6.5. A Sampling of Enzymes: Proteases
A set of model enzymes, proteases, is explored to highlight context and variability in enzymatic reactions. All proteases catalyze the breaking of amide bonds as part of the digestion of peptide polymers. However, there are a variety of ways that this is accomplished.
6.5.1. Why are there Different Types of Proteases?
The digestion of peptides/proteins is a common process that is required for life to exist. For example, being able to break peptide polymers back into their amino acid subunits allows recycling of old/unneeded peptides, incorporation of new amino acids (e.g. protein that was consumed), or provides a local source of amino acids if none are available (e.g. during starvation). Because the process is needed for life, many different organisms separately evolved ways to perform do it. This is sometimes referred to as “convergent evolution” and explains some of the variability and variety of proteases.
At the same time, most organisms produce multiple kinds of proteases within their cells. Some organisms produce hundreds of different kinds with small or large changes in their behaviour and selectivity. For example, different protease enzymes may use the same catalytic approach but, because of the rest of the structure, be selective for different peptide polymers. Others may use different catalytic approaches to allow the same process to occur under different conditions (e.g. at a different pH, at a different temperature, etc.). Finally, having variability in the structures and approaches allows cells to adjust the speed of the digestion.
6.5.2. Why Catalyze Reactions that Break Amides?
Recall that amides have resonance stabilization (see Section 4.4.4) and are generally far less reactive than may otherwise be expected (see Section 4.5.1); amides are very stable functional groups and do not easily undergo chemical reactions. In neutral aqueous conditions (e.g. water around pH 7) the half-life of an amide is approximately 500 years (Scheme 6.7). Because standard physiological conditions are very similar (pH of approximately 7.3) the half-life of amides in cells would be very similar.
Scheme 6.7 – Half-life Decomposition of an Amide Bond.
While this is beneficial for cell maintenance it is a problem for peptide recycling/digestion (most organisms do not have a few millennia to digest their meals). Several issues need to be addressed to allow the enzyme(s) to break amide bonds at a more appropriate speed for life to occur.
6.5.2.1. Problem: Nucleophilicity of Water
The uncatalyzed reaction mechanism involves water (a weak/moderate nucleophile) attacking an amide (a weak electrophile). One of the common strategies is to improve the nucleophilicity of the nucleophile (Scheme 6.8). This may involve base catalysis (i.e. form hydroxide or similar) or may involve changing the nucleophile to an amino acid side chain.
Scheme 6.8 – Strategies for Improving Amide Bond Cleavage with Enzymes: Improving the Nucleophile.
6.5.2.2. Problem: Electrophilicity of Amides
The uncatalyzed reaction mechanism involves water (a weak/moderate nucleophile) attacking an amide (a weak electrophile). One of the common strategies is to improve the electrophilicity of the electrophile (Scheme 6.9). This may involve (Lewis) acid catalysis (i.e. form oxonium or similar) or may involve several hydrogen bonds with amino acid side chains/NH’s.
Scheme 6.9 – Strategies for Improving Amide Bond Cleavage with Enzymes: Improving the Electrophile.
6.5.2.3. Problem: Leaving Group Ability of Anionic Nitrogen
The uncatalyzed reaction mechanism involves anionic nitrogen (a VERY poor leaving group) acting as a leaving group. One of the common strategies is to improve the leaving group ability of the leaving group (Scheme 6.10). This could involve protonating it before leaving (i.e. change the order of steps) but more commonly makes the protonation/deprotonation occur at the same time as the leaving group is leaving (i.e. combine two steps into one).
Scheme 6.10 – Strategies for Improving Amide Bond Cleavage with Enzymes: Improving the Leaving Group.
The uncatalyzed reaction mechanism involves two molecules colliding at a specific angle (and with a specific amount of energy). One of the common strategies is to improve the likelihood of a viable collision. This is catalysis by approximation (see Section 6.3.2.1). Bringing both molecules into a pocket within the enzyme already drastically improves the frequency of collision because they are forced to be close together. However, another factor is also typically addressed.
Recall that when two orbitals overlap constructively they form a covalent bond (σ or π). However, when they overlap deconstructively they form an anti-bonding orbital (σ* or π*). Usually, in order to break a covalent bond electron density must be put into its corresponding anti-bonding orbital. During the nucleophilic attack the covalent bond being broken is the carbon-oxygen π-bond, which is broken by putting electron density from the nucleophile into the corresponding anti-bonding π* orbital.
