Chymotrypsin Lab Report
The Kinetics of α-Chymotrypsin
Introduction
Chymotrypsin is a protease which cleaves proteins by a hydrolysis reaction, it does this by adding a molecule of water to a peptide bond. Although the hydrolysis reaction is thermodynamically favoured in the absence of a catalyst the half-life for a typical hydrolysis reaction by a protease is between 10 and 100 years, needless to say it is extremely slow1. Though this is true peptide bonds are hydrolysed within milliseconds in the body in the presence of catalysts. The kinetic stability of chymotrypsin which gives it hydrolysis resistance is due to the resonance structure that accounts for the planarity of the peptide bond. Such is the strength of this resonance structure that it confers partial …show more content…
double bond character. The carbonyl carbon atom in peptides is less electrophilic and therefore less susceptible to nucleophilic attack than carbonyl carbon atoms in most other compounds. This means that for a protease to function it has to cleave peptide-bonds at an unreactive carbonyl group1.
For this experiment we looked at the kinetics of chymotrypsin. We calculated the extinction co-efficient of p-nitrophenoxide which was important for the enzyme kinetic assays we carried out later in the experiment. We then measured the concentration of some stock solutions of chymotrypsin using both the Folin and BIORAD assays so we could get an idea of which assay is more accurate when measuring enzyme concentration. Finally we looked at the kinetics of chymotrypsin by carrying out various enzyme kinetic assays. We used different enzyme and substrate concentrations in our assay so we could determine the mechanism of action of chymotrypsin.
Chymotrypsin breaks down proteins in the digestive systems of mammals as well as other organisms. It selectively cleaves peptide bonds on the carboxyl-terminal side of the large hydrophobic amino acids such as tryptophan, tyrosine, phenylalanine and methionine1. Chymotrypsin operates by covalent catalysis whereby the serine residue acts as a nucleophile to attack the unreactive carbonyl carbon atom of the substrate so that it becomes briefly attached.
The kinetics of chymotrypsin action can be monitored by measuring the absorbance of a coloured product when the enzyme is acting on a substrate analog. In this experiment the coloured product is p-nitrophenoxide and the substrate is p-nitrophenyl trimethylacetate. Under steady state conditions, the reaction is known to favour Michaelis-Menten kinetics with a KM of 20µM and a kcat of 77s-1. The hydrolysis begins with an initial rapid burst phase followed by a steady state phase1.
The two phases of the reaction are caused because a covalently bound enzyme-substrate intermediate is formed. The acyl group initially becomes covalently attached to the enzyme as p-nitrophenoxide is released. This acyl-enzyme intermediate is then hydrolysed to release the carboxylic acid component of the substrate and regenerate the free enzyme1. This means that one molecule of p-nitrophenoxide is produced rapidly from each enzyme molecule as the acyl-enzyme intermediate is formed. Both phases are needed for enzyme turnover however it takes a lot longer to effectively free the enzyme for use by the hydrolysis of the acyl-enzyme intermediate1.
Chymotrypsin is spherical and is made up of 3 polypeptide chains conjoined by disulphide bonds. It is synthesized as chymotrypsinogen which is a single polypeptide which yields 3 chains after it is activated by proteolytic cleavage. Serine 195 marks the active site of chymotrypsin and lies in a cleft on the surface of the enzyme1. The unusually high reactivity of serine 195 is determined by the structure of the active site. There is a hydrogen bond between the imidazole ring of histidine 57 and the side chain of serine 195. The NH group of the imidazole ring is also hydrogen bonded to the carboxylate group of aspartate 102. The three aforementioned residues together make up the catalytic triad1.
The histidine residue polarizes the hydroxyl group of the serine side chain so that it is ready for deprotonation. When the substrate is present the histidine residue accepts the proton from the serine 195 hydroxyl group whereby the histidine residue effectively acts as a general base catalyst. The withdrawal of the proton from the hydroxyl group furthermore generates an alkoxide ion which is a much more powerful nucleophile than an alcohol. The aspartate residue is partly responsible for orienting the histidine residue to make it a better proton acceptor through hydrogen bonding and electrostatic effects1.
After the substrate has binded, the oxygen atom of the side chain of the serine 195 makes a nucleophilic attack on the carbonyl carbon atom of the target peptide bond. At this stage there are four atoms bonded to the carbonyl carbon arranged in a tetrahedron which is an unstable structure with a negative charge on the oxygen atom derived from the carbonyl group. This charge is stabilized by interactions with NH groups from the protein in a site called the oxyanion hole. The tetrahedral intermediate then collapses to form the acyl-enzyme intermediate which is also stabilised by the same interactions. This stage involves the histidine residue transferring a proton to the amino group formed by the cleavage of the peptide bond. The amine component then is freed from the enzyme which is the last step of the acylation of the enzyme1.
Deacylation occurs when a water molecule occupies the place where the amine component previously was.
The ester group of the acyl-enzyme is then hydrolysed by nucleophilic attack of serine on the peptide carbonyl group, collapse of the tetrahedral intermediate and the release of the amine component. Histidine 57 then acts as a general acid catalyst and draws a proton away from the water molecule. The OH- ion then attacks the carbonyl carbon atom of the acyl group which forms another tetrahedral intermediate which breaks down to form the carboxylic acid product. The release of this acid then frees up the enzyme to continue …show more content…
catalysis1.
Methods
The extinction coefficient of the p-nitrophenoxide ion was determined by measuring the absorbance twice in a sodium hydroxide solution containing p-nitrophenyl trimethylacetate. The average result was taken.
