Analysis Assignment 6

1.  Using the data of measured absorbance vs. time (min) for each substrate concentration, determine the slope of the linear portion of this curve.  The value of the slope is the initial velocity (Vo) in Abs/min.  Convert AU/min to mM/sec using the extinction coefficient you calculated for 5-TNB.  Use these values to construct a table in Excel with the following column titles.

 Trial # Sample # [E] mM [S] mM 1/[S] mM-1 Vo (mM/sec) 1/Vo (sec/mM)

• Print copies of each table

2.  Plot the initial velocity as a function of substrate concentration. This is a Michaelis-Menton plot. Fit the curve the best way you can. Estimate Vmax and KM from this plot. Note: The curve may not level off to give an accurate Vmax, but please try your best.  Print a copy of this plot with proper labels.  Add additional curves for each additional substrate.

3.  Plot (1/V0) as a function of (1/[S]). This is a Lineweaver - Burk plot or a double reciprocal plot. The data should fit a straight line. Calculate Vmax and KM from this plot.  Print a copy of this plot.  Complete plots and calculations for each additional substrate.

4.  Complete the table on the back of this page for easy comparison.  Use additional tables if needed.

5.  Write a descriptive paragraph interpreting your results.  Relate the numbers to your hypothesis (as best you can) and to the mechanism for specificity and chemistry of the enzyme substrate interaction.  Remember our goal is not to just generate numbers, but to give these numbers meaning in terms of how the enzyme shows specificity and enhances the rate of the reaction.

6.  Turn in your analysis by Friday at 5:00.

Name

 Substrate: Michaelis-Menton Lineweaver- Burke Enzyme Conc. (mM) pH Km (mM) Vmax (mM/sec) kcat (sec-1) Km (mM) Vmax (mM/sec) kcat (sec-1)

 Substrate: Michaelis-Menton Lineweaver- Burke Enzyme Conc. (mM) pH Km (mM) Vmax (mM/sec) kcat (sec-1) Km (mM) Vmax (mM/sec) kcat (sec-1)

 Substrate: Michaelis-Menton Lineweaver- Burke Enzyme Conc. (mM) pH Km (mM) Vmax (mM/sec) kcat (sec-1) Km (mM) Vmax (mM/sec) kcat (sec-1)

Disclaimer