by Andrew Dombrowski
Michaelis-Menten enzyme kinetics, and especially keeping track of the various kinds of reversible enzyme inhibition, is one of the most challenging areas of biochemistry for many PCAT students. It’s extremely easy to get lost in the details of memorizing terminology, types of graphs, x-intercepts, y-intercepts, and so forth – but superficially memorized information is very fragile on Test Day, because it’s easy to momentarily space out on details like that under stress.
So, what to do? The answer is to focus on the basics of types of inhibition, make sure that you have a solid understanding of how they work mechanistically and how we measure their effects, and only then to memorize their effects in tabular or graphical form. In this post, we’ll systematically explore how focusing on the basics first can help set you up for longer-term success with this topic. Note, though, that in order to get the most out of this post, you should already be familiar with the definition of enzymes, the lock-and-key versus induced fit models, and the basics of Michaelis-Menten and Lineweaver-Burk plots.
There are two main quantities that we care about when it comes to enzyme kinetics: vmax and Km. vmax, which was named to remind you of “maximum velocity,” refers to the maximum reaction rate, which you get by completely saturating the reaction mixture with substrate, to the point that all of the molecules of enzyme are busy all the time. Km is a little bit more confusing for many students, perhaps because biochemists tend to use the letter K (as well as lowercase k) in many different contexts. Km is not an equilibrium constant, or a constant of any kind for that matter. Instead, it’s a measure of substrate concentration; more specifically, it’s the substrate concentration that gets you halfway to vmax.
A very honest follow-up question might be: why on earth do we care so much about this particular concentration value? Essentially, it’s a way of measuring enzyme affinity. If Km is low, that suggests that the enzyme doesn’t need much available substrate to do its job, which means that it is very efficient at binding the available substrate. If Km is high, that means you basically have to flood the reaction mixture with substrate to get the enzyme to do its job, which means that it must not be very good at binding the substrate. An analogy would be how skillful someone is at swatting flies. If a single fly enters a room and you can swat it immediately, you must be a pretty good flyswatter – but if you have to have a room full of flies in order to successfully swat any, you’d be by definition pretty terrible at flyswatting.
Now that we’ve invested some time in really understanding how we measure enzyme kinetics and the effects of inhibitors, we can turn to analyzing different types of reversible inhibition. It’s all downhill from here!
Competitive inhibition is when an inhibitor interacts with the active site of an enzyme to block it from binding the substrate. The inhibitor can be thought of as competing with the substrate. However, you can out-compete the inhibitor by flooding the reaction mixture with substrate. Doing so makes the effect of the inhibitor negligible, meaning that vmax doesn’t change. However, the fact that we need more substrate to get the effects we want means that Km must increase.
Noncompetitive inhibition is when an inhibitor interacts with a non-active (allosteric) site on the enzyme. Changing the substrate concentration will make no difference here; if a noncompetitive inhibitor binds an enzyme, it is effectively knocked out of commission. This means that vmax decreases. However, the enzymes that aren’t affected by the inhibitor continue to do their job just as effectively as they did before, such that Km remains the same.
Uncompetitive inhibition occurs when an inhibitor interacts with the enzyme-substrate complex, blocking the enzyme from releasing the substrate. This effectively inactivates the enzyme, meaning that vmax is decreased. However, somewhat surprisingly, Km decreases too – this is because the inhibitor stabilizes the enzyme-substrate complex, effectively increasing the affinity of the enzyme for the substrate.
If you can clearly visualize how these inhibitors interact with enzymes, and have a solid understanding of what vmax and Km mean, then predicting the effects of different types of inhibition becomes straightforward. This is an excellent example of how investing time and energy into understanding the basics backwards and forwards before jumping straight to memorization can pay off.
We’d suggest that you try to apply this approach to other subjects you’re studying for the PCAT – it will pay off! We wish you the best of luck in your test prep journey!