Alkaline Phosphatase Kinetics

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BCM 254 & BCM 256

Alkaline Phosphatase Kinetics

Ina Keyser 10144383

University of Pretoria

14 May 2011

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INDEX INDEX 1. 1. INTRODUCTION INTRODUCTION 33 a) a) Aim Aim 44 2. 2. METHOD METHOD 55 a)

a) Materials Materials and and Reagents Reagents 55 b)

b) ProcedureProcedure i.

i. Determining Determining the the Optimal Optimal pH pH 55 ii.

ii. Determining Determining the the Optimal Optimal Temperature Temperature 66 iii.

iii. Determining Determining the the Vmax Vmax and and Km Km Values Values 66 3.

3. RESULTS RESULTS 77

a)

a) Optimum Optimum pH pH 77

b)

b) Optimum Optimum Temperature Temperature 88 c)

c) Optimum Optimum Substrate Substrate Concentration Concentration 1010 d)

d) Calculation Calculation of of Vmax Vmax and and Km Km Values Values 1111 4.

4. DISCUSSION DISCUSSION 1313

a)

a) Optimum Optimum pH pH 1313

b)

b) Optimum Optimum Temperature Temperature 1313 c)

c) Optimum Optimum Substrate Substrate Concentration Concentration 1414 5.

5. CONCLUTION CONCLUTION 1414

6.

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1.

1. INTRODUCTIONINTRODUCTION

Enzymes make up a large and diverse group of (mainly) proteins that are essential Enzymes make up a large and diverse group of (mainly) proteins that are essential for metabolic

for metabolic functions in functions in cells. cells. These subsThese substrate-specific mtrate-specific molecules functolecules function asion as catalysts in virtually all biochemical reactions by decreasing the activation energy catalysts in virtually all biochemical reactions by decreasing the activation energy required for the conversion of the substrates (or reagents) to products.

required for the conversion of the substrates (or reagents) to products.

Enzymes are usually composed from amino-acids with R-groups, each with defining Enzymes are usually composed from amino-acids with R-groups, each with defining characteristics

characteristics. The degree . The degree to which the R-groups of to which the R-groups of amino acids are protonated at aamino acids are protonated at a specific pH, determines the charge on those amino acids. And when present in an specific pH, determines the charge on those amino acids. And when present in an enzyme, the level of protonation of the various R-groups determines the degree of enzyme, the level of protonation of the various R-groups determines the degree of protonation and, therefore, the charge of the enzyme as a whole. The level of protonation and, therefore, the charge of the enzyme as a whole. The level of protonation of the individual R-groups, however, are responsible for the number and protonation of the individual R-groups, however, are responsible for the number and types of electrostatic interactions and hydrogen bonding; and in turn the types of electrostatic interactions and hydrogen bonding; and in turn the three-dimensional structure of the enzyme. Should the pH of the existing environment dimensional structure of the enzyme. Should the pH of the existing environment change, the protonation of the R-groups will vary and the electrostatic interactions change, the protonation of the R-groups will vary and the electrostatic interactions and hydrogen bonding will be redistributed so that the enzyme conformation and hydrogen bonding will be redistributed so that the enzyme conformation changes as a whole. Not all the enzymes in the population undergo these changes in changes as a whole. Not all the enzymes in the population undergo these changes in conformation. This result in either an increase or decrease in the number conformation. This result in either an increase or decrease in the number (concentration) of enzyme molecules present in the active conformation and can be (concentration) of enzyme molecules present in the active conformation and can be used as a measure for the rate of

used as a measure for the rate of the reaction.the reaction.

For many enzymes the rate of catalysis, defined as the number of moles (n) of For many enzymes the rate of catalysis, defined as the number of moles (n) of product formed per second (s), varies with the substrate (S) concentration. The product formed per second (s), varies with the substrate (S) concentration. The extent of product (P) formation is determined as a function of time for a series of extent of product (P) formation is determined as a function of time for a series of substrate concentrations. As expected in each

substrate concentrations. As expected in each case, the amount of product case, the amount of product increasesincreases with time, although eventually a time is reached when there is no net change in the with time, although eventually a time is reached when there is no net change in the concentrations of S or P. The enzyme is still actively converting S into P and concentrations of S or P. The enzyme is still actively converting S into P and vice-versa, but the reaction has attained a dynamic equilibrium. [1]

versa, but the reaction has attained a dynamic equilibrium. [1]

The reaction to be investigated is that catalysed by alkaline phosphatase (AP). The reaction to be investigated is that catalysed by alkaline phosphatase (AP). Alkaline phosphase is a hydrolase present in all tissues throughout the human body, Alkaline phosphase is a hydrolase present in all tissues throughout the human body, but particularly concentrated in the liver, bile duct, kidneys, bone and in pregnant but particularly concentrated in the liver, bile duct, kidneys, bone and in pregnant females

females – – the placenta. It is responsible for the hydrolysis of phosphor-ester bonds,the placenta. It is responsible for the hydrolysis of phosphor-ester bonds, releasing a phosphate group and the alcohol derivative of the substrate. [1, 3] releasing a phosphate group and the alcohol derivative of the substrate. [1, 3] Alkaline phosphase

