaelis-

The values of and can be determined from the Michaelis- In order to calculate the rate
The values of and can be determined from the Michaelis- In order to calculate the rate

In order to calculate the rate of reaction, we use the following formula (Watson, 2014):

of reaction, we use the following formula (Watson, 2014): Table 9 Theoretical values of rate of

Table 9 Theoretical values of rate of reaction at different PNPP concentration.

Theoretical [PNPP] (mM)

Theoretical rate of reaction

0.034

0.012

0.056

0.018

0.087

0.024

0.170

0.037

0.360

0.051

0.764

0.063

1.460

0.070

1.780

0.072

2.570

0.074

2.900

0.075

3.400

0.076

0.051 0.764 0.063 1.460 0.070 1.780 0.072 2.570 0.074 2.900 0.075 3.400 0.076 12

Student number: 14003336

By comparing experimental and theoretical rate of reaction, it can be noticed that

curves are very similar.

of reaction, it can be noticed that curves are very similar. for both However, to get

for both

However, to get more accurate values of

converted into linear Lineweaver-Burk plot. To do so, reciprocals of rate of reaction and PNPP concentration were calculated.

, the Michaelis-Menten equation must be

were calculated. , the Michaelis-Menten equation must be and Table 10 Values needed to plot the

and

were calculated. , the Michaelis-Menten equation must be and Table 10 Values needed to plot the

Table 10 Values needed to plot the Lineweaver-Burk plot.

V

[PNPP] (mM)

V [PNPP] (mM)
V [PNPP] (mM)

( mole/min)

0.019

0.033

52.363

30.000

0.026

0.050

38.161

20.000

0.029

0.083

33.973

12.000

0.039

0.167

25.699

6.000

0.051

0.333

19.702

3.000

0.063

0.667

15.757

1.500

0.071

1.667

14.041

0.600

0.079

3.333

12.697

0.300

14.041 0.600 0.079 3.333 12.697 0.300 From this graph, much more accurate values of and can

From this graph, much more accurate values of

0.600 0.079 3.333 12.697 0.300 From this graph, much more accurate values of and can be

and

0.600 0.079 3.333 12.697 0.300 From this graph, much more accurate values of and can be

can be obtained.

0.600 0.079 3.333 12.697 0.300 From this graph, much more accurate values of and can be
0.600 0.079 3.333 12.697 0.300 From this graph, much more accurate values of and can be

Student number: 14003336

The effect of inhibitor on the enzyme activity

In this part of the experiment, instead of adding normal citrate buffer, a phosphate-containing buffer was added. This buffer will inhibit the enzymatic reaction. To determine the influence of inhibitor on enzyme activity, solutions with different amount of 10mM PNPP were made and the absorbance values were measured.

Table 11 Absorbance at different 10mM PNPP volumes in the presence of inhibitor.

Test

Volume of reagent (ml)

 

Absorbance

tube

10mM

Distilled water

Phosphate containing citrate buffer pH=4.8

PNPP

1

0.010

0.990

1.000

0.017

3

0.050

0.950

1.000

0.030

5

0.100

0.900

1.000

0.095

7

0.200

0.800

1.000

0.141

9

0.500

0.500

1.000

0.199

11

1.000

0.000

1.000

0.206

Using the absorbance values and the calibration graph the amount of product and therefore the rate of the reaction can be obtained.

Table 12 The amount of PNP produced and the rate of reaction in each tube in the presence of inhibitor.

 

Amount of reagent (ml)

     

Test

   

Phosphate-

containing citrate buffer pH=4.8

PNP ( mole)

rate

tube

10mM

PNPP

Distilled

water

( mole/min)

1

0.010

0.990

1.000

0.055

0.004

3

0.050

0.950

1.000

0.097

0.006

5

0.100

0.900

1.000

0.307

0.020

7

0.200

0.800

1.000

0.456

0.030

9

0.500

0.500

1.000

0.644

0.043

11

1.000

0.000

1.000

0.666

0.044

Student number: 14003336

From the Table 12, it can be easily noticed that with an increase of 10mM PNPP volume, the rate of the reaction increases from 0.004 mole/min to 0.044 mole/min.

reaction increases from 0.004 mole/min to 0.044 mole/min. Graph 7 shows the relationship between the concentration

Graph 7 shows the relationship between the concentration of PNPP and its rate of reaction. The increase at the beginning is not as steep as in the Graph 4. However to get more accurate values, as in previous part of the experiment, the concentration of PNPP, in solutions with different PNPP volume and in the presence of inhibitor, was calculated.

