The Michaelis-Menten Model Accounts for the Kinetic Properties of Many Enzymes

The primary function of enzymes is to enhance rates of reactions so that they are compatible with the needs of the organism. To understand how enzymes function, we need a kinetic description of their activity. For many enzymes, the rate of catalysis V0, which is defined as the number of moles of product formed per second, varies with the substrate concentration [S] in a manner shown in Figure 8.11. The rate of catalysis rises linearly as substrate concentration increases and then begins to level off and approach a maximum at higher substrate concentrations.For more plz read at following link.

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http://www.ncbi.nlm.nih.gov/books/NBK22430/

To use the standard approach to measuring kinetic constants Km and Vmax, it is crucial to measure initial velocity. You will make a graph of the amount of product formed on the y-axis versus the reaction time on the x-axis. Collect as many time points as you can, as close together as you can. Starting at the origin of the graph, draw a straight line through the earliest time points. The points should all lie on the line (allowing for experimental error) for at least a little while before they start to drop below it as the reaction slows down. The slope of this line is the initial velocity.

Follow the above procedure for a range of substrate concentrations. Plot the initial velocity on the y-axis versus the substrate concentration on the x-axis. If you have covered a wide enough range of substrate concentrations, the graph should have the shape of a square hyperbola. Use a computer program to perform nonlinear least squares regression of the data in this graph to the M-M equation:

V = Vmax[S]/(Km + [S]), where V is the initial velocity and [S] is the substrate concentration.

This is the most basic situation, and it only applies to the simplest case of a one-substrate enzyme. Unfortunately for you, the enzyme thiopurine S-methyltransferase is a 2-substrate enzyme. You can still use the above technique, but you will have to do it for one of the substrates and then repeat it for a range of concentrations of the other substrate. Then, you can fit the entire set of data to the 2-substrate rate equation that pertains to TPMT. There are several possible rate equations, so investigate the literature to find out which one to use.

A shortcut that some people use is to measure the kinetic constants for one substrate at a saturating concentration of the other one, then switch substrates and do it again. There are some potential pitfalls to this approach (substrate inhibition, insufficient saturation) that can lead to error. Nevertheless, it is a good way to get started without having to do anything complicated.

You have probably seen the use of double-reciprocal replots like the Lineweaver-Burk plot to measure kinetic constants. My recommendation is to avoid these plots and use nonlinear regression computer programs.

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