LABORATORY SCHEDULE

Professor Charles E. McKenna
Department of Chemistry
Last updated October 10, 2003

All labs are in SGM 121

Week of October 13: Enzyme Kinetics using a Spreadsheet
Due date: Your lab date plus one week

In Lab 4, you learned to draw organic molecules in both two and three dimensions. In particular, you created the structure of an HIV-1 protease inhibitor. In Lab 5, you studied several protein and nucleic acid 3D structures. You paid special attention to HIV-1 protease and explored how a peptide inhibitor binds to the active site. In this lab, you will analyze experimental drug inhibition data from actual kinetic inhibition experiments done on the enzyme and will draw conclusions concerning mechanisms of drug action and location of the inhibitor binding site.

In order to complete this assignment, you must construct a Lineweaver-Burk plot, also known as a double reciprocal form of the Michaelis-Menten Equation, as discussed in class. In this plot, the y-axis is 1/V and the x-axis is 1/[S]. The slope of the line is Km/ V max and the intercept, i.e. the y-axis intercept is 1/V max; the extrapolated1/[S] intercept, i.e. the x-axis intercept is -1/Km. Finally, (1+I/Ki ) is equivalent to the slope for the inhibited enzyme divided by the slope for the enzyme without any inhibitor presented where I is the concentration of drug . To get Ki for each inhibitor, find the ratio of the slope of the inhibition curve to the slope of the control (no I) curve. Set this ratio equal to (1+I/K i), plug in the value of 'I' and solve for Ki. Ask your TA if you need more help with this.

A handout is provided online that guides you though the use of Microsoft Exel 5.0 or the iMac Appleworks spreadsheets, specifically for this assignment. Here is a link for Help Using Microsoft Excel 5.0 Graphing .

Please note that 1 millimolar (mM) =1000 micromolar (mM) and 1 micromolar (mM) = 1000 nanomolar (nM).

 Part 1: Lineweaver-Burk plot

The kinetics of HIV-1 protease-catalyzed hydrolysis of a substrate (Lys-Ala-Arg-Val-Nle-Nph-Glu-Ala-Nle-NH2) has been studied at various concentrations of a certain inhibitor. The short peptide substrate simulates the real viral protein substrate by mimicking the cleaved area. The data presented in Table 1 show the effect of substrate inhibitor concentration on the rate of substrate cleavage by the enzyme. (Note: Watch out for UNITS!)

i. Using Excel©, construct Lineweaver-Burk plots for the data. Find the x-axis intercept(s) (3 points).

ii. Calculate Km and Vmax (2 points).

iii. Determine the type of inhibition. (1 point).

iv. Evaluate Ki (2 points).

Recently, a different inhibitor for the same HIV-1 protease was investigated. The kinetic data were obtained by measuring the rate of cleavage of another peptide substrate (Lys-Ala-Arg-Val-Leu-Nph-Glu-Ala-Met-NH2). The data are presented in Table 2.

i. Using Excel©, construct a Lineweaver-Burk plots for the data. Find the x-axis intercept(s) (3 points).

ii. Calculate Km and Vmax (2 points).

iii. Determine the type of inhibition. (1 point).

iv. Evaluate Ki (2 points).

Part 2: Relationship between Kinetics and Binding Mode.

Figure 1 and 2 represent HIV-1 proteases complexed with two different inhibitors, A and B. Figure 1 illustrates a tripeptide Glu-Asp-Leu (GAL) inhibitor bound at the active site of the enzyme. Figure 2 represents a multiple theoretical docking of inhibitor B with HIV-1 protease. B binds to two symmetrical sites located at the periphery of the enzyme.

i. Predict the inhibition kinetics (competitive or non-competitive) for A and B (4 points).

(C) CE McKenna, Ph.D. USC, Chemistry Dept., 2003