Bke2 Biochemistry Exercises

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Group exercise: Enzyme kinetics

What is meant by the term "enzyme kinetics"? What kind of information do kinetic studies give?
Write down the rate equation for a simple non-enzymatic reaction in solution (with a substrate S being converted to a product, P). What does the rate constant in this reaction indicate? Now, write down the rate equation for a simple enzyme-catalyzed conversion of S to P. What is the rate equation for this reaction? Describe in a graph, and with your own words, what happens to the reaction velocity when you add substrate to an enzyme at increasing concentrations. How is this different from what happens when enzyme is not there?
The reaction velocity of a simple enzyme-catalyzed reaction, such as that in the previous question, is described by the Michaelis-Menten equation. Write down this equation. Under what conditions is this equation valid (i.e. what assumptions are made in deriving the equation)? By inspecting the equation, write down what the reaction velocity tends to as the substrate concentration becomes much larger than the K_m for that substrate. What is the velocity of the reaction when the substrate concentration is equal to the K_m?
What is the difference between a "big" K, like in K_m, and a "small" k, like in k_cat?
K_m is frequently equated with K_S, the [ES] dissociation constant. However, there is usually a difference between those values. Why? Under what conditions are K_m and K_S equivalent?
Assume that an enzyme-catalysed reaction follows Michaelis-Menten kinetics with a K_m of 1 uM. The initial velocity is 0.1 uM/min at 10 mM substrate. Calculate the initial velocity at 1 mM, 10 uM and 1 uM substrate. If the substrate concentration were increased to 20 mM would the initial velocity double? Why or why not?
Why are k_cat/K_m values useful to describe the specificity, or preference for different substrates, of an enzyme?
You think you may have found a new inhibitor (I) of the HIV protease, so you perform a few experiments to characterize its behavior. You find that if you add an excess of synthetic substrate to the protease in the presence of the inhibitor, enzyme actitivity increases but only reaches 50% of the V_max observed in the absence of the inhibitor. The affinity of the substrate for the enzyme is unaffected by the inhibitor. Furthermore, if you treat the enzyme with the inhibitor, then dialyze the protein, you find that full activity can be restored. What class of inhibitor is I?
One of the effects of aspirin is to slow blood clotting by inhibiting the enzyme cycloxygenase in blood platelets. The effects of aspirin wear off on the same time-scale as the platelets themselves are broken down (and new ones made). What kind of inhibitor is aspirin?
Hexokinase catalyses the following reaction:

Which compound below would you choose as the best competitive inhibitor of the hexokinase reaction, and why?

Ribose Galactose

Fructose 6-deoxyglucose
It is often convenient in experimental work to consider the Michaelis-Menten equation in terms of 1/v. Rewrite the equation in this form. Does it have the form of a straight line? If so, where does the line cross the x and y axes and what is its slope?

Reading material: Horton, chapters 5 & 6

Lectures:
Introduction to enzymes
Lecture: Enzyme kinetics

Links:
Suggested answers

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Ribose	Galactose

Fructose	6-deoxyglucose