Enzymes V - Enzyme inhibition

Most diseases do their damage by using, at different stages, different enzymes. You have probably heard about HIV-protease, an enzyme that cleaves certain HIV proteins crucial to the life cycle of the HIV virus. There are many other examples like this one, and therefore a lot of the effort from pharmaceutical companies is directed toward the development of compounds that can knock these enzymes inactive, because this means that the life cycle of the bug is disrupted.

Also, we may encounter molecules that inactivate enzymes that we need. These ones would be 'poisons', as they would kill enzymes that are required for normal life.

All these compounds, be them good or bad, are known as enzyme inhibitors. We can study enzyme inhibitors using the concepts we have learned so far. That means, we can describe what they do chemically, and we can also describe how they affect the kintetics of enzymatic reactions.

Types of inhibition

As with everything in life, we will have different types of inhibition, depending on the type of interaction they have with the enzyme and how they affect the binding of the normal substrate and enzyme catalysis. In the first section we will be describing reversible inhibition - That means, there will be an equilibrium between the enzyme and the inhibitor, and there won't be a permanent covalent bond formed. There are three main types of inhibition: competitive, noncompetitive, and uncompetitive.

Competitive inhibition

This type of inhibition is the simplest to understand: We can have a molecule that looks a lot like the substrate (or the transition state) and will therefore bind to the same active site in the enzyme. Instead of getting the enzyme-substrate (ES) complex, we get an enzyme-ihibitor (EI) complex.

A competitive inhibitor will bind the enzyme's active site, establish an equilibrium (K_I), and basically sit these doing nothing productive. i.e., there is no chemistry after the EI complex forms:

The equilibria involved in the process are the following:

In macroscopic terms, the inhibitor will lower the ammount of free enzyme capable of binding to the correct substrate. However, since the EI complex is in equilibrium with free E, increasing the concentration of substrate will eventually shift the equilibrium to formation of ES. That means that when we saturate the system with S, the enzyme will be primarily as ES.

We can tell pretty easily that an a compound is a competitive inhibitor of an enzyme using kinetic measurements of initial velocities. As we explained for bisubstrate enzymes, these measurements are done several times, each using a different concentration of inhibitor I.

For a certain concentration of inhibitor I at low concentrations of S the ammount of free enzyme to bind S will be small. Therefore, instead of seeing the normal affinity of the enzyme (K_M), we will see a lowered affinity - A larger K_M.

As we increase [S], more enzyme will be in the ES form, because it will displace I from the binding site. At a certain point, most of the enzyme will be in the ES form, the same as if we had no inhibitor I around. Therefore, the V_max (V_max = K₂ [E]_T) is the same as for the enzyme in the absence of I.

If we repeat the runs for higer concentrations of I, we will see the same. An even larger apparent K_M, but at very high [S] we will still see the same rates - V_max is the same. This is very simple to see in a Lineweaver-Burke plot. The slope of the different lines (K_M / V_max) will increase for higher [I], but the Y intercept (1 / K_M) will be the same. We get an intersecting pattern that looks like this:

This is very easy to see with this kind of plot, but a little harder with a normal V_o vs [S] plot. We can also do the whole derivation of our rate (V_o) equations, this time taking into account that [E]T = [ES] + [EI] + [E]. What we get in the end is the follwoing:

The apparent K_M we have been talking about is K_M ( 1 + [I] / K_I ). So, from kinetic measurements we can also calculate the K_I constant, or the inhibitor affinity for the enzyme.

Noncompetitive inhibition

We encounter another case of inhibition in cases in which the inhibitor does not bind the same active site, but a binding site remote to the active site. In this cases, the inhibitor does not block the active site from the substrate, but affects the enzyme in such a way that even if it is bound to substrate it will no longer produce any catalytic products. These are called noncompetitive inhibitors:

The inhibitor can bind both to the free enzyme or to the ES complex. After binding, the enzyme dies. The equilibria involved in this type of inhibition are:

Again, we can analyze the system in macroscopic terms by making use of kinetic measurements. Since the inhibitor does not block the active site but lowers its catalytic power when bound, see an apparent k_cat that is smaller than in the absence of inhibitor. Therefore, the appartent V_max of the system goes down. A simplifying assumption is that the affinity of E and EI for S is the same, and therefore the following equilibria have the same K_M:

Assuming this, we can see that V_max is affected (k_cat is affected), but not K_M. Therefore, if we do a series of Lineweaver-Burke plots for different concentrations of inhibitor, we get an intersecting pattern that looks like this:

In this case, since V_max gets smaller for higher [I], the slope (K_M / V_max) goes up. However, we do not affect K_M, so the X intercept will be the same in all plots. Again, if we considered all the species involved in the different equilibria and solved the rate equations, we get in the end something like this:

In this case, the apparent V_max is V_max / ( 1 + [I] / K_I ). As was the case for competitive inhibition, we can calculate the K_I of the noncompetitive inhibitors from kinetic measurements.

