Enzymes II

Last time we saw how is that enzymes increase the rates of biochemical reactions. We saw that enzymes catalyze reactions by lowering the activation energy barrier of the reactions (DG^R), therfore increasing the value of k_forward, or reaction rate:

We briefly saw that the initial kick given to the substrate by the enzyme to make it adopt the transition state structure was due to a combination of a number of favourable weak interactions (hydrophobic, electrostatic, h-bonds), or complementarity, between the substrate and the enzyme's binding site, and that the principal decrease in activation energy came from the complementarity between the structure of the transition state and the enzyme active site. These favourable interactions between the enzyme and substrate are collectively known as the binding energy, and today we will start by disecting the different contributions to the binding energy.

Components of the binding energy

As we said before, the substrate (or substrates) will bind to the active site with good complementarity, but not extremely good, because if they do, they will basically sit there and no reaction will occur. However, the DG liberated in substrate binding (binding energy) will give the substrate enough extra energy to deform itself and adopt the structure of a transition state.

Therefore, the binding energy is one of the principal factors that will drive a reaction, and has a profound effect in the activity (how fast the enzyme can do what we want it to do). Poor binding of the substrate to the enzyme (i.e., poor complementarity between the substrate and the enzyme means less free energy for the substrate to go to the transition state, and longer reaction times.

What contributes to the binding energy? As we said, all the interactions between substrate and  binding site are the same type of interactions we saw in isolated proteins. If we think that we can have ~ 5 to 10 interactions between the substrate and the active site, and that they vary from 1 to 15 Kcal/mol (the range from vdW to salt-bridge), we will get anything from 5 to 150 Kcal/mol. On average, the binding energy will be ~ 50 Kca/mol. If you substract this number from DG^R and plug the result into the equation for k_forward, you will get an increase of 10 million in the rate of the reaction. However, this is a very approximate figure, as there are other things that we have to take into account:

- The obvious ones are the ones we mentioned several times already: van der Waals, h-bonds, hydrophobic, and electrostatic interactions, which will all favour the ES intermediate and the E[TS]^R complex. This energy will compensate for any unfavourable strain energy that the substrate runs into when going into its transition state.

- As you all know, two molecules in solution will try to be as disorganized as possible, because this is favourable entropically. If the molecules are always next to each other in an optimal orientation to react, the entropy will go down (decrease in DS), and DG will increase. Therefore, entropy will work to our disadvantage. Now, if both substrates have high affinity for the active site, they will be bound most of the time, because this is favourable energetically. Since we now have both of the bound, they will be close to one another and in the right orientation more time than when they are in solution. Therefore, we have an effective reduction of unfavourable entropic factors related to the reaction. In other way, we can consider that by bringing both molecules together we are increasing their effective concentration. It has been shown that constraining the movements of two substrates in a reaction to the active site has an increase of up to 10⁸ M in the rate, which is equivalent to increase the concentration of one of them to 10⁸ M (100,000,000 M!).

- Any molecule in solution is surrounded by solvent, i.e., they will have a solvation shell. In order for two molecules to react, both will have to get rid of their of their solvation shells, and another solvation shell will form around the transition state. The same applies with an enzyme around: First, the molecules will desolvate to get into the active site. Second, water that may have been lying around the active site has to be spitted out before the substrates get in. What happens is that all the non-specific interactions between the solvent and the substrates will be replaced by specific interactions between the active site and the substrate.

Since we are usually in water, we call these energies (or differences in energies) hydration energy. Usually, what we gain when we form specific interactions with the active site is very close to what we loose in interactions with the solvent: h-bonds will be replaced by h-bonds to amino acid residues in the active site, etc., etc. However, unfavourable hydration energies will weigh unfavourably in the activity of the enzyme (in the affinity for the substrate). Something that is very happy in solution won't go into an active site that easily, unless there are a lot of favourable interactions with the binding site.

Types of reactions in enzyme catalysis

OK, so now the molecule (or molecules) are bound to the active site, and it is easy for them to go into their transition state due to a number of energetically favourable contributions from the substrate-active site complex. Is this the only way in which an enzyme catalyzes a reaction? By no means. Apart from all the non-covalent interactions between substrate (and transition state) and the active site of the enzyme, there are an important number of specific interactions between functional groups present in the active site of the enzyme and the substrate that will help in the breaking of bonds, formation of bonds, group transfers, isomerizations, etc. These modes of catalysis can be cathegorized according to the types of reactions involved:

Acid-base catalysis

Many reactions, like to formation or cleavage of a peptide bond, require the formation of charged species (charged transition states). In theabsence of an appropriate stabilizing agent, these transition states will go back to their reactant species (the stuff to the left of the equilibrium equation), because they are unstable. For the reaction:

We have the following transition state:

If we don't have something that stabilizes this transition state (the specie to the right), the reaction won't go. In this case, we have to remove the positive charge from the oxygen atom, otherwise water (a very good leaving group) will fall off the transition state and give us reactants without product.