Alkaline phosphase needs Mg²⁺ ions to function and is inhibited by chelating agentsneeds Mg²⁺ ions to function and is inhibited by chelating agents (e.g. EDTA) and inorganic phosphate. In this experiment p-nitro phenol-phosphate (e.g. EDTA) and inorganic phosphate. In this experiment p-nitro phenol-phosphate will be used as a substrate for the synthesis of p-nitro phenol. P-Nitro phenol is will be used as a substrate for the synthesis of p-nitro phenol. P-Nitro phenol is yellow and p-nitro phenol-phosphate is colourless. It is therefore easy to perform a yellow and p-nitro phenol-phosphate is colourless. It is therefore easy to perform a spectrophotometric enzyme assay.

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A spectrophotometer is a device used to measure light absorbency of a solution at a A spectrophotometer is a device used to measure light absorbency of a solution at a specific wavelength. When the enzymes act on the substrate to form product, the specific wavelength. When the enzymes act on the substrate to form product, the product causes the solution to display yellow. A sample of the solution can then product causes the solution to display yellow. A sample of the solution can then absorb the light passing through the cuvette in the spectrophotometer and absorb the light passing through the cuvette in the spectrophotometer and absorbency (dependent on the various [P]) can be

absorbency (dependent on the various [P]) can be obtained.obtained.

To produce an effective enzyme

To produce an effective enzyme assay, the optimum pH and temperature (conditionsassay, the optimum pH and temperature (conditions at which enzyme function is optimal and therefore the highest [P] can be obtained) at which enzyme function is optimal and therefore the highest [P] can be obtained) must be known. The product concentration value can be determined by using the must be known. The product concentration value can be determined by using the

Beer-Beer-Lambert’s Law. This law constitutes the empirical relationship between theLambert’s Law. This law constitutes the empirical relationship between the absorption of light to the properties of the material it is directed through. With the absorption of light to the properties of the material it is directed through. With the environmental factors, pH and temperature, values an experiment van be

environmental factors, pH and temperature, values an experiment van be conductedconducted to determine the optimal [S].

to determine the optimal [S].

The Michaelis-Menten and Lineweaver-Burke graphs can be plotted with the The Michaelis-Menten and Lineweaver-Burke graphs can be plotted with the abovementioned data in order to calculate the Vmax and Km.

abovementioned data in order to calculate the Vmax and Km. Vmax is the theoreticalVmax is the theoretical maximum velocity of the reaction, since it never reaches this maximum, but maximum velocity of the reaction, since it never reaches this maximum, but approaches it asymptotically and therefore the [S] equals zero at this point. Half of approaches it asymptotically and therefore the [S] equals zero at this point. Half of the Vmax is taken to determine the corresponding [S] and is called the Km. Km, also the Vmax is taken to determine the corresponding [S] and is called the Km. Km, also referred to as the Michaelis constant is the [S] at which half of the enzyme active referred to as the Michaelis constant is the [S] at which half of the enzyme active sites are occupied (or filled). The Km thus provides a measure of the [S] required for sites are occupied (or filled). The Km thus provides a measure of the [S] required for significant catalysis to occur. [2, 3]

significant catalysis to occur. [2, 3]

a)

a) AimAim

To determine the kinetic parameters Vmax and Km of the non-allosteric To determine the kinetic parameters Vmax and Km of the non-allosteric enzyme, alkaline phosphatase, by investigating the optimum pH and enzyme, alkaline phosphatase, by investigating the optimum pH and temperature at which this enzyme

temperature at which this enzyme functions.functions.

2.

2. METHODMETHOD

To conduct the experiment, it was very important to keep the laboratory work To conduct the experiment, it was very important to keep the laboratory work station neat and tidy, to

station neat and tidy, to clean all apparatus prior to the clean all apparatus prior to the experiment with alcohol andexperiment with alcohol and distilled water and to dry these apparatus before starting with the experiment

distilled water and to dry these apparatus before starting with the experiment – – it isit is of the utmost importance to uphold the principles of Good Lab Practice (GLP).

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a)

a) Materials and ReagentsMaterials and Reagents

Materials: Materials:



 SpectrophotometerSpectrophotometer 

 “Handy Step”“Handy Step” 

 12 test tubes (in a12 test tubes (in a stand)

stand) 

 1 cuvette1 cuvette 

 1 “Handy Step” tip1 “Handy Step” tip (5ml)

(5ml) 

 2 beakers (20ml)2 beakers (20ml) 

 Incubator (water bath)Incubator (water bath) with resettable with resettable temperature dial temperature dial Reagents: Reagents: 

 5mM p-nitro phenol5mM p-nitro phenol phosphate substrate phosphate substrate standard

standard 

 Buffers (with pH varyingBuffers (with pH varying from 2 to 12)

from 2 to 12) 