The concentration of α-chymotrypsin was determined by a Folin assay as well as a BIORAD assay. Both required collecting results from a BSA standard in order to work out the values for the concentration for α-chymotrypsin.
The enzyme assays were done at three different substrate concentrations and two enzyme concentrations. The absorbance was read from a spectrometer at different time intervals. p-nitrophenyl tri-methylacetate was used as the substrate instead of p-nitrophenyl acetate which would have occurred to fast and would have required the stopped-flow spectrophotometric technique to follow.
Results
Extinction Coefficient (ε) of p-nitrophenoxide
This was calculated using the Beer-Lambert law:
The two absorbance readings were 1.092 and 1.114. (1.092+1.114)/2=1.103 which gave the mean absorbance.
We rearranging the Beer-Lambert law to work out the extinction coefficient: ε=A/(lc).
By substituting A for 1.103, l for 1cm and c for 5.83 x 10-5 we worked out ε to be 18919mol-1Lcm-1.
Measurement of a-chymotrypsin concentration
Folin Assay
Figure 1
We took the average of two absorbance measurements from each α-chymotrypsin sample which were at estimated concentrations of 0.02mg/ml, 0.03mg/ml and 0.04mg/ml. We used the equation y=0.5671x-0.0085 which was the line of best fit from the BSA concentrations to find out the actual concentration of α-chymotrypsin.
Since y represented the absorbance at 680nm, x was used to represent the actual concentration of α-chymotrypsin in mg/ml. We rearranged the equation to x=(y+0.0085)/0.5671.
We substituted each mean absorbance measurement for y in the rearranged equation to obtain the actual concentration of α-chymotrypsin.
The estimated concentration results were diluted differently, the 0.02mg/ml sample was diluted 30 fold, the 0.03mg/ml sample was diluted 20 fold and the 0.04mg/ml sample was diluted 40/3 fold. The corresponding actual concentration results were respectively multiplied by 30, 20 and 40/3 to obtain the actual concentration of α-chymotrypsin in the original solution.
The results were as follows:
Estimated Concentration of α-Chymotrypsin (mg/ml)
Mean Absorbance
Actual Concentration of α-Chymotrypsin (mg/ml)
Actual Concentration of α-Chymotrypsin in the Original Solution(mg/ml)
0.02
0.02
0.050255687
1.507670605
0.03
0.028
0.064362546
1.287250926
0.04
0.049
0.101393052
1.351907365
Figure 2
The average actual concentration of α-chymotrypsin in the original solution was 1.382276299 mg/ml.
BIORAD Assay
Figure 3
We took the mean of two absorbance measurements for each estimated α-chymotrypsin sample. We used the equation y=61.7x-0.0153 from the line of best fit from Figure 3 to work out the actual α-chymotrypsin concentrations in each sample by rearranging it to x=(y+0.0153)/61.7. We substituted the mean absorbance for y to obtain the actual concentration of α-chymotrypsin.
The estimated 0.004mg/ml and 0.008mg/ml concentrations were diluted 250 fold and 125 fold respectively. So the results obtained by the rearranged equation were multiplied by 250 and 125 respectively.
The results were as follows:
Estimated Concentration of α-Chymotrypsin (mg/ml)
Mean Absorbance
Actual Concentration of α-Chymotrypsin (mg/ml)
Actual Concentration of α-Chymotrypsin in the Original Solution(mg/ml)
0.004
0.1805
0.00317342
0.793354943
0.008
0.3135
0.005329011
0.666126418
Figure 4
The average actual concentration of α-Chymotrypsin in the original solution was 0.729740681.
Enzyme Assays of α-chymotrypsin
Figure 5
Figure 5 displays the cumulative production of the p-nitrophenoxide ion which is produced by the chymotrypsin catalysed reaction. During the reaction the enzyme concentration remained at 50µl whilst the substrate concentration was varied by addition of either 25, 50 or 75µl aliquots of substrate. At all the substrate concentrations we can witness the initial burst phase followed by the steady state phase which indicates the biphasic nature of the reaction. As the substrate concentration was increased so did the absorbance as was expected. We can see that the gradient and so therefore the rate of the steady state increased gradually as the amount of substrate increased. Likewise when looking at the initial burst phase we can see that the gradient also becomes more steep at increasing levels of substrate.
Figure 6
Figure 6 represents the production of the p-nitrophenoxide ion similarly to Figure 5 however 25µl of enzyme was used instead of 50µl. Again we can witness the initial burst phase followed by the steady state phase which indicates the biphasic nature of the reaction. There was an increase in absorbance after 25µl of substrate but after that the readings for 50µl and 75µl were very similar. The steady state rate and the initial burst rate for these two substrate levels were also very similar. At 25µl of substrate the rate at the steady state was greater than it was at higher substrate levels. Figure 8 Figure 7
Figure 9 Figure 10
From looking at Figure 7 we can see that the A constants for 50µl of enzyme were significantly higher than for 25µl of enzyme for all the substrate amounts. There was not a clear correlation between the substrate amount and the A constant since for 25µl of enzyme, higher substrate amounts decreased the A constant and at 50µl of enzyme, lower substrate amounts increased the A constant. It has to be noted that the scale is relatively small, suggesting that the effect of the enzyme was minimal.
From looking at Figure 8 we can see that increasing the amount of enzyme increased the B constant for all the different substrate amounts. The B constant showed little variation at 25µl of enzyme and a lot more variation at 50µl of enzyme. There seems to be a positive correlation between the substrate amount and the B constant.