The carbon and oxygen are sp2 hybridized and trigonal planar. The p orbitals which make up the π bond are located both above and below the carbonyl. This is also true for the corresponding anti-bonding orbitals (π*), which simply point in the opposite directions (Figure 6.10).
Figure 6.10 – Representations of Pi (π) Bonding and Pi* (π*) Anti-Bonding Orbitals for an Amide.
Because of the shape of the anti-bonding orbital there is an ideal angle of approach for the nucleophile which leads to the best overlap of orbitals (Figure 6.11). Some sources may refer to this as the Bürgi-Dunitz angle. Approaching from this angle makes the reaction “cost” the least amount of energy, which makes the reaction faster. Often the enzyme organizes the relative positions of the nucleophile and electrophile so that this is the angle of approach.
Figure 6.11 – Representations of Viability of Different Angles of Approach for Nucleophilic Attack of the Carbonyl of an Amide Highlighting the Bürgi-Dunitz Angle.
This is difficult to represent in two dimensional mechanisms and is often omitted from discussion. However, it is one of the very common strategies proteases use to accelerate their reactions.
6.5.3. Example: Serine Proteases
One general class of proteases are the so-called “Serine Proteases”. These use the side chain of a serine for their catalytic activity. Two other nearby amino acids, aspartic acid and histidine, help create a local environment that allows the serine to be acidic enough for its role. Some sources refer to this as a “catalytic triad”, a group of three amino acid side chains working together to catalyze the reaction. In many serine proteases there are also other interactions that make the reaction faster, such as hydrogen bonding to other side chains and/or amide NH’s.
Interestingly, often the three amino acids (aspartic acid, histidine, and serine) are far apart from one another in the peptide chain itself. They are brought in close proximity only in the tertiary structure (or rarely, only after the substrate binds). Once together the three are very precisely oriented and spaced with respect to one another inside the enzyme; the exact distances and angles between groups are highly controlled to optimize the way they need to work together.
Serine proteases follow the standard three-phase approach: the first step is always binding of the substrates, the last step is always release of the products, and the mechanistic steps of the reactions happen between these two phases.
First the enzyme and substrates bind to form the enzyme-substrate complex (Scheme 6.11). Hydrogen bonds help organize the positions and orientations of the water and amide and increases their relative reactivities for the next phase.
Second, the reaction occurs in a series of steps.
The side chain of histidine (base) removes a proton from the side chain of serine (acid). The anionic charge and hydrogen bonding from the side chain of aspartic acid help increase the basicity of the histidine side chain. At the same time, the oxygen of the side chain of serine (nucleophile) attacks the carbon of the carbonyl in the amide (electrophile). This forms a new bond and adds a lone pair to the oxygen. Hydrogen bonding from nearby groups helps increase the electrophilicity of the carbonyl.
A lone pair of the oxygen forms a π bond with the carbon, ejecting the leaving group (“amine”). At the same time, the nitrogen (base) removes a proton from the side chain of histidine (the former base) regenerating the base. Having this occur at the same time greatly increases its leaving group ability.
The side chain of histidine (base) removes a proton from water (acid). The anionic charge and hydrogen bonding from the side chain of aspartic acid help increase the basicity of the histidine side chain. At the same time, the oxygen of the water (nucleophile) attacks the carbon of the carbonyl in the ester intermediate (electrophile). This forms a new bond and adds a lone pair to the oxygen. Hydrogen bonding from nearby groups helps increase the electrophilicity of the carbonyl.
A lone pair of the oxygen forms a π bond with the carbon, ejecting the leaving group (serine’s side chain). At the same time, the oxygen (base) removes a proton from the side chain of histidine (the former base) regenerating both the histidine and serine side chains. Having this occur at the same time greatly increases its leaving group ability. This is equivalent to regenerating the catalyst.
Finally, the enzyme releases the final products. This empties the enzyme and allows it to begin the catalytic cycle again.
Scheme 6.11 – Example of a Catalytic Cycle/Mechanism for Serine Proteases.
