Enzyme inhibition II

Last time we discussed different types of reversible inhibition from a kinetic point of view, and we also saw some examples of irreversible inhibitors. Today we will discuss how we make inhibitors in order to better match (and therefore bind tightly) the shape (or conformation) of the transition state.

Transition state analogs

When we started our discussion of enzymes we mentioned that the affinity of the substrate for an enzyme depnded in non-covalent interactions between the enzyme active site and the substrate. The collection of interactions were called the binding energy, and it was the binding energy what gave the substrate some of the energy it needed to go into its transition state.

Therefore, our initial ide to make a molecule that binds the active site of certain enzyme would be to make something that has the same shape (conformation) and electronic structure (positioning of functional groups) so that it will bind to the active site. We would also try to leave some key part of the molecule that is involved in the chemical catalysis performed by the enzyme - In that way, the enzyme would bind it, but no chemistry occurs.

Now, we also saw that although we had a number of favourable interactions between the enzyme and the substrate in the enzyme-substrate complex, there were a lot more interactions between the enzyme and the substrate's trnasition state. These interactions were in charge of lowering the activation energy (DG#) and therefore rising the rates of the enzyme.

What if we them make molecules that instead of looking like the substrate look more like the transition state that will sit in the enzyme? This is the way to go. Since there are a lot more interactions, and therefore a lot more stabilizing forces, between the enzyme and TS, we will be far better off if we make a molecule that resembles, both structurally (shape) and electronically (functional groups), the transition state.

These requires that we not only know the structure of the substrate, but also what reaction the enzyme is catalyzing. Depending on the reaction we will have different transition states, and thus we have to know what type of chemistry goes on.

As we did for suicide inhibitors, the best way to analyze these are by examples. Perhaps the most 'juicy' example these days is HIV-protease.

HIV-protease inhibitors

The HIV virus creates some of the peptides it needs by splicing up larger proteins. The virus uses a protease, HIV-protease (HIV-PR), to do this. HIV-PR belongs to a larger family of proteases called the aspartyl proteases. If we think of the name, it probably means that aspartate residues play an important role.

Indeed they do. All aspartyl proteases use two aspartate residues. Several studies have shown that the two aspartates are used for general acid-base catalysis. That means, there is no chemical catalysis, and no intermediates covalently attached to the enzyme are formed.

HIV-PR is an homodimer (two identical subunits) with 100 amino acids per subunit. The two Asp residues critical in HIV-PR correspond the Asp 25 in both chains. The enzyme cleaves peptide bonds between a bulky aromtic residue and a proline. The 'normal' substrate for HIV-PR is the following:

If we have no chemical catalysis and no covalent intermediates (that is, no acyl-enzyme intermediate as with chymotrypsin), then the attack to the carbonyl of the peptide is from a water molecule. This water molecule is made a better nucleophile with the aid of one of the Aps 25 residues, which pulls away a proton. The first step in the reaction (formation of the transition state) is:

This compound is the transition state of the reaction. If it wasn't for the stabilization of the negative charge in the oxygen by the aspartate residue, the thing would fall apart in no time.Thus, apart from the favourable interactions between the peptide substrate and the enzyme (many of which are hydrophobic in nature), the electrostatic interactions between Asp 25 and the transition state are what make things go. The next step in the reaction is the loss of the amine (the carboxylate forms), because the C-N bond becomes very weak.

One thing that we note in the transition state is that the hybridization of the carbon on which we had the attack by water changed: It was sp² hybridized, and now is a run-of-the-mill tetrahedral (sp³) carbon.

Now, if we make something that looks like this we will have very good binding with the enzyme active site. How can we make something that looks like this, but which won't fall apart? We have to follow some rules:

1. It has to have a tetrahedral center, so that it will have the same shape than the transition state.

2. We want to have an hydroxyl pointing in the right direction, so that we can take advantage of the interactions with Asp 25.

3. We need something that is more stable than the TS and won't come apart. One of the reasons the TS breaks up is that it has three electronegative atoms on the same carbon. We can solve this by removing two of the electronegative groups - A nitrogen and an oxygen.