What we do is add acids and bases (species that provide and abstract protons) to stabilize the TS. Twe have two different cases. In one, water (as the H₃O⁺/OH^- duo) can come to the rescue:

If the catalysis goes this way, using simply H₃O⁺/OH^-, it is called specific acid-base catalysis, meaning that the source of protons or hydroxyls come first from water, not from any other group. The stabilization of the TS is done through the interactions with water.

Now, if we are in the active site of an enzyme, we will usually have several functional groups from amino acid residue side chains that can donate or accept protons as acids or bases: if :B is a basic group (note the 'lone pair'), and HA is an acid that belong to the enzyme, we can have the following:

In this case, the catalysis is known as general acid-base catalysis. In both cases, the stabilized TS (the TS with redistributed charges) will now break down to the desired products. In either case, the proton is not on the oxygen, but on the nitrogen, so the charged amine becomes a good leaving group. The carboxyl reforms and kicks out the amine.

What groups in proteins can aid acid-base catalysis? A bunch! All the side chains of amino acid residues that have ionizable groups (Lys, Arg, His, Glu, Asp in particular) will serve as general acid-base catalysts. In most cases, the same group will act as an acid when it is protonated, and as a base wharn it is deprotonated. This property is due to the pK_R values of the ionizable groups in these side chains, which will be close to the optimal pH at which the reaction takes place. A list of these goes here:

A nice example of genral acid-base catalysis are the several enzymatic mechanisms of keto-enol isomerization. The enolization of a ketone to an enol, which happens over and over in sugars, involves the following reaction, here shown uncatalyzed:

We see that there are species with a shared negative charge and the proton which is positivelly changred, making ther TS unstable. In the enzyme active site, we can have, for example, a general acid that donates a proton to aid in the stabilization of the negative oxygen that forms during the reaction:

We can also pick to use a general base to pull the hydrogen off, ans thus neutralize the positive charge that the proton generates:

Usually, the protonated general bases or deprotonated general acids will regain/lose the proton from water to go back to their original state.

Covalent catalysis

In this catalysis mechanism, the functional groups in the enzyme (either from amino acid residue side chains or from prosthetic groups) will form transient species in which there is a covalent bond between the enzyme and the substrate. This is a clear example of how the enzyme can carry the reaction through a completelly different transition state than the one operating when we have no enzyme around.

For example, for the hydrolysis of an ester or an amide, we have an uncatalyzed reaction that goes like this:

I used the same example than before for general acid base for simplicity. We can rewrite the mechanism by including a functional group from the active site of the enzyme:

In this case, there is a direct attack from a group in the active site, acting as a nucleophile (^:Nu-R), that attacks an electropositive center. Other times, the covalent intermediate can change the electronic properties of a region of the molecule, making it, for example, more electron withdrawing. A nice example of this are imines of Schiff bases, which increase the electron defficiency of carbonyls, transforming them into electron sinks:

An example of this is an enzyme that catalyzes the conversion of aceto-acetate into acetone and carbon dioxide. The uncatalyzed reaction is actually not that bad, because we can form resonant structures (which ones?):

If we use an amine in the enzyme to for a Schiff base, things fly, because we don't have the negative charge around, and nitrogen is happier being protonated than oxygen hanging a negative charge:

We will see several reactions of this type when we talk in detail about the mechanisms of certain enzymes. The nucleophiles in these reactions will be electronegative groups of the protein (hydroxyls, thiols, unprotonated amines and imidazole). Remember carboxylates are really bad nucleophiles. Electrophiles are protons, metals, carbonyls, and Schiff bases.

Metal ion catalysis

The last mechanism of enzyme catalysis that we will analyze is metal ion catalysis. As we mentioned in the past, some metal ions (in small ammounts) are crucial for life. These ions are usually involved in this type of catalysis. A third of all known enzymes require a metal ion or more to function properly. The metal ions can be either tightly bound to the enzyme (as a cofactor), or come in and out of the binding site with the substrate.

The enzymes that bind the metal tightly, as part of the active site, are called metalloenzymes, and the metals are usually Fe^2+/3+, Cu²⁺, Zn²⁺, Mn²⁺, or Co²⁺, which are all transition metals. In this enzymes, the metal usually participates in the reaction, either by orienting the reacting species, by participating in oxidation-reduction reactions, or by shielding negative charges of the intermediates.

The second type, the ones in which the metal goes in and out with the substrate, are called metal-activated enzymes. The metals are alkaline metals (Na⁺, K⁺, Mg²⁺, or Ca²⁺). Usually these are to electropositive to participate in the reaction itself, but they will coordinate with charged atoms in the TS and lock its conformation.

A nice example of a metalloenzyme is an enzyme we mentioned before, carbonic anhydrase. Zn²⁺ is crucial in this enzyme, because it stabilizes a hydroxyl group that participates as a nucleophile in the reaction. The hydroxyl group attacks the electropositive carbonyl of carbon dioxide to generate HCO₃^-:

The zinc cation is held in place in the active binding site through coordination with three imidazoles from histidine residues, making this a metalloenzyme.

Next time we will start discussing how we can peek into enzyme mechanism by studying enzyme kinetics.

Prepared by Guillermo Moyna, 1999.