Table 13 Concentration of PNPP at different 10mM PNPP volumes.

Test

 

Amount of reagent (ml)

Absorbance

[PNPP]

tube

10mM

Distilled water

citrate buffer

(mM)

PNPP

pH=4.8

1

0.010

0.990

1.000

0.017

0.033

3

0.050

0.950

1.000

0.030

0.167

5

0.100

0.900

1.000

0.095

0.333

7

0.200

0.800

1.000

0.141

0.667

9

0.500

0.500

1.000

0.199

1.667

11

1.000

0.000

1.000

0.206

3.333

1.667 11 1.000 0.000 1.000 0.206 3.333 As previously, to get more accurate values of and
1.667 11 1.000 0.000 1.000 0.206 3.333 As previously, to get more accurate values of and

As previously, to get more accurate values of and , the Michaelis-Menten equation

must be converted into linear Lineweaver-Burk plot. Reciprocals of the rate of reaction and PNPP concentration were calculated.

Student number: 14003336

Table 14 Values needed to make the Lineweaver-Burk plot.

V

[PNPP] (mM)

V [PNPP] (mM)
V [PNPP] (mM)

(mole/min)

0.004

0.033

250.000

30.303

0.008

0.167

125.000

5.988

0.020

0.333

50.000

3.003

0.030

0.667

33.333

1.499

0.043

1.667

23.256

0.600

0.044

3.333

22.727

0.300

3.003 0.030 0.667 33.333 1.499 0.043 1.667 23.256 0.600 0.044 3.333 22.727 0.300 16

16

Student number: 14003336

From the Graph 8, values of

Student number: 14003336 From the Graph 8, values of and can be obtained. Effects of temperature

and

Student number: 14003336 From the Graph 8, values of and can be obtained. Effects of temperature

can be obtained.

14003336 From the Graph 8, values of and can be obtained. Effects of temperature on enzyme
14003336 From the Graph 8, values of and can be obtained. Effects of temperature on enzyme

Effects of temperature on enzyme activity

In order to determine the effect of temperature on enzyme activity, samples were kept for 15 minutes at different temperatures (range: 5 o C-75 o C) and after the addition of NaOH to experimental test tubes, the absorbance was measured. The amount of citrate buffer and substrate were constant.

Table 15 Absorbance value at different temperatures in control test tubes.

controls

test tuba number

2

4

6

8

10

12

5.0mM PNPP (ml)

1

1

1

1

1

1

Citrate buffer pH=4.8 (ml)

1

1

1

1

1

1

Distilled water (ml)

1

1

1

1

1

1

Incubation temp ( o C)

5

20

25

37

50

75

Absorbance

0.082

0.092

0.092

0.099

0.166

1.896

Table 16 Absorbance value at different temperatures in experimental test tubes.

 

experimental

test tuba number

1

3

5

7

9

11

5.0mM PNPP (ml)

1

1

1

1

1

1

Citrate buffer pH=4.8 (ml)

1

1

1

1

1

1

Incubation temp ( o C)

5

20

25

37

50

75

Absorbance

0.121

0.264

0.332

0.621

1.025

1.794

To calculate the optimum temperature, the difference in absorbance of the corresponding test tubes must be calculated as well as PNP amount and the rate of reaction.

Student number: 14003336

Table 17 The amount of PNP and the rate of reaction.

corresponding test tubes

Difference

in

Amount

PNP

rate

absorbance

(mole)

(mole/min)

1&2

0.039

0.126

 

0.008

3&4

0.172

0.556

 

0.032

5&6

0.240

0.776

 

0.052

7&8

0.522

1.688

 

0.113

9&10

0.859

2.778

 

0.185

11&12

0.003

0.010

 

0.001

Table 18 Temperature against the rate of reaction.

 

Corresponding

test

Rate (mole/min)

Temperature

 

tubes

(

o C)

1&2

0.008

5

3&4

0.032

20

 

5&6

0.052

25

 

7&8

0.113

37

 

9&10

0.185

50

 

11&12

0.001

75

 
0.185 50   11&12 0.001 75   With the increase in temperature there is an increase

With the increase in temperature there is an increase in the rate of reaction. The trend remains until the optimum temperature when the highest rate is observed. With further increase in temperature, the rate falls quickly. From the Graph 9, we can see that the peak value is reached at temperature around 40.8 o C. On the other hand the graph is not very accurate and therefore the best way is to determine the optimum range. In this case, it is 40.4-50.2 o C.

Student number: 14003336

Table 19 Values of log(V) and 1/temperature.