For Figure 9, we can see that there is a positive correlation between the substrate amount and the b constant, apart from the unexpected high result at 25µl of enzyme and 50µl of substrate. When the amount of enzyme is doubled it does not affect the b constant.
For Figure 10 at 25µl of enzyme there is a negative correlation between the amount of substrate and the [E0]/[E] value. There was a generally higher [E0]/[E] value when the amount of enzyme is doubled. There seems to be no correlation between the amount of substrate and the [E0]/[E] value of 50µl of enzyme.
The average value of K2/KS constant in our experiments came to be 127.2857s-1, this is close to published value of the K2/KS constant of 231.25s-1. The average value of the K3 constant was 2 x 10-3 which is quite similar to the published value of 1.3 x 10-4.
Discussion
We worked out the extinction co-efficient of p-nitrophenoxide to be 18919mol-1Lcm-1 and the value we got was close to the published value2 of 18000mol-1Lcm-1. Our result is within 1 standard deviation of the class mean. This suggests that the value we obtained is reliable since similar results have been achieved by so many others.
For measuring the concentration of chymotrypsin in a stock solution both the BIORAD and the Folin methods determined that the protein concentration of chymotrypsin was close to the sample region at approximately 1mg/ml. The Folin Lowry assay calculated a concentration of 1.382276299mg/ml for chymotrypsin whereas the BIORAD method calculated a concentration of 0.729740681mg/ml.
The Folin method involves the reduction of the Folin reagent by tryptophan, tyrosine, cysteine, cystine and histidine which means that the absorbance is going to be affected by the number of the aforementioned exposed amino acid residues on the proteins.
The Folin assay overestimated the chymotrypsin concentration because of the difference in the properties of BSA and chymotrypsin. The different amino acid composition could have had an effect on how much the Folin reagent was reduced which causes the observed colour change. Too many of these amino acids which have this reducing ability in chymotrypsin could have led to the higher observed concentration seen in this
experiment.
The BIORAD method relies on binding of Coomassie Brilliant Blue G-250 to the protein and the hydrophobic interaction between the dye and the protein. The reliance on hydrophobic interaction means that the binding is dependent on the number of exposed hydrophobic residues on the protein as well as the temperature. The BIORAD assay underestimated the protein concentrations. Not all proteins have the same hydrophobic properties, chymotrypsin may have not had very potent hydrophobic properties which would have meant there is not as much colour change and would have caused the low concentration of chymotrypsin that was observed. It is possible that chymotrypsin has fewer hydrophobic regions which Coomassie Brilliant Blue can bind to which would have caused a reduction in chymotrypsin concentration.
After comparing the results to the class averages we saw that the values we obtained were within 1 standard deviation of the class mean values gained by each method. This is testament to the accuracy of our results. The class results also showed that the Folin assay overestimated the chymotrypsin concentration at 1.10mg/ml and that the BIORAD method underestimated the chymotrypsin concentration at 0.56mg/ml. The Folin assay had a standard deviation of 0.40mg/ml which was larger than the BIORAD assay standard deviation of 0.19mg/ml. This lower standard deviation suggested that the BIORAD assay was a more precise method. However if we assumed that the actual concentration of chymotrypsin was exactly 1mg/ml then the Folin assay would have been more accurate than the BIORAD assay. Table 2 in the appendix of the lab manual also showed that the Folin method overestimated the concentration of chymotrypsin at 11.6mg/ml in a 10mg/ml stock solution whereas the BIORAD method underestimated it at 7.8mg/ml. This only further agreed with our conclusions so far. The class data disproved our null hypothesis that the mean of Folin is equal to the mean of BIORAD with a probability of less than 0.001. This was to be expected since two completely different methods were employed.
Finally, we looked at the kinetics of the chymotrypsin catalysed hydrolysis. The hydrolysis of p-nitrophenyl tri-methylacetate is slower than the hydrolysis of p-nitrophenyl acetate because of the steric hindrance of its bulky tri-methylacetate group which slowed down the reaction. During the reaction a serine residue attacks the anti-bonding orbital of the carbon group, which results in the hydrolysis of the ester. This requires the serine nucleophile to overlap with the anti-bonding orbital which relies on close alignment. The bulky tri-methylacetate group reduces the chance that the nucleophile comes into the correct orientations to perform the catalytic reaction, therefore causing a much slower reaction.
By looking at Figure 5 we can see that the rate of the burst phase was positively correlated with the substrate concentration at 50µl of enzyme. This is because at this phase increased amounts of the substrate will mean that there will be more successful collisions between the enzyme and the substrate which will in turn result in more product being observed. However this same increase was not observed by looking at Figure 6 where two very similar burst phase and steady state rates were obtained at 50µl and 75µl of substrate. At 25µl of substrate the rate at the steady state was greater than it was at higher substrate levels, which suggests that a different factor other than the amount of substrate had become limiting. I would have expected a higher amount of substrate to result in a higher burst phase and steady state rate as in Figure 5. However since in Figure 6 half as much enzyme was used this suggests that the enzyme became saturated at lower levels of substrate. The level of enzyme was the limiting factor. The enzyme became saturated when 50µl of substrate was added which is why higher amounts of substrate had no effect on the reaction. The lower rate of reaction when 75ul of substrate was added may have also been due to experimental error most likely by a lack of pipetting accuracy.