 Alkaline Alkaline phosphatephosphate enzyme mixture

enzyme mixture 

 Distilled water (cleaningDistilled water (cleaning purpose)

purpose) 

 Ethanol Ethanol (cleaning(cleaning purpose)

purpose)

b)

b) ProcedureProcedure

i.

i. Determining the Optimal pHDetermining the Optimal pH

The spectrophotometer (SPM) was switched on and allowed to warm up The spectrophotometer (SPM) was switched on and allowed to warm up for about 20 minutes. After warming up, the wavelength was set to for about 20 minutes. After warming up, the wavelength was set to 405nm. To determine the optimum pH, the temperature was kept 405nm. To determine the optimum pH, the temperature was kept constant at 35

constant at 35ooC throughout the experiment and the substrateC throughout the experiment and the substrate concentration in all the test tubes, 5mM of p-nitro phenol. The concentration in all the test tubes, 5mM of p-nitro phenol. The absorbance reading was zeroed

absorbance reading was zeroed by using a “blank”by using a “blank”, i.e. distilled water., i.e. distilled water. Eleven test tubes were used to conduct the experiment. For the first test Eleven test tubes were used to conduct the experiment. For the first test tube p-nitro phenol phosphate (5mM), a buffer (with a pH of 2) and 10 tube p-nitro phenol phosphate (5mM), a buffer (with a pH of 2) and 10 units of the enzyme AP was added to make a final volume of 3ml. For the units of the enzyme AP was added to make a final volume of 3ml. For the second test tube p-nitro phenol phosphate (5mM), a buffer (pH of 3) and second test tube p-nitro phenol phosphate (5mM), a buffer (pH of 3) and 10 units of the enzyme AP was added to make a final volume of 3ml. The 10 units of the enzyme AP was added to make a final volume of 3ml. The steps were repeated for the rest of the

steps were repeated for the rest of the test tubes but each time test tubes but each time a buffer,a buffer, with a pH of 1

with a pH of 1 higher than the previous was addedhigher than the previous was added – – resulting in test tuberesulting in test tube 11 ending with a pH of 12.

11 ending with a pH of 12.

Once the enzyme was added to a test tube, the experiment for that Once the enzyme was added to a test tube, the experiment for that particular test tube was immediately carried out. This ensured that the particular test tube was immediately carried out. This ensured that the enzyme did not have extra time to allow for unaccounted formation of enzyme did not have extra time to allow for unaccounted formation of product, as this could lead to skewed results. Only when results are product, as this could lead to skewed results. Only when results are obtained at a particular pH can one then go on to make the next test obtained at a particular pH can one then go on to make the next test substance at the differing pH.

substance at the differing pH.

The content of each test tube was then poured into the cuvette (one at a The content of each test tube was then poured into the cuvette (one at a time) to determine the absorbencies of each test tube, respectively. The time) to determine the absorbencies of each test tube, respectively. The cuvette was placed into the spectrophotometer and the absorbance of cuvette was placed into the spectrophotometer and the absorbance of

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each tube was recorded every

each tube was recorded every minute for 10 minutes. After obtaining theminute for 10 minutes. After obtaining the results for each test tube, the product concentration was determined results for each test tube, the product concentration was determined using

Beer-using Beer-Lamberts’ law:Lamberts’ law: A=ƐCL.A=ƐCL. (where A was the absorbance,(where A was the absorbance, ƐƐ thethe molar absorptivity, C the product concentration and L the wavelength). molar absorptivity, C the product concentration and L the wavelength). The product concentration was then plotted against pH and a standard The product concentration was then plotted against pH and a standard curve was drawn. The peak of the curve indicated the optimum pH of curve was drawn. The peak of the curve indicated the optimum pH of alkaline phosphatase.

alkaline phosphatase.

ii.

ii. Determining the Optimal TemperatureDetermining the Optimal Temperature

The spectrophotometer was switched on and allowed to warm up for 20 The spectrophotometer was switched on and allowed to warm up for 20 minutes. After warming up, the wavelength was set to 405nm. For minutes. After warming up, the wavelength was set to 405nm. For determining the optimum temperature, the pH was kept constant at the determining the optimum temperature, the pH was kept constant at the newly found optimum pH (which was 8) throughout the experiment as newly found optimum pH (which was 8) throughout the experiment as well as the substrate concentration at 5mM. The absorbance reading on well as the substrate concentration at 5mM. The absorbance reading on the spectrophotometer was zeroed using

the spectrophotometer was zeroed using distilled waterdistilled water (the “blank”).(the “blank”).