Corresponding

Rate

Temperature

log(v)

1/Temperature

test tubes

(mole/min)

o (
o
(
o (
o
(

1&2

0.008

278.200

-2.097

0.0036

3&4

0.032

293.200

-1.495

0.0034

5&6

0.052

298.200

-1.284

0.0034

7&8

0.113

310.200

-0.947

0.0032

9&10

0.185

323.200

-0.733

0.0031

11&12

0.001

348.200

-3.000

0.0029

0.0031 11&12 0.001 348.200 -3.000 0.0029 The effect of pH on enzyme activity. To measure how

The effect of pH on enzyme activity.

To measure how the rate of reaction is affected by the pH, the absorbance was measured. In this part of the experiment, the amount of substrate and citrate buffer were constant. The independent variable are pH values of citrate buffer.

Student number: 14003336

Table 20 Absorbance value at different pH of 1.0ml citrate buffer.

Test tuba number

1

3

5

7

9

11

13

Enzyme solution (ml)

1.0

1.0

1.0

1.0

5.5

1.0

1.0

pH of 1.0ml Citrate buffer

3.0

4.0

4.5

5.0

5.5

6.0

7.0

Absorbance

0.036

0.214

0.291

0.363

0.328

0.253

0.05

With the increase of pH, in the test tubes 1-7 there is an increase in absorbance. However in the test tubes 9-13, a decrease in absorbance can be observed. From this data, at pH=5.0, the highest absorbance was recorded. In order to determine the optimum pH range, the amount of PNP and the rate of reaction were calculated.

Table 21 Rate of reaction at different pH of 1.0 ml citrate buffer.

Test tube

pH

of

1.0

ml

Amount of PNP (mole)

Rate (mole/min)

citrate buffer

1

3.0

0.116

0.008

3

4.0

0.692

0.032

5

4.5

0.941

0.063

7

5.0

1.174

0.078

9

5.5

1.061

0.071

11

6.0

0.818

0.055

13

7.0

0.162

0.011

5.0 1.174 0.078 9 5.5 1.061 0.071 11 6.0 0.818 0.055 13 7.0 0.162 0.011 20

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The Graph 11 presents the relationship between pH of 1.0ml citrate buffer and its rate of reaction. As in the case of temperature, with the increase in pH, the rate increases up to a certain point where it reaches the highest value. With further increase in pH, the rate of reaction decreases. From the curve, the optimum pH range for acid phosphate is between 4.8 and 5.3.

Discussion

In these experiments, it has been demonstrated that there is a variety of factors that have a tremendous effect on the rate of the enzymatic reaction. Temperature, amount of substrate, presence of inhibitor and pH were investigated.

In order to confirm that the increase in substrate concentration results in the increase in the rate of reaction up to a certain point, rate of the reaction was determined for different volumes of 10mM PNPP in the solution. The results presented in the Michaelis-Menten equation confirmed that hypothesis. Using the Lineweaver-Burk plot, the and were

hypothesis. Using the Lineweaver-Burk plot, the and were equal to and respectively. In the Michaelis-Menten equation,
hypothesis. Using the Lineweaver-Burk plot, the and were equal to and respectively. In the Michaelis-Menten equation,
hypothesis. Using the Lineweaver-Burk plot, the and were equal to and respectively. In the Michaelis-Menten equation,
hypothesis. Using the Lineweaver-Burk plot, the and were equal to and respectively. In the Michaelis-Menten equation,

equal to and respectively. In the Michaelis-Menten equation,

equal to and respectively. In the Michaelis-Menten equation, and The difference in results obtained from the
equal to and respectively. In the Michaelis-Menten equation, and The difference in results obtained from the

and The difference in results obtained from the Graph

4 and 6, gives the evidence how the real values can be underestimated using the first graph. By comparing experimental and theoretical rate of reaction, it can be noticed that for both

rate of reaction, it can be noticed that for both curves are very similar. This suggests

curves are very similar. This suggests that the experimental curve is correct with the theoretical data and the results obtained are correct.

After reaching a maximal velocity, when more substrate (PNPP) is added, molecules have to wait for free enzyme to undergo catalytic reaction (Berg, Tymoczko, Stryer, 2012). Unlike the , is independent on the acid phosphatase concentration and therefore is an

on the acid phosphatase concentration and therefore is an excellent enzyme affinity indicator. A low suggests
on the acid phosphatase concentration and therefore is an excellent enzyme affinity indicator. A low suggests
on the acid phosphatase concentration and therefore is an excellent enzyme affinity indicator. A low suggests

excellent enzyme affinity indicator. A low suggests weak enzyme-substrate binding and

therefore a low substrate concentration may be enough to reach a maximum rate (Hames, Hooper, 2009).