From comparing Figures 5&6 it could also be seen that a higher concentration of product was produced after the burst phase when higher concentrations of enzyme were used. This is expected since there is much more free enzyme to which the substrate can bind to. At a lower enzyme concentration, there would be less active sites to occupy which would result in less reactions and therefore less product. This is why the steady state rates in Figure 6 occur at a much lower absorbance than the steady state rates in Figure 5.
For Figure 7 when the amount of enzyme was doubled the constant A increased, although the constant was affected by the amount of substrate due to experimental error most likely by inaccurate pipetting. However, this does show that constant A is dependent on enzyme concentration since A=k3[E0] even though it is not completely independent of the substrate. For Figure 8 we can see that apart from the anomaly the doubled amount of enzyme increased the B constant. Similarly the substrate showed interference most likely caused by the same way. This suggests that constant B is dependent on enzyme concentration since B=[E0]. For Figure 9 we can see that the doubled enzyme amount does not affect the b constant and the higher amount of enzyme there was a positive correlation between the amount of substrate and the b constant. This proves that constant b is independent of the enzyme and suggests that it is dependent on the substrate since b=(k2[S0]/KS).
For Figure 9 the fraction of active enzyme [E0]/[E] was worked out for each reactions. As seen and expected as the amount of enzyme was doubled the fraction of active enzyme increased. This value can be affected by temperature, pH, and salt content. If the temperature and pH are not optimal the enzymes will not function at their maximum capability. If these conditions stray too far from the optimum the enzymes may even become denatured if the bonds which make up the active sites become altered. The ions in extremely high salt concentrations would interfere with the weak ionic bonds of proteins which would also inhibit the functionality of the enzyme. Furthermore when there are other macromolecules in the solution macromolecular crowding can occur which can alter the rates and equilibrium constants of enzyme reactions which will definitely have a knock on effect on the fraction of active enzyme.
References
1. Berg, J., Tymoczko, J., and Stryer, L. (2012) Biochemistry, 7th edition, New York: W. H. Freeman and Company.
2. Bender et al. (1967) Journal of Chemical Education 44, 84-88.
Further Calculations
50μl enzyme
25μl enzyme
75μl substrate
50μl substrate
25μl substrate
75μl substrate
50μl substrate
25μl substrate
15
0.7629
0.545
0.3368
0.27445
0.24725
0.22905
30
0.5809
0.4365
0.2893
0.2582
0.186
0.1851
45
0.4059
0.344
0.2438
0.18695
0.14575
0.17515
60
0.3049
0.2925
0.2133
0.1567
0.1125
0.1552
75
0.2369
0.256
0.1848
0.13145
0.07325
0.13425
90
0.1869
0.2165
0.1573
0.1122
0.064
0.1183
LN
LN
LN
LN
LN
LN
15
-0.270628318
-0.606969484
-1.088265997
-1.292986184
-1.397355308
-1.473814959
30
-0.543176654
-0.828966904
-1.240291067
-1.354020801
-1.682008605
-1.686859059
45
-0.901648455
-1.067113622
-1.411407062
-1.676914078
-1.925862454
-1.742112529
60
-1.187771425
-1.229290612
-1.545055654
-1.85342213
-2.184802057
-1.863040671
75
-1.440117168
-1.362577835
-1.68848112
-2.029128728
-2.613877031
-2.008051546
90
-1.677181565
-1.530164732
-1.849600469
-2.187472286
-2.748872196
-2.134531508
b b b b b b 0.0191
0.0122
0.0101
0.0127
0.0187
0.0084
A
A
A
A
A
A
1.05714E-08
5.28569E-09
5.28569E-09
2.64285E-09
2.64285E-09
3.69998E-09
B
B
B
B
B
B
1.1269
0.6585
0.4483
0.3997
0.3805
0.289
E0
E0
E0
E0
E0
E0
5.95645E-05
3.48063E-05
2.36958E-05
2.11269E-05
2.01121E-05
1.52756E-05
18919
[substrate]
[substrate]
[substrate]
[substrate]
[substrate]
[substrate]
0.000175
0.000116667
5.83333E-05
0.000175
0.000116667
5.83333E-05
[Emax]
[Emax]
[Emax]
[Emax]
[Emax]
[Emax]
0.000175
0.000116667
5.83333E-05
0.000175
0.000116667
5.83333E-05
K2/Ks S-1
K2/Ks S-1
K2/Ks S-1
K2/Ks S-1
K2/Ks S-1
K2/Ks S-1
109.1428571
104.5714286
173.1428571
72.57142857
160.2857143
144
127.2857
K3
K3
K3
K3
K3
K3
0.000177478
0.00015186
0.000223065
0.000125094
0.000131406
0.000242215
0.000175
[Eo]/[E]
[Eo]/[E]
[Eo]/[E]
[Eo]/[E]
[Eo]/[E]
[Eo]/[E]
0.340368337
0.298339538
0.406212953
0.120725197
0.172389057
0.261868265
1. The difference in the absorption between the steady state and the burst phase was noted.
2. The natural log of these values was taken.
3. We then plotted a graph of In(ΔAbs) against time and from this found the value of b.
4. From the equation of the steady state we knew A to be the gradient and B to be the y-intercept. A was divided by the extinction coefficient.
5. E0 was B divided by the extinction coefficient
6. The concentration of substrate was found by multiplying the 7mM value and the amount of substrate (75-25µl) present to found the moles. This was divided by the total volume of the mixture (3ml).
7. [Emax]=[Substrate]
8. K2/Ks=b/S0
9. K3=A/[E0]
Appendix
See scans for data.
