Virtual Investigation of an Enzyme. A new protease (ref. no PR/66-430-010), isolated from B.yorkii has been characterised. A series of experiments were run on the enzyme to determine the optimum pH for the enzyme to run at, the effect of pH on KM and Vmax

Results

Fig.1 shows that the optimum pH at which the enzyme works at was pH 7.0.

Fig.2 is a Lineweaver-Burk plot of the reciprocal values of the enzyme recorded at ph 7.0, with all values constant other than the concentration of the substrate.

The Vmax and Km of the enzyme under inhibitor influence, double inhibitor and a control of no inhibitor were calculated from the lines of best fit on the plot and are as follows.

Control                Vmax - 1/0.35 = 2.9

                            Km – 1/15 = 0.06

Inhibitor               Vmax - 1/0.35 = 2.9

                            Km – 1/4 = 0.25

Double Inhibitor  Vmax - 1/0.35 = 2.9

                            Km – 1/2.5 = 0.4

Discussion

The precision of the assay procedure was determined using the following data:

The mean of the velocity was calculated as 1.19075 and the standard deviation (SD) was calculated to be 0.047842439.

The effect of pH on the enzyme was determined when the following results were obtained and then plotted as in Fig. 1

From these results it can be seen that pH 7.0 was the optimum pH for the enzyme to work under. The velocity of the reaction was at it’s highest under this pH. When the pH was raised the velocity of the reaction dropped and above pH 9, the reaction was too slow to record the velocity. The same was true for the velocity when the pH was lowered, with the velocity again dropping.

The effect of the inhibitor azide can be seen from Fig.2 to slow the velocity and reduce the Km of the reaction. When the concentration of azide was at 0.2µmol the inhibition was at it’s highest and when the concentration of the inhibitor was doubled, the inhibition rate was reduced slightly. Also, from the Lineweaver-Burk plot is was determined that azide is a competitive inhibitor. This was determined because the lines of best fit of the solutions which contained the inhibitor crossed at the same point on the y-axis and met with the control experiment at this point.