Seven test tubes were prepared by adding 5mM p-nitro phenol Seven test tubes were prepared by adding 5mM p-nitro phenol phosphate, a buffer which had a pH of 8 and 10 units of the enzyme phosphate, a buffer which had a pH of 8 and 10 units of the enzyme alkaline phosphate into the test tubes - final volume was 3ml. Once the alkaline phosphate into the test tubes - final volume was 3ml. Once the enzyme was added to test tube, the experiment for that particular test enzyme was added to test tube, the experiment for that particular test tube was carried out. This ensured that the enzyme did not have extra tube was carried out. This ensured that the enzyme did not have extra time to allow for unaccounted formation of product as this could lead to time to allow for unaccounted formation of product as this could lead to skewed results. Only when the results were obtained at a particular skewed results. Only when the results were obtained at a particular temperature can one go on to make the next test substance for the next temperature can one go on to make the next test substance for the next temperature to be tested.

temperature to be tested.

The first test tube was incubated at 34

The first test tube was incubated at 34ooC, the second at 35C, the second at 35ooC, and theC, and the rest of the test tubes were incubated at 1

rest of the test tubes were incubated at 1 oo C higher than the previousC higher than the previous tube, up until 40

tube, up until 40ooC. The cuvette was filled with each test tube substance,C. The cuvette was filled with each test tube substance, respectively, and then placed into the spectrophotometer for the respectively, and then placed into the spectrophotometer for the absorbance of each tube to be recorded every 10 minutes. After absorbance of each tube to be recorded every 10 minutes. After obtaining the results for each test tube, the product concentration was obtaining the results for each test tube, the product concentration was determined using

Beer-determined using Beer-Lamberts’ laLamberts’ law (as detailed in i. Determining thew (as detailed in i. Determining the Optimal pH). The product concentration was then plotted against the Optimal pH). The product concentration was then plotted against the temperature and a standard curve was constructed. The peak of the temperature and a standard curve was constructed. The peak of the curve indicated the optimum temperature

curve indicated the optimum temperature of alkaline phosphatase.of alkaline phosphatase.

iii.

iii. Determining the Vmax and Km ValuesDetermining the Vmax and Km Values

The spectrophotometer was switched on and allowed to warm up for 20 The spectrophotometer was switched on and allowed to warm up for 20 minutes. After warming up, the wavelength was set

minutes. After warming up, the wavelength was set to 405nm. During theto 405nm. During the experiment the pH of the buffer was kept constant at 8 and the experiment the pH of the buffer was kept constant at 8 and the temperature at 37

temperature at 37ooC. The absorbance reading on the spectrophotometerC. The absorbance reading on the spectrophotometer was zeroed

was zeroed by using a “blank”by using a “blank”. Eight test tubes were used to conduct the. Eight test tubes were used to conduct the experiment. The first tube was used as a control as no alkaline experiment. The first tube was used as a control as no alkaline phosphatase was added. For the remaining seven test tubes, the phosphatase was added. For the remaining seven test tubes, the

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substrate concentrations were changed. The second test tube was filled substrate concentrations were changed. The second test tube was filled with 5mM p-nitro phenol phosphate, a buffer (pH 8) and 10 units of with 5mM p-nitro phenol phosphate, a buffer (pH 8) and 10 units of alkaline phosphatase, to make a total volume of 3 ml. The third test tube alkaline phosphatase, to make a total volume of 3 ml. The third test tube contained 10mM of p-nitro phenol and the rest of the test tubes 20mM, contained 10mM of p-nitro phenol and the rest of the test tubes 20mM, 30mM, 40mM, 50mM, 75mM and 100mM

30mM, 40mM, 50mM, 75mM and 100mM, respectively., respectively.

The absorbance of each test tube was recorded every minute for 10 The absorbance of each test tube was recorded every minute for 10 minutes. After obtaining the results for each test tube, the product minutes. After obtaining the results for each test tube, the product concentration was determined using

Beer-concentration was determined using Beer-Lambert’s law.Lambert’s law. The productThe product concentration was then plotted against time and the gradients of the concentration was then plotted against time and the gradients of the seven test tubes were determined to give the

seven test tubes were determined to give the velocity of the reaction.velocity of the reaction.

The velocity was then plotted against the corresponding substrate The velocity was then plotted against the corresponding substrate concentration, giving a graph known as a Michaelis-Menten graph. The concentration, giving a graph known as a Michaelis-Menten graph. The Vmax

Vmax and ⅟ and ⅟ 22 Vmax values were determined from the peak of theVmax values were determined from the peak of the

Michaelis-Menten graph. The Km

Michaelis-Menten graph. The Km value was determined using thevalue was determined using the ⅟ ⅟ 22

Vmax

Vmax value. The Lineweaver-Burke graph was then plotted using thevalue. The Lineweaver-Burke graph was then plotted using the same data used to obtain the Michaelis-Menton graph. The graph is a same data used to obtain the Michaelis-Menton graph. The graph is a double reciprocal graph - as it is the

double reciprocal graph - as it is the inverse of the velocity of inverse of the velocity of the reactionthe reaction vs. the inverse of

vs. the inverse of the substrate concentratiothe substrate concentration.n.

3.