6.5.3.2. Catalytic Principles Used
Of the four general catalytic principles, serine proteases use three: catalysis by approximation (e.g. binding, hydrogen bonding for organization), catalysis by general acid/base reactivity (e.g. histidine side chain base/acid), and catalysis by covalent bonding (e.g. serine side chain nucleophile/leaving group). Catalysis by metal ion interactions is not used.
The catalytic principles were used directly to address the problems with the reaction (see Section 6.5.2 and subsections therein). The nucleophilicity of water was improved (hydrogen bonding, deprotonation occurring at the same time as the nucleophilic attack [acts like hydroxide]). The electrophilicity of the amide was improved (hydrogen bonding of the amide, changing the electrophile to an ester intermediate). The leaving group ability of the anionic nitrogen was improved (protonation occurring at the same time [acts like neutral leaving group]). The issues with entropy were improved (bringing compounds close together beforehand, controlling positions/orientations/angles).
A simplified reaction coordinate may be helpful to compare the improvements/changes (Figure 6.12).
The uncatalyzed reaction mechanism has three steps: nucleophile attacks electrophile, leaving group leaves, and an acid-base step. The first two steps have very high activation energies and are consequently very slow. The overall reaction is thus also very slow.
The enzyme changes the mechanism of the reaction. The mechanism now has significantly more steps but all of the steps have much smaller activation energies and are much faster. The overall reaction is thus also much faster.
Figure 6.12 – Simplified Comparison of Uncatalyzed and Catalyzed Reaction Coordinates for Serine Protease Catalyzed Amide Cleavage.
6.5.4. Example: Cysteine Proteases
One general class of proteases are the so-called “Cysteine Proteases”. These use the side chain of a cysteine for their catalytic activity. One other nearby amino acids, histidine, helps create a local environment that allows the cysteine to be acidic enough for its role. Some sources refer to this as a “catalytic diad”, a group of two amino acid side chains working together to catalyze the reaction. In many cysteine proteases there are also other interactions that make the reaction faster, such as hydrogen bonding to other side chains and/or amide NH’s.
Cysteine proteases work the same way as serine proteases. The side chain of cysteine is inherently more acidic than serine’s. As a result, the extra stabilization from aspartic acid is not needed. All other considerations, including the mechanistic steps, are identical.
Cysteine proteases follow the exact same mechanism as serine proteases (see Section 6.5.3.1; Scheme 6.12). The only differences are the use of cysteine instead of serine (viz. replacing oxygen with sulfur) and the lack of a nearby aspartic acid for stabilization.
Scheme 6.12 – Example of a Catalytic Cycle/Mechanism for Cysteine Proteases.
6.5.4.2. Catalytic Principles Used
All catalytic principles used by serine proteases are also used by cysteine proteases: catalysis by approximation (e.g. binding, hydrogen bonding for organization), catalysis by general acid/base reactivity (e.g. histidine side chain base/acid), and catalysis by covalent bonding (e.g. cystein side chain nucleophile/leaving group). Catalysis by metal ion interactions is not used.
As before, the catalytic principles were used directly to address the problems with the reaction (see Section 6.5.2 and subsections therein).
6.5.5. Example: Metallo-Proteases
One general class of proteases are the so-called “Metallo-Proteases”. These use a metal ion for their catalytic activity. Several different metal ions are possible, though cationic zinc is the most commonly used. Depending on the metal ion one or more nearby amino acids help to bind and orient the metal and substrates. The actual catalysis comes from other nearby amino acid side chains, often histidine and glutamic or aspartic acid; the metal ion only helps to organize the compounds and acts as a Lewis acid to increase reactivity.
The standard mechanism for a zinc metallo-protease is discussed as an example (Scheme 6.13). Other metallo-proteases often follow similar mechanisms.
Zinc metallo-proteases follow the standard three-phase approach: the first step is always binding of the substrates, the last step is always release of the products, and the mechanistic steps of the reactions happen between these two phases.
First the enzyme and substrates bind to form the enzyme-substrate complex. The metal ion is already bound in the enzyme and remains in its position after the reaction. Lewis acid-base interactions and hydrogen bonds (not shown) help organize the positions and orientations of the water and amide and increase their relative reactivities for the next phase.
Second, the reaction occurs in a series of steps.
