Km And Vmax Lab Report

Determining the of value of Km & Vmax at 37°C and 65°C using Michaelis-Menten

Introduction

Biological reactions involves the use of enzymes, which acts as catalyst. Reactions only occur if the substrate fits into the active site of an enzyme and so changes in temperature can denature the enzyme and therefore makes it void[1]. Trypsin is a digestive enzyme used to breakdown proteins such as BAPNA in the body, It has a optimum temperature of 37 °C[2]. The aim of this experiment is to calculate Km and Vmax at 37 and 65 °C by using Michaelis-Menten and Eadie-Hofstee Graph in order to determine how temperature affects the rate of digestion of BAPNA by trypsin.

Method

Eight ml of six different substrate concentrations (2000, 1500, 1250, 1000,

Figure 1: Michaelis-Menten graph used to estimate the Km and Vmax at 37 & 65 °C Table 3: showing the values of Km and Vmax calculated from graphpad

Km and Vmax For Michaelis-Menten From Graphpad at 37 And 65°C

37°C

65°C

Km

6284

6106

Vmax

0.2585 0.14 00

Table 4: Shows the values used to produce a Eadie-Hofstee graph at 65°C

Initial Velocity/[S] Against Initial Velocity Concentration At 37°C

Initial velocity V0 / [S] x 10-5( µMmin-1dm3mol-1)

Initial velocity V0 (µMmin-1)

3.4821

0.03980

3.6215

0.03104

3.7085

0.02649

3.7102

0.02120

4.0357

0.01153

Figure 2: Eadie-Hofstee linear graph to determine Km and Vmax at 37°C

Table 5: shows the values used to produce a Eadie-Hofstee graph at 65°C

Initial Velocity/[S] Against Initial Velocity Concentration At 65°C

Initial velocity V0 / [S] x 10-6 (µMmin-1dm3mol-1)

Initial velocity V0 (µMmin-1)

3.8688

0.004422

3.7335

0.003200

4.4491

0.003178

4.3945

0.002511

3.9657

0.001133

Figure 3: Eadie-Hofstee linear graph to determine Km and Vmax 65°C

Km and Vmax For Eadie-Hofstee Kinetic Graph at 37 And

Km and Vmax could not be determined accurately using the Michaelis-Menten graph as it doesn’t plateau at 37/65°C , because a high enough substrate concentration was not used at regular intervals. Table 6 shows that Km value for 65°C is lower than 37°C which suggest that a higher affinity of trypsin to BAPNA at 65°C; because a higher collision frequency would occur due to an increase in kinetic energy and seems more efficient. However, a higher Vmax at 37°C indicates that trypsin was saturated with BAPNA because the optimum temperature of trypsin is 37°C. In figure 1 at higher temperatures the reaction plateaus at a faster rate as the enzymes active sites become denatured. The Eadie-Hofstee kinetics graphs shows the value for Km at 37°C is higher than at 65°C, which supports calculations of Km and Vmax found from graphpad. Lineweaver- Burk can be used to find Km and Vmax however it’s inaccurate and outdated . Eadie hofstee is more accurate than the Michaelis-Menten as it has a linear relationship, thus it is prefered. From the values of Km and Vmax it can be concluded that trypsin is more efficient at 37°C. When the temperature is increased Km is larger therefore the affinity also increases but Vmax decreases so rate of product formation is lowered .However the data gathered is unreliable as the test was not