3. RESULTSRESULTS

a)

a) Optimum pHOptimum pH

The absorbencies, and pH and the calculated p-nitro phenol concentrations The absorbencies, and pH and the calculated p-nitro phenol concentrations are tabulated below (

are tabulated below (Table 1Table 1) and) and Graph 1Graph 1 illustrates the relationshipillustrates the relationship between the concentrations of p-nitro phenol in the eleven test tubes, between the concentrations of p-nitro phenol in the eleven test tubes, respectively, and their corresponding pH.

respectively, and their corresponding pH.

Controls (constants): Controls (constants):



 p-nitro phenol phosphate substrate concentration = 5mMp-nitro phenol phosphate substrate concentration = 5mM 

 Temperature = 37°CTemperature = 37°C 

 Alkaline phosphatase enzyme units used = 10Alkaline phosphatase enzyme units used = 10

C = A/

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0 0 1 1 2 2 3 3 4 4 5 5 6 6 0 0 22 44 66 88 1100 1122 1144 p p - - N N i i t t r r o o p p h h e e n n o o l l C C o o n n c c e e n n t t r r a a t t i i o o n n ( ( m m o o l l / / L L x x 1 1 0 0 ¯ ¯ ⁸ ⁸ ) ) pH pH

The Relationship between

The Relationship between p-Nitrophenol

p-Nitrophenol

Concentration (mol/L x

10¯⁸

) and pH

Table1:

Table1:Absorbance, pH and p-Nitro Phenol Absorbance, pH and p-Nitro Phenol Concentration of each Test TubeConcentration of each Test Tube

Test Test Tube Tube Wavelength Wavelength (nm) (nm) Temperature Temperature (°C) (°C) Absorbance Absorbance pHpH [p-Nitro phenol] [p-Nitro phenol] (mole/L) (mole/L) 1 1 405 405 37 37 0.275 0.275 2 2 3.613.61 2 2 405 405 37 37 0.275 0.275 3 3 3.613.61 3 3 405 405 37 37 0.275 0.275 4 4 3.613.61 4 4 405 405 37 37 0.337 0.337 5 5 4.434.43 5 405 5 405 37 37 0.39 0.39 6 6 5.125.12 6 405 6 405 37 37 0.41 0.41 7 7 5.385.38 7 405 7 405 37 37 0.43 0.43 8 8 5.655.65 8 8 405 405 37 37 0.412 0.412 9 9 5.415.41 9 9 405 405 37 37 0.43 0.43 10 10 5.655.65 10 10 405 405 37 37 0.41 0.41 11 11 5.385.38 11 11 405 405 37 37 0.39 0.39 12 12 5.125.12 Graph 1:

Graph 1:The Relationship between Product Concentration (mole/L xThe Relationship between Product Concentration (mole/L x 10¯⁸10¯⁸) and pH) and pH The optimum pH was found to be 8.

The optimum pH was found to be 8.

b)

b) Optimum TemperatureOptimum Temperature

The absorbencies, and pH and the calculated p-nitro phenol concentrations The absorbencies, and pH and the calculated p-nitro phenol concentrations are tabulated below (

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5.3 5.3 5.35 5.35 5.4 5.4 5.45 5.45 5.5 5.5 5.55 5.55 5.6 5.6 5.65 5.65 5.7 5.7 3 333 3344 3355 3366 3377 3388 3399 4400 4411 p p - - N N i i t t r r o o p p h h e e n n o o l l C C o o n n c c e e n n t t r r a a t t i i o o n n ( ( m m o o l l / / L L x x 1 1 0 0 ¯ ¯ ⁸ ⁸ ) ) Temperature (°C) Temperature (°C)

The Relationship between

The Relationship between p-Nitrophenol

p-Nitrophenol

Concentration (mol/Lx10¯⁸) and Temperature

(°C)

between the concentrations of p-nitro phenol in the seven test tubes, between the concentrations of p-nitro phenol in the seven test tubes, respectively, and their corresponding temperature.

respectively, and their corresponding temperature.

Controls (constants): Controls (constants):



 p-nitro phenol phosphate substrate concentration = 5mMp-nitro phenol phosphate substrate concentration = 5mM 

 pH = 8pH = 8 

Table2:

Table2:Absorbance, pH, Temperature and p-Nitro phenol Concentration of Absorbance, pH, Temperature and p-Nitro phenol Concentration of each Test Tubeeach Test Tube

Test Test Tube Tube Wavelength Wavelength (nm) (nm) Temperature Temperature (°C) (°C) Absorbance Absorbance pHpH [p-Nitro phenol] [p-Nitro phenol] (mole (mole/Lx10¯⁸)/Lx10¯⁸) 1 405 1 405 34 34 0.412 0.412 8 8 5.45.4 2 405 2 405 35 35 0.405 0.405 8 8 5.325.32 3 405 3 405 36 36 0.417 0.417 8 8 5.485.48 4 4 405 405 37 37 0.43 0.43 8 8 5.655.65 5 405 5 405 38 38 0.417 0.417 8 8 5.485.48 6 405 6 405 39 39 0.405 0.405 8 8 5.325.32 7 405 7 405 40 40 0.412 0.412 8 8 5.45.4 Graph 2:

Graph 2: The Relationship between Product Concentration (mole/L xThe Relationship between Product Concentration (mole/L x 10¯⁸10¯⁸) and Temperature) and Temperature (°C)

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0 0 2 2 4 4 6 6 8 8 10 10 12 12 14 14 16 16 0 0 22 44 66 88 1100 1122 p p - - N N i i t t r r o o P P h h e e n n o o l l C C o o n n c c e e n n t t r r a a t t i i o o n n ( ( m m M M ) ) Time (minutes) Time (minutes)

The Relasionship between p-Nitro Phenol

Phosphate (mM), p-Nitro Phenol Concentration

(mole/Lx10¯⁸) and Time (minutes)

5mM 5mM 10mM 10mM 20mM 20mM 30mM 30mM 50mM 50mM 75mM 75mM 100mM 100mM

The optimum temperature was found to be 37°C. The optimum temperature was found to be 37°C.

c)

c) Optimum SubstrateOptimum Substrate

Graph 2

Graph 2 illustrates the relationship between the concentrations of p-nitroillustrates the relationship between the concentrations of p-nitro phenol phosphate and p-nitro phenol over a specific time period.

phenol phosphate and p-nitro phenol over a specific time period.

Controls (constants): Controls (constants):   Temperature = 37°CTemperature = 37°C   pH = 8pH = 8 

Graph 3:

Graph 3: The Relationship between p-Nitro Phenol Phosphate (mM), p-Nitro PhenolThe Relationship between p-Nitro Phenol Phosphate (mM), p-Nitro Phenol Concentration (mole/Lx10¯⁸) and Time (minutes)

Concentration (mole/Lx10¯⁸) and Time (minutes)

The optimum substrate concentration has been determined, is 100mM. The optimum substrate concentration has been determined, is 100mM.

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0 0 0.2 0.2 0.4 0.4 0.6 0.6 0.8 0.8 1 1 1.2 1.2 1.4 1.4 0 0 2200 4400 6600 8800 110000 112200 R R e e a a c c t t i i o o n n V V e e l l o o c c i i t t y y ( ( m m o o l l e e / / L L / / m m i i n n u u t t e e x x 1 1 0 0 ¯ ¯ ⁸ ⁸ ) )

p-p-Nitro Phenol Phosphate (mole/L x10¯⁸)Nitro Phenol Phosphate (mole/L x10¯⁸)

Michaelis-Menten Graph to illustrate the

relationship between Vmax and Km

1/2Vmax = 0.595 x10 1/2Vmax = 0.595 x10¯⁸¯⁸ Vmax = 1.19x10 Vmax = 1.19x10¯⁸¯⁸ Km = 18 Km = 18 d)

d) Calculation of Vmax and Km ValuesCalculation of Vmax and Km Values

The initial substrate concentrations and the correlating reaction velocities The initial substrate concentrations and the correlating reaction velocities (mole/L/minute x10¯⁸) are tabulated in

(mole/L/minute x10¯⁸) are tabulated in Table 3,Table 3, and the relationship of theseand the relationship of these factors are illustrated with a Michaelis-Menten graph

factors are illustrated with a Michaelis-Menten graph ((Graph 4Graph 4).).

Table 3:

Table 3: Initial substrate concentrations (mM) and the correlating reaction velocitiesInitial substrate concentrations (mM) and the correlating reaction velocities (mole/L/minute x10¯⁸) of each Test Tube

(mole/L/minute x10¯⁸) of each Test Tube

Substrate Concentration (mM)

Substrate Concentration (mM) Reaction Velocity (mole/L/minute Reaction Velocity (mole/L/minute x10¯⁸)x10¯⁸)

5 0.28 5 0.28 10 0.47 10 0.47 20 0.66 20 0.66 30 0.86 30 0.86 50 0.98 50 0.98 75 1.12 75 1.12 100 1.19 100 1.19 Graph 4:

Graph 4:Michaelis-Menten Graph to illustrate the relationship between Vmax and Michaelis-Menten Graph to illustrate the relationship between Vmax and KmKm

The Michaelis-Menten graph relates the initial rate Vo to the [S]. The above The Michaelis-Menten graph relates the initial rate Vo to the [S]. The above graph is a hyperbolic function, where the maximum rate is described as graph is a hyperbolic function, where the maximum rate is described as Vmax.