The deprotonated side chain of glutamic acid (base) removes a proton from the side chain of histidine (acid). At the same time, the other nitrogen of the side chain of histidine (base) removes a proton from water (acid) regenerating the histidine side chain as the other tautomer (see Scheme 4.6). At the same time, the oxygen of the water (nucleophile) attacks the carbon of the carbonyl in the amide (electrophile). This forms a new bond and adds a lone pair to the oxygen. Lewis acid interactions help increase the electrophilicity of the carbonyl.
The intermediate coordinates to the metal ion from multiple atoms. A lone pair of the oxygen forms a π bond with the carbon, ejecting the leaving group (“amine”). At the same time, the nitrogen (base) removes a proton from the side chain of histidine (acid). Having this occur at the same time greatly increases the nitrogen’s leaving group ability. At the same time, the other nitrogen of the side chain of histidine (base) removes a proton from the side chain of glutamic acid (acid), regenerating the histidine side chain as the original tautomer. This also re-forms the deprotonated glutamic acid side chain and is equivalent to regenerating the catalyst.
Finally, the enzyme releases the final products. This empties the enzyme and allows it to begin the catalytic cycle again.
Scheme 6.13 – Example of a Catalytic Cycle/Mechanism for a Zinc Metallo-Protease.
6.5.5.2. Catalytic Principles Used
Of the four general catalytic principles, zinc metallo-proteases use three: catalysis by approximation (e.g. binding), catalysis by general acid/base reactivity (e.g. histidine and glutamic acid side chain base/acid reactivities), and catalysis by metal ion interactions (e.g. Lewis acid/base interactions for increased reactivity and organization). Catalysis by covalent bonding is not used.
As before, the catalytic principles were used directly to address the problems with the reaction (see Section 6.5.2 and subsections therein).
6.5.6. Example: Aspartyl Proteases
One general class of proteases are the so-called “Aspartyl Proteases”. These use the side chains of two aspartic acids for their catalytic activity. The local environment is highly controlled such that one side chain may act as a base and the other as an acid. Some, but not all, aspartyl proteases also have other interactions that make the reaction faster, such as hydrogen bonding to other side chains and/or amide NH’s.
Interestingly, often the two amino acids (aspartic acids) are physically close together in the peptide chain itself. The folding of the chain helps create the local environment but does not typically bring the two catalytic parts together. This is not always true (e.g. in some aspartyl proteases the two amino acids are on different peptide chains).
Aspartyl proteases follow the standard three-phase approach: the first step is always binding of the substrates, the last step is always release of the products, and the mechanistic steps of the reactions happen between these two phases.
First the enzyme and substrates bind to form the enzyme-substrate complex (Scheme 6.14). Hydrogen bonds help organize the positions and orientations of the water and amide and increases their relative reactivities for the next phase (not shown).
Second, the reaction occurs in a series of steps.
The deprotonated side chain of aspartic acid (base) removes a proton from water (acid). At the same time, the oxygen of the water (nucleophile) attacks the carbon of the carbonyl in the amide (electrophile). This forms a new bond and adds a lone pair to the oxygen. At the same time, the oxygen of carbonyl (base) removes a proton from the side chain of aspartic acid (acid). Having this occur at the same time greatly increases the electrophilicity of the carbonyl.
The deprotonated side chain of aspartic acid (base) removes a proton from an OH of the intermediate (acid), regenerating the original acid side chain. At the same time, a lone pair of that oxygen forms a π bond with the carbon, ejecting the leaving group (“amine”). At the same time, the nitrogen (base) removes a proton from the side chain of aspartic acid (acid), regenerating the original base. Having this occur at the same time greatly increases its leaving group ability. This is equivalent to regenerating the catalyst.
Finally, the enzyme releases the final products. This empties the enzyme and allows it to begin the catalytic cycle again.
Scheme 6.14 – Example of a Catalytic Cycle/Mechanism for Aspartyl Proteases.
6.5.6.2. Catalytic Principles Used
Of the four general catalytic principles, aspartyl proteases use two: catalysis by approximation (e.g. binding, hydrogen bonding for organization) and catalysis by general acid/base reactivity (e.g. aspartic acid and deprotonated aspartic acid side chains). Catalysis by covalent bonding and catalysis by metal ion interactions are not used.
As before, the catalytic principles were used directly to address the problems with the reaction (see Section 6.5.2 and subsections therein).