Secondly, the effects of the inhibitor presence on the enzyme activity were noticed. Literature states that buffer will inhibit the enzymatic reaction (Martini, Nath, Bartholomew, 2012). To prove that, a constant amount (1ml) of phosphate-containing citrate buffer was added to the solutions with different amount of 10mM PNPP and the absorbance values were measured. At the beginning in the Michaelis-Menten equation, the increase in the rate is slower

Student number: 14003336

than in inhibitor free reaction, however with time the maximum velocity is reached, This supports the hypothesis that the substrate increase overcome the acid phosphatase. From the

Graph 8, values of

overcome the acid phosphatase. From the Graph 8, values of and were calculated.   Without Inhibitor

and

the acid phosphatase. From the Graph 8, values of and were calculated.   Without Inhibitor With

were calculated.

 

Without Inhibitor

With Inhibitor

  Without Inhibitor With Inhibitor
  Without Inhibitor With Inhibitor
  Without Inhibitor With Inhibitor
  Without Inhibitor With Inhibitor
  Without Inhibitor With Inhibitor
  Without Inhibitor With Inhibitor

By comparing the results for the reaction in the presence and without an inhibitor, it can

in reaction with inhibitor and a bigger increase

in , equal to 0.145mM. This suggest that a phosphate-containing citrate buffer is an

be noticed that there is a small decrease in

buffer is an be noticed that there is a small decrease in competitive inhibitor. The relationship
buffer is an be noticed that there is a small decrease in competitive inhibitor. The relationship

competitive inhibitor.

The relationship between temperature and enzyme activity was determined by taking the absorbance measurement for the solutions at different temperatures: 5, 20, 25, 37, 50, 75 o C. The amount of substarte and citrate buffer were constant. The Table 18 shows that for the first five sets of corresponding test tubes, there is an increase in the rate of reaction, reaching at 50 o C the velocity of 0.185 mole/min. In the last set, sharp decrease was noticed. This supports the theoretical information that with the increase in temperature there is an increase in the rate of reaction by increasing the thermal energy of the substrate molecules. It reaches the peak value and then enzyme starts to denature, non-covalent interactions are disrupted and the reaction rate rapidly falls down (Roberts, Reiss, 2000). Plotting the data (Graph 9), indicated the optimum temperature range of 40.4-50.2 o C for this reaction.

Calculating the values of log(V) and 1/temperature, presented the relationship between the temperature and the reaction velocity in an linear pattern. The Graph 10 clearly shows almost directly proportional dependence from which the optimum temperature can be more accurately determined. On the other hand, in this experiment there are too big differences between the following temperatures to safely determine the optimum value. From the same graph, it can be observed that one result is separated from the others and does not follow the linear pattern. This is a denatured enzyme and due to lost enzymatic activity it has a very low

rate of reaction (

lost enzymatic activity it has a very low rate of reaction ( ). Finally, pH affects

).

Finally, pH affects the rate of an enzyme- catalysed reaction by changing the charges carried by ionisable side chains. This will result in disruption of tertiary structure of an enzyme and finally in the denaturation. Even the small pH deviations lead to change in the rate of the reaction (Martini, Nath, Bartholomew, 2012). As recorded in the Table 20, the change of pH from 4.0 to 4.5 resulted in pH change of 0.077. The results of the experiment shows that with the increase of pH, in the test tubes 1-7 there is an increase in absorbance. However in the test

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tubes 9-13, a decrease in absorbance can be observed. As in case of temperature, the Graph 11 is not enough accurate to determine the optimum pH therefore, the optimum pH range for acid phosphate is between 4.8 and 5.3.

The results of the experiment were correct with the information stated in literature. However, conducting the same experiment for different types of enzymatic reaction and comparing the results might give an interesting feedback.

References

1. Hames, D., Hooper, N. 2009. Biochemistry, 3 rd edn. Abingdon: Taylor & Francis Group.

2. Kiskines, A.M.P., Klibanov, A.M. 1996. Enzymatic reactions in organic media. Glasgow: Blackie Academic & Proffesional.

3. Martini, F.H., Nath, J.L., Bartholomew E.F. 2012. Fundaments of anatomy & physiology, 10 th edn. Edinburgh: Pearson.

4. Roberts, M., Reiss, M. 2000. Advanced Biology, 2 nd edn. UK: Nelson.

5. Watson, D. 2014. Year 1Biochemistry and Biomedical ScienceProtocol Booklet 2014/14.Keele University.