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0 0 0.5 0.5 1 1 1.5 1.5 2 2 2.5 2.5 3 3 3.5 3.5 4 4 --00..11 --00..0055 00 00..0055 00..11 00..1155 00..22 00..2255 I I n n v v e e r r s s e e o o f f t t h h e e R R e e a a c c t t i i o o n n V V e e l l o o c c i i t t y y ( ( 1 1 / / m m o o l l e e / / L L / / m m i i n n u u t t e e

Inverse p-Nitro phenol phosphate (1/mM) Inverse p-Nitro phenol phosphate (1/mM)

Linewaever-Burke Graph to illustrate the

relation

relationship between the 1/[S]

ship between the 1/[S] (1/mM) and

(1/mM) and

1/V (1/mole/L/minute x10¯⁸)

1/Vmax = 0.840 1/Vmax = 0.840 -1/Km = -0.06 -1/Km = -0.06

The mathematical relationship is as follows: The mathematical relationship is as follows:

The following graph relates the inverse of the substrate concentrations The following graph relates the inverse of the substrate concentrations (1/[S])

(1/[S]) with the inverse of the reaction velocities (1/mole/L/minutes x10¯⁸).with the inverse of the reaction velocities (1/mole/L/minutes x10¯⁸). This

Lineweaver-This Lineweaver-Burke graph gives a constant relationship as a “straight line”Burke graph gives a constant relationship as a “straight line” (constant) function.

(constant) function.

Graph 5:

Graph 5: Lineweaver-Burke graph gives a constant relationship between the inverse of theLineweaver-Burke graph gives a constant relationship between the inverse of the substrate concentrations (1/mM) with the inverse of the reaction velocities substrate concentrations (1/mM) with the inverse of the reaction velocities (1/mole/L/minutes x10¯⁸)

(1/mole/L/minutes x10¯⁸)

The Km was found to

The Km was found to be 18mM and the be 18mM and the Vmax 1.19x10¯⁸mM/minute.Vmax 1.19x10¯⁸mM/minute.

Vmax [S] Vmax [S] Vo

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1

1 Km Km + + [S] [S] ( ( Km) Km) (1) (1) 11 V

V = = Vmax Vmax [S] [S] = (Vmax) = (Vmax) x [S] + x [S] + VmaxVmax

The mathematical relationship is as follows: The mathematical relationship is as follows:

4.

4. DISCUSSIONDISCUSSION

a)

a) Optimum pHOptimum pH

The name alkaline phosphate indicates that the enzyme functions optimally The name alkaline phosphate indicates that the enzyme functions optimally at alkaline conditions. In determining the optimum pH, the enzyme and at alkaline conditions. In determining the optimum pH, the enzyme and substrate concentration as well as the temperature were kept

substrate concentration as well as the temperature were kept constant at theconstant at the same time altering the pH and determining the highest product same time altering the pH and determining the highest product concentration by testing the absorbance of the solution containing p-nitro concentration by testing the absorbance of the solution containing p-nitro phenol and then using the

Beer-phenol and then using the Beer-Lambert’s law.Lambert’s law. The graph to obtain theThe graph to obtain the optimum pH was drawn and the optimum pH was und to be at pH 8 and 10. optimum pH was drawn and the optimum pH was und to be at pH 8 and 10. Since alkaline phosphatase functions within the body (living tissue conditions) Since alkaline phosphatase functions within the body (living tissue conditions) with an approximate pH of 8, a pH

with an approximate pH of 8, a pH of 8 was used throughout the experiment.of 8 was used throughout the experiment.

Enzymes are composed from amino acids with defined characteristics. Enzymes are composed from amino acids with defined characteristics. Interactions such as hydrogen bonds, disulphide bonds,

Interactions such as hydrogen bonds, disulphide bonds, ionic interactions andionic interactions and hydrophobic interactions between different amino acids side chains in the hydrophobic interactions between different amino acids side chains in the protein determine the specific three dimensional structure of the enzyme. protein determine the specific three dimensional structure of the enzyme. Since an increase in pH will lower the hydrogen ion concentration and vice Since an increase in pH will lower the hydrogen ion concentration and vice versa, this results in a disturbing of the bonds keeping the enzyme in a versa, this results in a disturbing of the bonds keeping the enzyme in a correct conformation and thereby changing the three dimensional structure correct conformation and thereby changing the three dimensional structure of the enzyme and so

of the enzyme and so affecting the binding ability of the substrate too.affecting the binding ability of the substrate too.

b)

b) Optimum TemperatureOptimum Temperature

Alkaline phosphatase is found in the human body and will therefore be Alkaline phosphatase is found in the human body and will therefore be expected to function optimally at or around 37

expected to function optimally at or around 3700C, since this is theC, since this is the temperature of the body under normal living conditions. In determining the temperature of the body under normal living conditions. In determining the optimum temperature, the substrate and enzyme concentrations were kept optimum temperature, the substrate and enzyme concentrations were kept constant as well as the pH (at 8),m while varying the values of the constant as well as the pH (at 8),m while varying the values of the temperature. The Beer-Lambert

temperature. The Beer-Lambert’s’s law was used to calculate the productlaw was used to calculate the product concentrations which were then plotted against the corresponding concentrations which were then plotted against the corresponding temperature. The optimum temperature was found to be 37°C, which temperature. The optimum temperature was found to be 37°C, which corresponds with the theoretical (human body) temperature, as expected. corresponds with the theoretical (human body) temperature, as expected.

