Question

You are studying a new muscle enzyme, M, and need to characterize its kinetic properties. a) Experimentally, how will you determine the M’s VMAX and KM for its substrate , A? EXPLAIN in detail, including any limitations your mathematical methods may have. b) Now consider that it is known that another molecule, D, also binds to M, and interferes with the reaction that converts A to product Q, but you don't know where or how. What kind of kinetic evidence would indicate that D binds to the active site of M, to the exclusion of A? EXPLAIN. How could you prove it? c) You ultimately figure out that the inhibitor, D, and substrate, A, bind to the enzyme M independently. What kind of inhibition is this? How would it be characterized kinetically? d) It turns out that M catalyzes the conversion of Q to A in liver tissue. Explain how this is possible.

Answer 1

We can calculate the Vmax and Km of an enzyme by determining the rate of product formation per unit time.
i.e. take different substrate concentrations and measure the rate of reaction at each substrate concentration.
Plot [S] values on the X-axis and rate values on the Y-axis.
If it is a Michelis-Menton enzyme, it will give a hyperbolic curve. The limitation of this curve is that it is asymptotic i.e. we cannot determine the exact Vmax value.

To avoid this problem, take 1/[S] and 1/v values and plot graph i.e. Lineweaver-Burk plot.
Y-intercept = 1/Vmax
X-intercept = -1/Km

Km is the substrate concentration at which the reaction rate is 1/2 Vmax.

See the image for variation in response to different regulatory molecules.

If the regulatory molecule binds to the same site on the enzyme as that of the substrate, it is competitive inhibition.
If the regulatory molecule binds at a different site other than the substrate binding site, it is an allosteric effector molecule.