Enzyme Kinetics Lab Report

Protein Lab

Module IV

Determination of Kcat and Km of Trypsin hydrolyzed reaction

Minor Lab Report

Presented to

Dr. Claire Komives

San José State University

ChE - 194

by

Priyanka Tiwari

March 14, 2012

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1 Objective To measure enzyme activity of Trypsin (Km and kcat) which is used to hydrolyse benzoyl-DL-arginine-4-nitroanilide hydrochloride and the variation of enzyme activity with change in pH.

2 ResultsThe experiment was carried out using Tris-HCl buffer at different pH of 7.5, 8.0 and 8.5. The initial rate of reaction was plotted with substrate concentration for all pH values. A non-linear regression method was used to find maximum rate forward velocity Vm and Michaelis-Menten constant (Km) using the following equation.

V=Vm× SKm+S (1)

Where S is the substrate concentration, V is the rate of reaction and Vm & Km are maximum forward rate velocity and Michaelis-Menten constant respectively.

2.1 Results at pH = 7.5

Figure 1 shows the variation of initial rate versus substrate concentration at pH 7.5. The values of Vm and Km obtained from Polymath are 2.92 mMol/L/hr and 15 mM respectivley. Kcat was calculated using these values and it was found to be equal to 0.11 mMol/g/min. The Polymath results are shown in Figure 2 and Figure 3. The value of R2 is 0. 97 and variance is 0.001. The standard error was calculated as 0.0345 in Excel.

0 0.5 1 1.5 2 2.5 3 3.5 4 4.50

0.1

0.2

0.3

0.4

0.5

0.6

0.7

pH = 7.5

pH = 7.5

Substrate DL-BAPA, [S] (mM)

Initial rate, V, mMol/L/hr

Figure 1. Initial rate versus substrate concentration at pH = 7.5

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Figure 2. The results of Polymath for a pH of 7.5

Figure 3. Shows the difference between Calculated initial rate versus experimental initial rate. The two curves are following each other closely. Thus it is a good fit to Michealis-Menten equation


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Figure 4 shows the variation of initial rate versus substrate concentration at pH 8.0. The values of Vm and Km obtained from Polymath are 0.6 mMol/L/hr and 1.08 mM respectivley. kcat was calculated using these values and it was found to be equal to 0.02 mMol/g/min. The Polymath results are shown in Figure 5 and Figure 6. The value of R2 is 0.29 and variance is 0.03. The standard error was calculated as 0.195 in Excel.

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5-0.10

0.10.20.30.40.50.60.70.8

pH=8

pH=8

Substrate DL-BAPA, , [S] (mM)




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Figure 6. Shows the difference between Calculated initial rate versus experimental initial rate.


Figure 7 shows the variation of initial rate versus substrate concentration at pH 8.5. The values of Vm and Km obtained from Polymath are 37 mMol/L/hr and 303 mM respectivley. kcat was calculated using these values and it was found to be equal to 1.41 mMol/g/min. The Polymath results are shown in Figure 8 and Figure 9. The value of R2 is 0.72 and variance is 0.011. The standard error was calculated as 0.109 in Excel.

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5-0.2

0

0.2

0.4

0.6

0.8

pH = 8.5

pH = 8.5

Substrate DL-BAPA, [S] (mM)



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Figure 9. Shows the difference between calculated initial rate versus experimental initial rate. The difference between the calculated and experimental value is large.

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3 Discussion

1.1 Interpretation of results and comparison with published literature From the results we can conclude that the activity of enzyme is affected with variation in pH. The above results show that Km is low at low pH. A low Km indicates a higher affinity of the enzyme towards the substrate.

The value of Km obtained at pH 7.5 was 15mM. From figure 2 and figure 3, we can conclude that the results fit the Michaelis-Menten model equation well. A similar study was carried out by Tokura et al in 1971. On hydrolysis of DL-BAPA with enzyme trypsin, the researches got a value of 2.8 mM at a pH of 7.2 [1].In another experiment by Sears et al., the researchers calculated a value of 0.98 min-1[2]. In our experiment, Km and kcat were found to be equal to 15 mM and 0.11 mMol/g/min respectively at a pH of 7.5. The reason for this variation could be different working conditions for both the experiments. The value of kcat is reported as mol/g/min. The value can be expressed in min-1 if it is multiplied with the molecular weight of Trypsin.

At higher pH values of 8.0 and 8.5, it is observed that there is a significant error in polymath results. The R2 values are low (0.6 and 0.7). This means that the experimental results don’t fit Michaelis-Menten model well. The reason can be autohydrolysis of enzyme that has brought the error in initial rates measurement. There are also chances of pipetting errors that might have led to the errors in the experiment.

To summarize we can say that the effect of pH is clearly seen in the experiment. At low values of pH a higher affinity of enzyme towards substrate was observed and the affinity was decreased as seen by the increased value of Km at higher pH (8.5).

3.1 Sources of Error Autohydrolysis of enzyme can result if the enzyme solution is prepared before the

substrate solution. The enzyme solution should be added to substrate just before one is ready to take the

readings in the UV-vis spectrophotometer. This is because we are interested in measuring the initial rate of reaction.

Pipetting mistake is also a source of error. If the enzyme solution is not kept cold during the reaction, it may result in autohydrolysis

of enzyme. Buffer solution should be prepared carefully. If a buffer is not prepared well, the readings

for reaction rate will be erroneous.

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4 Conclusions

The results of hydrolysis of DL-BAPA by trypsin at different pH are as follows.

pH Km (mM) kcat (mMol/g/min) Standard Error7.5 15 0.11 0.03458 1.08 0.02 0.195

8.5 303.991 1.41 0.109

Therefore we can see that the activity of the enzyme got changed with a change in pH. This is an expected behavior. We know that at low pH, the enzyme activity is high. This is also shown from the experimental results at pH=7.5. In the experiment the standard error is minimum at pH=7.5. The experimental results also fit the Michaelis-Menten model well as the value of R2 is close to 1 and the variance is less than 0.01. A similar study by Tokura et al. and Sears et al. also show that the optimum pH for Trypsin activity is 7.2. Thus the result is in agreement with the published literature.

5 References

1. S. Tokura, N. Nishi and J. Noguchi, "A new substrate for papain, benzoyl-L-arginine-p-nitroanilide (L-BAPA)", Journal of Biochemistry, 69,. 599 (1971).

2. P.S. Sears, D.S. Clark, “Comparison of soluble and Immobilized Trypsin kinetics : Impliction for peptide Synthesis”, Biotech. & Bioengineering, 42, 118-124 (1993).

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Enzyme Kinetics Lab Report