There are several more now, but many are still experimental and awaiting FDA approval. In these compounds, the alcohol needs to have the correct stereochemistry, because since the TS is tetrahedral, it will also have a certain stereochemistry that depends on the direction from which the water molecule was added from.

Also, these and other compounds in which there are chunks of peptide but some non-peptidic components are called peptidomimetics. These are compounds that look like peptides, but lack some crucial characteristic from peptides (an amide bond, for example). This make them behave as peptide-like, but will not be hydrolized by enzymes that easily.

Proline racemase

Another enzyme that provides us with a good example of transition state analogs is proline racemase, and enzyme that catalyzes the conversion of L- (or D-) proline into a racemic (D,L)-mixture:

The reaction reaction is catalyzed by a general acid-base pair. At this point you should be able to figure out how this works. In any case, the transition state of the reaction is a planar resonant structure. So, what can we use that resembles proline and is planar?

There are compounds known as pyrroles (remember heme?) which have some of the carachteristics we need. Two known TS inhibitors of proline racemase are pyrrole-2-carboxylate and pyrroline-2-carboxylate:

There are many other examples of transition state inhibitors. What we have to remember is that they work by resembling the transition state of the reaction that the normal substrate undergoes in the enzyme active site, but they lack certain parts of the substrate which are necessary to complete the normal reaction. They take advantage of the favourable interactions between TS and enzyme, but they don't undergo the normal reaction after they are bound.

Detailed enzyme mechanisms

The best way to fully understand how an enzyme works is to study its mechanism in detail, which includes point-miutation studies, kinetic studies, inhibition studies, preparation and testing of possible transition state analogs, and, finally, X-ray or NMR studies of the free enzyme and the enzyme-substrate intermediate.

Although there are thousands of enzymes known, only for hundreds we do have enough data to draw a full picture of the mechanism. One of the families of enzymes for which extensive studies have been performed are the serine proteases. We have mention one serine protease, chymotrypsin, when we discussed suicide inhibitors. These family of enzymes use a serine residue in chemical catalysis to break peptide bonds, and are digestive enzymes. We will discuss chymotrypsin in detail.

Chymotrypsin

The first step in determining the amino acid residues forming part of an active site usually involves chemical labeling studies. We treat the enzyme with certain reactive (usually organic) compounds which will attach to exposed amino acid residues (those in the active site), and then we sequence the labelled protein. Some of the amino acid residues will change from the native protein, and in this way we can tell which ones were tagged by the label.

We already saw how one of the important amino acids in chymotrypsin was identified: treatement with diisopropylphosphofluoridate (DIPF), the suicide inhibitor we saw last time, labels a serine residue, Ser 195:

Another important residue was determined by labelling with tosyl-L-phenylalanine cloromethylketone (TPCK). This compound labels a histidine residue, His 57:

So we have two residues which are in the active site, and crucial for activity. By studying the compounds they form, we can also guess what the residues would do in the normal reaction. Since Ser 195 reacts with a very electrophilic center (the P in DIPF), it was beleived that this group was in charge of the chemical catalysis - that is, attacking the carbonyl center in the peptide bond to cleave.

His 57, on the other hand, acts mainly as a general base, which pulls the proton from Ser 195 and activates it:

Other studies also showed that an aspartyl residue, Asp 102, was also crucial for proper activiy - mutation of this residues to something else gave very slow enzyme. The function of this did not come until X-ray studies of the enzyme with its substrate were perfomed. These studies showed that Asp 102, which is in its deprotonated form, forms a salt bridge with the protonated nitrogen of His 57, making its other nitrogen atom to point in the right direction - toward the Ser 195:

These three amino acids are conserved in alll serine proteases, and they are known as the catalytic triad. The rest of the reaction goes in the way we discussed before: We have cleavage of the peptide bond and release of the amine-end of the cleaved peptide, formation of an acyl-enzyme intermediate, and then release of the carboxylic acid-end of the cleaved peptide. In all the steps of the reaction, His 57 act as a general acid-base, donating and pulling protons from different charged species.

Next time we will see the machanism of chymotrypsin in detail, as well as the machanism of acetoacetate decarboxylase, and enzyme which uses a Schiff base in chemical catalysis. We will conclude enzymes with a discussion of allosteric enzymes.

Prepared by Guillermo Moyna, 1999.