Uncompetitive inhibition

The last type of inhibition we will analyze involves also binding of the inhibitor at a binding site different from the active site, but only after the enzyme has bound to the propers substrate. Why could this happen this way?

This type of inhibition is termed uncompetitive inhibition. The inhibitor only inactivates the enzyme after it has formed the ES complex. The equilibria involved in this type of inhibition are:

Again, we can analyze the inhibition by looking at its kinetics. In the same way that competitive inhibition lowered the amount of productive enzyme, uncompetitive inhibitors lower the concentration of ES complex that will produce products. However, increasing the [S] does not help here, because we are not competing for the same active site.

In terms of double-reciprocal plots, an uncompetitive inhibitor will do two things. First, the V_max is lowered because we will have less enzyme that will produce products, no matter how much we increase [S]. Second, the apparent K_M also decreases, because it the data will look as if we have less productive enzyme binding more the same ammount of substrate in the presence of the inhibitor, therefore increasing the apparent affinity of the enzyme for the substrate. In other words, it looks as if we were increasing the ammount of substrate that we can bind (smaller K_M), but the rate does not increase (smaller V_max).

Fortunately (?) both are affected equally (in both cases it will depend on the affinity of the enzyme for the inhibitor). Therefore, we get a parallel double-reciprocal pattern:

If we consider again all the species involved and solve the rate equations, we get the following:

One important thing to note - Despite that all this double-reciprocal plots look a lot like the ones we saw for bisubstrate binding, the change in the slopes goes the other way. This makes sense, because in bisubstrate binding, increasing the concentration of the second substrate increases the activity. With inhibitors, increasing the concentration of the inhibitor decreases the activity of the enzyme.

Suicide inhibition

All these discussions we did made the assumption that the inhibitor binds the enzyme reversibly. That is, the inhibitor just sits there, either in the active site or in a second binding site, but it is free to go back to solution. There is another type of inhibitors that undergo irreversible chemistry with certain key amino acid residues (or prosthetic groups) in the active site, therefore killing the enzyme's activity, and preventing the inhibitor from being released out of the active site. This inhibitors kill the enzyme for good, but since they also 'die' in the process, they are called suicide or mechanism-based inhibitors.

Many of these molecules are very effective drugs, because they are targeted specifically for a certain enzyme (we have to look for good complementarity with the active site and the enzyme-transition state interactions), and kill the enzymes for good.

The only way to understand them is by a) understanding first the mechanism of the enzymatic reaction, and b) understanding how the enzyme becomes 'stuck' after doing part of the chemistry it needs to do with the normal substrate.

For example, lets look at serine proteases, a class of enzymes of which chymotrypsin forms part that catalyze the cleavage of peptide bonds. One of the steps in the reaction involves the attack of the hydroxyl in a serine residue (Ser195) to the carbonyl carbon of the peptide bond:

In the next step of the reaction, the carbonyl forms back, kicks out the amine (cleaves the N-CO bond), and we have an acyl-enzyme intermediate. Water then comes in and hydrolyzes the acyl-enzyme intermediate to products;

A nice suicide inhibitor of chymotrypsin is diisopropyl fluorophosphate (DIFP - below). This compound is a phosphate with a very labile P-F bond:

In the first step of the reaction between chymotrypsin and DIFP, Ser195 attacks the phospate, kicking the fluoride out:

That's it for the enzyme - The phosphate-enzyme intermediate that forms is now too stable, and it does not hydrolyze with water as the acyl-enzyme intermediate.

Another nice one is with triose phosphate isomerase (the one in the problem set). The normal substrate is triose phosphate:

We saw the reaction in recitation, and you better get it before turning in the problem set. Now, an inhibitor of this enzyme is the following epoxide:

How does it inactivate the enzyme? Here the general base that pulled the proton a to the carbonyl attacks the epoxide carbon, because it is very reactive due to strain. That's it for the enzyme...

There are thousands more out there...

Prepared by Guillermo Moyna, 1999.