Introduction
Chymotrypsin is a protease which cleaves proteins by a hydrolysis reaction, it does this by adding a molecule of water to a peptide bond. Although the hydrolysis reaction is thermodynamically favoured in the absence of a catalyst the half-life for a typical hydrolysis reaction by a protease is between 10 and 100 years, needless to say it is extremely slow1. Though this is true peptide bonds are hydrolysed within milliseconds in the body in the presence of catalysts. The kinetic stability of chymotrypsin which gives it hydrolysis resistance is due to the resonance structure that accounts for the planarity of the peptide bond. Such is the strength of this resonance structure that it confers partial …show more content…
double bond character. The carbonyl carbon atom in peptides is less electrophilic and therefore less susceptible to nucleophilic attack than carbonyl carbon atoms in most other compounds. This means that for a protease to function it has to cleave peptide-bonds at an unreactive carbonyl group1.
For this experiment we looked at the kinetics of chymotrypsin. We calculated the extinction co-efficient of p-nitrophenoxide which was important for the enzyme kinetic assays we carried out later in the experiment. We then measured the concentration of some stock solutions of chymotrypsin using both the Folin and BIORAD assays so we could get an idea of which assay is more accurate when measuring enzyme concentration. Finally we looked at the kinetics of chymotrypsin by carrying out various enzyme kinetic assays. We used different enzyme and substrate concentrations in our assay so we could determine the mechanism of action of chymotrypsin.
Chymotrypsin breaks down proteins in the digestive systems of mammals as well as other organisms. It selectively cleaves peptide bonds on the carboxyl-terminal side of the large hydrophobic amino acids such as tryptophan, tyrosine, phenylalanine and methionine1. Chymotrypsin operates by covalent catalysis whereby the serine residue acts as a nucleophile to attack the unreactive carbonyl carbon atom of the substrate so that it becomes briefly attached.
The kinetics of chymotrypsin action can be monitored by measuring the absorbance of a coloured product when the enzyme is acting on a substrate analog. In this experiment the coloured product is p-nitrophenoxide and the substrate is p-nitrophenyl trimethylacetate. Under steady state conditions, the reaction is known to favour Michaelis-Menten kinetics with a KM of 20µM and a kcat of 77s-1. The hydrolysis begins with an initial rapid burst phase followed by a steady state phase1.
The two phases of the reaction are caused because a covalently bound enzyme-substrate intermediate is formed. The acyl group initially becomes covalently attached to the enzyme as p-nitrophenoxide is released. This acyl-enzyme intermediate is then hydrolysed to release the carboxylic acid component of the substrate and regenerate the free enzyme1. This means that one molecule of p-nitrophenoxide is produced rapidly from each enzyme molecule as the acyl-enzyme intermediate is formed. Both phases are needed for enzyme turnover however it takes a lot longer to effectively free the enzyme for use by the hydrolysis of the acyl-enzyme intermediate1.
Chymotrypsin is spherical and is made up of 3 polypeptide chains conjoined by disulphide bonds. It is synthesized as chymotrypsinogen which is a single polypeptide which yields 3 chains after it is activated by proteolytic cleavage. Serine 195 marks the active site of chymotrypsin and lies in a cleft on the surface of the enzyme1. The unusually high reactivity of serine 195 is determined by the structure of the active site. There is a hydrogen bond between the imidazole ring of histidine 57 and the side chain of serine 195. The NH group of the imidazole ring is also hydrogen bonded to the carboxylate group of aspartate 102. The three aforementioned residues together make up the catalytic triad1.
The histidine residue polarizes the hydroxyl group of the serine side chain so that it is ready for deprotonation. When the substrate is present the histidine residue accepts the proton from the serine 195 hydroxyl group whereby the histidine residue effectively acts as a general base catalyst. The withdrawal of the proton from the hydroxyl group furthermore generates an alkoxide ion which is a much more powerful nucleophile than an alcohol. The aspartate residue is partly responsible for orienting the histidine residue to make it a better proton acceptor through hydrogen bonding and electrostatic effects1.
After the substrate has binded, the oxygen atom of the side chain of the serine 195 makes a nucleophilic attack on the carbonyl carbon atom of the target peptide bond. At this stage there are four atoms bonded to the carbonyl carbon arranged in a tetrahedron which is an unstable structure with a negative charge on the oxygen atom derived from the carbonyl group. This charge is stabilized by interactions with NH groups from the protein in a site called the oxyanion hole. The tetrahedral intermediate then collapses to form the acyl-enzyme intermediate which is also stabilised by the same interactions. This stage involves the histidine residue transferring a proton to the amino group formed by the cleavage of the peptide bond. The amine component then is freed from the enzyme which is the last step of the acylation of the enzyme1.
Deacylation occurs when a water molecule occupies the place where the amine component previously was.
The ester group of the acyl-enzyme is then hydrolysed by nucleophilic attack of serine on the peptide carbonyl group, collapse of the tetrahedral intermediate and the release of the amine component. Histidine 57 then acts as a general acid catalyst and draws a proton away from the water molecule. The OH- ion then attacks the carbonyl carbon atom of the acyl group which forms another tetrahedral intermediate which breaks down to form the carboxylic acid product. The release of this acid then frees up the enzyme to continue …show more content…
catalysis1.
Methods
The extinction coefficient of the p-nitrophenoxide ion was determined by measuring the absorbance twice in a sodium hydroxide solution containing p-nitrophenyl trimethylacetate. The average result was taken.