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Def

kk

22

+ k

-1-1

Km

Km =

= kk

11

≈ K

DD

c)

c) Optimum Substrate ConcentrationOptimum Substrate Concentration

In determining the optimum substrate concentration, the pH was kept In determining the optimum substrate concentration, the pH was kept constant at 8 and the temperature at 37

constant at 8 and the temperature at 3700C. In this part of the C. In this part of the experiment, theexperiment, the substrate concentration was the variable. The reaction velocities were substrate concentration was the variable. The reaction velocities were determined by calculating the gradients of the graph and the optimum determined by calculating the gradients of the graph and the optimum substrate concentration was found to be 100mM.

substrate concentration was found to be 100mM.

This makes sense, because the more substrate available for the enzyme to This makes sense, because the more substrate available for the enzyme to bind to, the more products can be synthetized

bind to, the more products can be synthetized – – therefore the absorbenciestherefore the absorbencies were read to determine the [P].

were read to determine the [P].

This process is summarized by the following equation: This process is summarized by the following equation:

5.

5. CONCLUTIONCONCLUTION

We were able to successfully accomplish our aim of determining K

We were able to successfully accomplish our aim of determining KMM(which we found(which we found

to be 18mM) and the V

to be 18mM) and the Vmaxmax(which we found as 1.19 x 10(which we found as 1.19 x 10-8-8mMminmMmin-1-1).).

We can therefore conclude that at a p-nitro phenol phosphate concentration of We can therefore conclude that at a p-nitro phenol phosphate concentration of 18nM, 50% of the alkaline phosphate enzyme active sites are occupied by substrate. 18nM, 50% of the alkaline phosphate enzyme active sites are occupied by substrate. (Km is a measure of how tightly substrate is bound to the enzyme. The greater the (Km is a measure of how tightly substrate is bound to the enzyme. The greater the Km, the less tightly the substrate is bound to

Km, the less tightly the substrate is bound to the enzyme.)the enzyme.)

Km is also the dissociation constant of the ES-complex, when we assume k

Km is also the dissociation constant of the ES-complex, when we assume k-1-1>> k>> k2.2.

Vmax is related to the turnover number of an enzyme, a quantity equal to the Vmax is related to the turnover number of an enzyme, a quantity equal to the catalytic constant k

catalytic constant k2.2. The constant is referred to as kThe constant is referred to as kcat.cat.The turnover number is theThe turnover number is the

moles p-nitro phenol phosphate that reacts to form p-nitro phenol per mole of moles p-nitro phenol phosphate that reacts to form p-nitro phenol per mole of alkaline phosphate per unit time (assuming that AP is fully saturated with p-nitro alkaline phosphate per unit time (assuming that AP is fully saturated with p-nitro phenol phosphate and thus the reaction is proceeding at a maximum rate).

phenol phosphate and thus the reaction is proceeding at a maximum rate).

K K11 kk22

E

E +

+ S

S

ES

E

E +

+ P

P

kk-1-1 Def Def

Vmax = k

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We have to note

We have to note that this methodology of determining enzymatic kinetic parametersthat this methodology of determining enzymatic kinetic parameters is only suitable when we investigate non-allosteric enzymes. When dealing with is only suitable when we investigate non-allosteric enzymes. When dealing with allosteric enzymes, it is difficult to account for the effect of cooperation displayed by allosteric enzymes, it is difficult to account for the effect of cooperation displayed by the enzyme.

the enzyme.

Lineweaver-Burke plots by virtue of their reciprocal nature are prone to error, as Lineweaver-Burke plots by virtue of their reciprocal nature are prone to error, as they increase small errors in measurement. Also most points on the graph are found they increase small errors in measurement. Also most points on the graph are found to the right of the y-axis (owing to the limiting solubility not allowing large values of to the right of the y-axis (owing to the limiting solubility not allowing large values of [S] and therefore a small 1/[S] value) calling for extrapolation to obtain the x and y [S] and therefore a small 1/[S] value) calling for extrapolation to obtain the x and y intercepts. This explains why the 1/Vmax values for 0

intercepts. This explains why the 1/Vmax values for 0 and 0.01 concentration had theand 0.01 concentration had the same value of 0.84. This is the only manner to allow for further extrapolation to same value of 0.84. This is the only manner to allow for further extrapolation to determine the x intercept (-1/Km).

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6.

6. REFERENCESREFERENCES

1.

1. Campbell, Farrell. Biochemistry. 6Campbell, Farrell. Biochemistry. 6ththEd. Belmont. Brooks/Cole. 2008: 144-164.Ed. Belmont. Brooks/Cole. 2008: 144-164. 2.

2. Wikipedia. Alkaline Phosphate [cited 2009 April 24].Wikipedia. Alkaline Phosphate [cited 2009 April 24]. http://en.wikipedia.org/wiki/Alkaline_phosphate

http://en.wikipedia.org/wiki/Alkaline_phosphate.. 3.

3. Berg Jm, Tymoczko JL, Stryer P and Lubert C. Berg Jm, Tymoczko JL, Stryer P and Lubert C. Biochemistry. 5Biochemistry. 5ththEd. New York:Ed. New York: W.H. Freeman and Co: 2002.