The concentration of α-chymotrypsin was determined by a Folin assay as well as a BIORAD assay. Both required collecting results from a BSA standard in order to work out the values for the concentration for α-chymotrypsin.
The enzyme assays were done at three different substrate concentrations and two enzyme concentrations. The absorbance was read from a spectrometer at different time intervals. p-nitrophenyl tri-methylacetate was used as the substrate instead of p-nitrophenyl acetate which would have occurred to fast and would have required the stopped-flow spectrophotometric technique to follow.
Results
Extinction Coefficient (ε) of p-nitrophenoxide
This was calculated using the Beer-Lambert law:
The two absorbance readings were 1.092 and 1.114. (1.092+1.114)/2=1.103 which gave the mean absorbance.
We rearranging the Beer-Lambert law to work out the extinction coefficient: ε=A/(lc).
By substituting A for 1.103, l for 1cm and c for 5.83 x 10-5 we worked out ε to be 18919mol-1Lcm-1.
Measurement of a-chymotrypsin concentration
Folin Assay
Figure 1
We took the average of two absorbance measurements from each α-chymotrypsin sample which were at estimated concentrations of 0.02mg/ml, 0.03mg/ml and 0.04mg/ml. We used the equation y=0.5671x-0.0085 which was the line of best fit from the BSA concentrations to find out the actual concentration of α-chymotrypsin.
Since y represented the absorbance at 680nm, x was used to represent the actual concentration of α-chymotrypsin in mg/ml. We rearranged the equation to x=(y+0.0085)/0.5671.
We substituted each mean absorbance measurement for y in the rearranged equation to obtain the actual concentration of α-chymotrypsin.
The estimated concentration results were diluted differently, the 0.02mg/ml sample was diluted 30 fold, the 0.03mg/ml sample was diluted 20 fold and the 0.04mg/ml sample was diluted 40/3 fold. The corresponding actual concentration results were respectively multiplied by 30, 20 and 40/3 to obtain the actual concentration of α-chymotrypsin in the original solution.
The results were as follows:
Estimated Concentration of α-Chymotrypsin (mg/ml)
Mean Absorbance
Actual Concentration of α-Chymotrypsin (mg/ml)
Actual Concentration of α-Chymotrypsin in the Original Solution(mg/ml)
0.02
0.02
0.050255687
1.507670605
0.03
0.028
0.064362546
1.287250926
0.04
0.049
0.101393052
1.351907365
Figure 2
The average actual concentration of α-chymotrypsin in the original solution was 1.382276299 mg/ml.
BIORAD Assay
Figure 3
We took the mean of two absorbance measurements for each estimated α-chymotrypsin sample. We used the equation y=61.7x-0.0153 from the line of best fit from Figure 3 to work out the actual α-chymotrypsin concentrations in each sample by rearranging it to x=(y+0.0153)/61.7. We substituted the mean absorbance for y to obtain the actual concentration of α-chymotrypsin.
The estimated 0.004mg/ml and 0.008mg/ml concentrations were diluted 250 fold and 125 fold respectively. So the results obtained by the rearranged equation were multiplied by 250 and 125 respectively.
The results were as follows:
Estimated Concentration of α-Chymotrypsin (mg/ml)
Mean Absorbance
Actual Concentration of α-Chymotrypsin (mg/ml)
Actual Concentration of α-Chymotrypsin in the Original Solution(mg/ml)
0.004
0.1805
0.00317342
0.793354943
0.008
0.3135
0.005329011
0.666126418
Figure 4
The average actual concentration of α-Chymotrypsin in the original solution was 0.729740681.
Enzyme Assays of α-chymotrypsin
Figure 5
Figure 5 displays the cumulative production of the p-nitrophenoxide ion which is produced by the chymotrypsin catalysed reaction. During the reaction the enzyme concentration remained at 50µl whilst the substrate concentration was varied by addition of either 25, 50 or 75µl aliquots of substrate. At all the substrate concentrations we can witness the initial burst phase followed by the steady state phase which indicates the biphasic nature of the reaction. As the substrate concentration was increased so did the absorbance as was expected. We can see that the gradient and so therefore the rate of the steady state increased gradually as the amount of substrate increased. Likewise when looking at the initial burst phase we can see that the gradient also becomes more steep at increasing levels of substrate.
Figure 6
Figure 6 represents the production of the p-nitrophenoxide ion similarly to Figure 5 however 25µl of enzyme was used instead of 50µl. Again we can witness the initial burst phase followed by the steady state phase which indicates the biphasic nature of the reaction. There was an increase in absorbance after 25µl of substrate but after that the readings for 50µl and 75µl were very similar. The steady state rate and the initial burst rate for these two substrate levels were also very similar. At 25µl of substrate the rate at the steady state was greater than it was at higher substrate levels. Figure 8 Figure 7
Figure 9 Figure 10
From looking at Figure 7 we can see that the A constants for 50µl of enzyme were significantly higher than for 25µl of enzyme for all the substrate amounts. There was not a clear correlation between the substrate amount and the A constant since for 25µl of enzyme, higher substrate amounts decreased the A constant and at 50µl of enzyme, lower substrate amounts increased the A constant. It has to be noted that the scale is relatively small, suggesting that the effect of the enzyme was minimal.
From looking at Figure 8 we can see that increasing the amount of enzyme increased the B constant for all the different substrate amounts. The B constant showed little variation at 25µl of enzyme and a lot more variation at 50µl of enzyme. There seems to be a positive correlation between the substrate amount and the B constant.
For Figure 9, we can see that there is a positive correlation between the substrate amount and the b constant, apart from the unexpected high result at 25µl of enzyme and 50µl of substrate. When the amount of enzyme is doubled it does not affect the b constant.
For Figure 10 at 25µl of enzyme there is a negative correlation between the amount of substrate and the [E0]/[E] value. There was a generally higher [E0]/[E] value when the amount of enzyme is doubled. There seems to be no correlation between the amount of substrate and the [E0]/[E] value of 50µl of enzyme.
The average value of K2/KS constant in our experiments came to be 127.2857s-1, this is close to published value of the K2/KS constant of 231.25s-1. The average value of the K3 constant was 2 x 10-3 which is quite similar to the published value of 1.3 x 10-4.
Discussion
We worked out the extinction co-efficient of p-nitrophenoxide to be 18919mol-1Lcm-1 and the value we got was close to the published value2 of 18000mol-1Lcm-1. Our result is within 1 standard deviation of the class mean. This suggests that the value we obtained is reliable since similar results have been achieved by so many others.
For measuring the concentration of chymotrypsin in a stock solution both the BIORAD and the Folin methods determined that the protein concentration of chymotrypsin was close to the sample region at approximately 1mg/ml. The Folin Lowry assay calculated a concentration of 1.382276299mg/ml for chymotrypsin whereas the BIORAD method calculated a concentration of 0.729740681mg/ml.
The Folin method involves the reduction of the Folin reagent by tryptophan, tyrosine, cysteine, cystine and histidine which means that the absorbance is going to be affected by the number of the aforementioned exposed amino acid residues on the proteins.
The Folin assay overestimated the chymotrypsin concentration because of the difference in the properties of BSA and chymotrypsin. The different amino acid composition could have had an effect on how much the Folin reagent was reduced which causes the observed colour change. Too many of these amino acids which have this reducing ability in chymotrypsin could have led to the higher observed concentration seen in this
experiment.
The BIORAD method relies on binding of Coomassie Brilliant Blue G-250 to the protein and the hydrophobic interaction between the dye and the protein. The reliance on hydrophobic interaction means that the binding is dependent on the number of exposed hydrophobic residues on the protein as well as the temperature. The BIORAD assay underestimated the protein concentrations. Not all proteins have the same hydrophobic properties, chymotrypsin may have not had very potent hydrophobic properties which would have meant there is not as much colour change and would have caused the low concentration of chymotrypsin that was observed. It is possible that chymotrypsin has fewer hydrophobic regions which Coomassie Brilliant Blue can bind to which would have caused a reduction in chymotrypsin concentration.
After comparing the results to the class averages we saw that the values we obtained were within 1 standard deviation of the class mean values gained by each method. This is testament to the accuracy of our results. The class results also showed that the Folin assay overestimated the chymotrypsin concentration at 1.10mg/ml and that the BIORAD method underestimated the chymotrypsin concentration at 0.56mg/ml. The Folin assay had a standard deviation of 0.40mg/ml which was larger than the BIORAD assay standard deviation of 0.19mg/ml. This lower standard deviation suggested that the BIORAD assay was a more precise method. However if we assumed that the actual concentration of chymotrypsin was exactly 1mg/ml then the Folin assay would have been more accurate than the BIORAD assay. Table 2 in the appendix of the lab manual also showed that the Folin method overestimated the concentration of chymotrypsin at 11.6mg/ml in a 10mg/ml stock solution whereas the BIORAD method underestimated it at 7.8mg/ml. This only further agreed with our conclusions so far. The class data disproved our null hypothesis that the mean of Folin is equal to the mean of BIORAD with a probability of less than 0.001. This was to be expected since two completely different methods were employed.
Finally, we looked at the kinetics of the chymotrypsin catalysed hydrolysis. The hydrolysis of p-nitrophenyl tri-methylacetate is slower than the hydrolysis of p-nitrophenyl acetate because of the steric hindrance of its bulky tri-methylacetate group which slowed down the reaction. During the reaction a serine residue attacks the anti-bonding orbital of the carbon group, which results in the hydrolysis of the ester. This requires the serine nucleophile to overlap with the anti-bonding orbital which relies on close alignment. The bulky tri-methylacetate group reduces the chance that the nucleophile comes into the correct orientations to perform the catalytic reaction, therefore causing a much slower reaction.
By looking at Figure 5 we can see that the rate of the burst phase was positively correlated with the substrate concentration at 50µl of enzyme. This is because at this phase increased amounts of the substrate will mean that there will be more successful collisions between the enzyme and the substrate which will in turn result in more product being observed. However this same increase was not observed by looking at Figure 6 where two very similar burst phase and steady state rates were obtained at 50µl and 75µl of substrate. At 25µl of substrate the rate at the steady state was greater than it was at higher substrate levels, which suggests that a different factor other than the amount of substrate had become limiting. I would have expected a higher amount of substrate to result in a higher burst phase and steady state rate as in Figure 5. However since in Figure 6 half as much enzyme was used this suggests that the enzyme became saturated at lower levels of substrate. The level of enzyme was the limiting factor. The enzyme became saturated when 50µl of substrate was added which is why higher amounts of substrate had no effect on the reaction. The lower rate of reaction when 75ul of substrate was added may have also been due to experimental error most likely by a lack of pipetting accuracy.
From comparing Figures 5&6 it could also be seen that a higher concentration of product was produced after the burst phase when higher concentrations of enzyme were used. This is expected since there is much more free enzyme to which the substrate can bind to. At a lower enzyme concentration, there would be less active sites to occupy which would result in less reactions and therefore less product. This is why the steady state rates in Figure 6 occur at a much lower absorbance than the steady state rates in Figure 5.
For Figure 7 when the amount of enzyme was doubled the constant A increased, although the constant was affected by the amount of substrate due to experimental error most likely by inaccurate pipetting. However, this does show that constant A is dependent on enzyme concentration since A=k3[E0] even though it is not completely independent of the substrate. For Figure 8 we can see that apart from the anomaly the doubled amount of enzyme increased the B constant. Similarly the substrate showed interference most likely caused by the same way. This suggests that constant B is dependent on enzyme concentration since B=[E0]. For Figure 9 we can see that the doubled enzyme amount does not affect the b constant and the higher amount of enzyme there was a positive correlation between the amount of substrate and the b constant. This proves that constant b is independent of the enzyme and suggests that it is dependent on the substrate since b=(k2[S0]/KS).
For Figure 9 the fraction of active enzyme [E0]/[E] was worked out for each reactions. As seen and expected as the amount of enzyme was doubled the fraction of active enzyme increased. This value can be affected by temperature, pH, and salt content. If the temperature and pH are not optimal the enzymes will not function at their maximum capability. If these conditions stray too far from the optimum the enzymes may even become denatured if the bonds which make up the active sites become altered. The ions in extremely high salt concentrations would interfere with the weak ionic bonds of proteins which would also inhibit the functionality of the enzyme. Furthermore when there are other macromolecules in the solution macromolecular crowding can occur which can alter the rates and equilibrium constants of enzyme reactions which will definitely have a knock on effect on the fraction of active enzyme.
References
1. Berg, J., Tymoczko, J., and Stryer, L. (2012) Biochemistry, 7th edition, New York: W. H. Freeman and Company.
2. Bender et al. (1967) Journal of Chemical Education 44, 84-88.
Further Calculations
50μl enzyme
25μl enzyme
75μl substrate
50μl substrate
25μl substrate
75μl substrate
50μl substrate
25μl substrate
15
0.7629
0.545
0.3368
0.27445
0.24725
0.22905
30
0.5809
0.4365
0.2893
0.2582
0.186
0.1851
45
0.4059
0.344
0.2438
0.18695
0.14575
0.17515
60
0.3049
0.2925
0.2133
0.1567
0.1125
0.1552
75
0.2369
0.256
0.1848
0.13145
0.07325
0.13425
90
0.1869
0.2165
0.1573
0.1122
0.064
0.1183
LN
LN
LN
LN
LN
LN
15
-0.270628318
-0.606969484
-1.088265997
-1.292986184
-1.397355308
-1.473814959
30
-0.543176654
-0.828966904
-1.240291067
-1.354020801
-1.682008605
-1.686859059
45
-0.901648455
-1.067113622
-1.411407062
-1.676914078
-1.925862454
-1.742112529
60
-1.187771425
-1.229290612
-1.545055654
-1.85342213
-2.184802057
-1.863040671
75
-1.440117168
-1.362577835
-1.68848112
-2.029128728
-2.613877031
-2.008051546
90
-1.677181565
-1.530164732
-1.849600469
-2.187472286
-2.748872196
-2.134531508
b b b b b b 0.0191
0.0122
0.0101
0.0127
0.0187
0.0084
A
A
A
A
A
A
1.05714E-08
5.28569E-09
5.28569E-09
2.64285E-09
2.64285E-09
3.69998E-09
B
B
B
B
B
B
1.1269
0.6585
0.4483
0.3997
0.3805
0.289
E0
E0
E0
E0
E0
E0
5.95645E-05
3.48063E-05
2.36958E-05
2.11269E-05
2.01121E-05
1.52756E-05
18919
[substrate]
[substrate]
[substrate]
[substrate]
[substrate]
[substrate]
0.000175
0.000116667
5.83333E-05
0.000175
0.000116667
5.83333E-05
[Emax]
[Emax]
[Emax]
[Emax]
[Emax]
[Emax]
0.000175
0.000116667
5.83333E-05
0.000175
0.000116667
5.83333E-05
K2/Ks S-1
K2/Ks S-1
K2/Ks S-1
K2/Ks S-1
K2/Ks S-1
K2/Ks S-1
109.1428571
104.5714286
173.1428571
72.57142857
160.2857143
144
127.2857
K3
K3
K3
K3
K3
K3
0.000177478
0.00015186
0.000223065
0.000125094
0.000131406
0.000242215
0.000175
[Eo]/[E]
[Eo]/[E]
[Eo]/[E]
[Eo]/[E]
[Eo]/[E]
[Eo]/[E]
0.340368337
0.298339538
0.406212953
0.120725197
0.172389057
0.261868265
1. The difference in the absorption between the steady state and the burst phase was noted.
2. The natural log of these values was taken.
3. We then plotted a graph of In(ΔAbs) against time and from this found the value of b.
4. From the equation of the steady state we knew A to be the gradient and B to be the y-intercept. A was divided by the extinction coefficient.
5. E0 was B divided by the extinction coefficient
6. The concentration of substrate was found by multiplying the 7mM value and the amount of substrate (75-25µl) present to found the moles. This was divided by the total volume of the mixture (3ml).
7. [Emax]=[Substrate]
8. K2/Ks=b/S0
9. K3=A/[E0]
Appendix
See scans for data.