6.3. Enzymes

Enzymes are proteins that catalyze chemical reactions. In general, each enzyme catalyses a single type of chemical reaction, often on a single type of molecule.

The study of enzymes (enzymology) is a very complex topic that often requires a wide array of techniques (including crystallography, radiolabelling, and kinetics/math) to fully understand what is happening. Exactly how enzymes perform their functions is incredibly interesting, but can rapidly become incredibly complicated. When working through these sections keep in mind that this is meant only as a foundation covering the fundamentals; the field of enzymology is far too complex and varied to be covered in-depth in any introductory course. A few examples of “simple” enzymes that work in straightforward ways are presented. However, while the underlying principles presented are accurate MANY advanced details are being omitted.

Most biochemical reactions occur through enzyme catalysis. Typically, the goal of the catalysis is to make the reaction(s) happen fast enough that life can occur. For example, the enzyme carbonic anhydrase helps regulate the pH inside cells. Water (H₂O) and carbon dioxide (CO₂) naturally combine to form carbonic acid (CH₂O₃; Scheme 6.1). This decreases the pH by adding an acid. The reverse reaction is also possible. This increases the pH by removing an acid. Uncatalyzed, the reaction has a rate around 0.1 s^-1 (one molecule is made every 10 seconds). When catalyzed by carbonic anhydrase the rate becomes approximately 100,000 s^-1 (~1,000,000 molecules are made every 10 seconds). The drastic increase allows the cell to quickly adjust the pH to the desired value and keep biological processes happening.

Scheme 6.1 – Uncatalyzed and Catalyzed Reaction Rates for Carbonic Acid Formation with Carbonic Anhydrase.

A second, but equally important, function of enzymes is to ensure stereoselectivity. Recall that it was possible to synthesize amino acids using a complicated rhodium catalyst with stereoselectivity (Figure 6.5; see Section 4.3.3).

Figure 6.5 – Simplified Reaction Coordinate for Synthesis of Amino Acids Using a Chiral Rhodium Catalyst Highlighting Stereochemical Outcomes.

Under ideal conditions this catalyst affords approximately 98.7% of the product as the (S) enantiomer. While this number seems high, for an enzyme performing this function this value would be unacceptably low; an enzyme making an equivalent amino acid would typically have incredibly high enantioselectivity, with >99.999% of the product in the correct enantiomer (Figure 6.6).

Figure 6.6 – Simplified Reaction Coordinate for Synthesis of Amino Acids Using an Enzyme Catalyst Highlighting Stereochemical Outcomes.

Again, enzymology deals with many other issues. Because they are typically resource- and energy-intensive to make, typically they must work well at very low concentrations. This is on top of the low concentrations of the compounds they are catalyzing the reactions of. They must also work at biological conditions (e.g. at body temperature, at a certain pH, etc.). Different organisms live under different conditions (e.g. fish in the arctic, lizards in the desert, etc.), which results in changes to the enzymes they produce to allow them to operate under the modified conditions. Finally, it is important to be aware that there are many other considerations (e.g. How does the enzyme turn on/off?, Can it be transported from one part of the cell to another?, Should it be made constantly, intermittently, or rarely?, etc.). Often the physical enzyme itself forms part of the systems that regulate these factors.

6.3.1. Enzyme-Substrate Complexes

Different enzymes perform their functions in different ways. However, in the vast majority of cases they follow a three-stage process (Scheme 6.2).

Scheme 6.2 – Simplified Three-Stage Process for Enzyme Catalysis: Binding, Reaction(s), and Release.

First, the enzyme and the starting material(s) bind together. Most sources refer to the starting material(s) in enzyme reactions as substrates. The binding may be covalent (covalent bond(s) formed between the enzyme and the substrate(s)) or result from non-covalent interactions (ionic bonds, hydrogen bonds, dispersion interactions, and/or pi-stacking). Although the binding step appears simple, in many cases the binding is caused by (and itself causes) many conformational changes to the enzyme itself, all of which are necessary for the enzyme to bind the substrate(s). Once bound together the enzyme and the substrate(s) form a so-called “enzyme-substrate complex” (often abbreviated as “ES” or “ES complex”; sometimes called the “Michaelis Complex” or “Michaelis-Menton Complex”).

Second, the enzyme performs the chemical reaction(s). Because of the wide variability in enzymes each will be different in exactly how this occurs; some will perform a single reaction in a single mechanistic step, some will perform multiple reactions with many mechanistic steps. Again, in reality this stage is often very complicated and may involve a variety of other processes, conformational changes, etc. during the reaction(s).

Finally, the enzyme releases the product(s). This also regenerates the catalyst (the enzyme) to allow it to perform another reaction. Again, although the release step appears simple, in many cases the release is caused by (and itself causes) many conformational changes to the enzyme itself, all of which are necessary for the enzyme to release the product(s) and revert to its original form.

Often the entire transformation is fully reversible; in many cases the same enzyme that converts molecule A into molecule B also converts B into A.

6.3.2. Enzymes and Basic Catalytic Principles

Although the exact ways that each enzyme catalyzes its reaction vary, there are four general strategies that are commonly involved. All enzymes use at least one of these strategies to help accelerate their reaction(s) and/or to increase stereoselectivity.

6.3.2.1. Catalysis by Approximation

One of the strategies is the act of binding the starting materials (substrates). This is sometimes referred to as “catalysis by approximation” and performs two primary functions.

Binding the starting materials and the catalyst together effectively changes the rection from an intermolecular process to an intramolecular process. This has a number of consequences but the principal benefit is a dramatic reduction in the entropy cost of the reaction. Because the molecules are already close together their relative concentrations do not affect the rate of reaction (as much). Because they are correctly oriented there are significantly fewer possible states/collisions. Reducing the entropy cost makes the reaction much faster. Use of the following formulae is not required, but recalling that ΔG = ΔH – TΔS and/or that entropy is a part of E_a in k=Ae^-Ea/RT may be helpful in seeing why reducing entropy will accelerate the reaction.

Additionally, because the binding is typically very specific and proceeds through multiple interactions the molecules are typically highly organized. This again has a number of consequences but the principal benefit is in reducing the number of possible conformations and/or approaches for chemical reaction. Exceptionally high stereoselectivity can be achieved by organizing the molecules in specific ways. Consider a hypothetical electrophile bound in an enzyme (Figure 6.7). Hydrogen bonding orients the carbonyl, another hydrogen bond orients the nitrogen of the amide, and a sterically bulky group blocks the bottom side of the electrophile from nucleophilic attack. Because of the high degree of organization, in theory any nucleophile has only one possible approach and will always attack the same conformation and orientation of the electrophile.

Figure 6.7 – Hypothetical Example of Organizing Starting Materials in an Enzyme to Select for a Stereochemical Outcome.

6.3.2.2. Catalysis by General Acid/Base Reactivity

One of the strategies is acid/base catalysis, typically using the side chain(s) of amino acids nearby.

Many of the common amino acids possess side chains with functional groups that can act directly as acid or base catalysts (Figure 6.8). Additionally, a large number may become acids or bases and then perform the same function. These catalyze reactions in exactly the same way as traditional acid/base catalysis by improving the electrophile, nucleophile, or leaving group.

Figure 6.8 – Examples of Amino Acid Side Chains That Could Act as General Acid or Base Catalysts.

In many cases the exact protonation state of all of the amino acid side chains in the enzyme is highly controlled and the side chains near the substrates affect their acidity/basicity (see Section 6.4). This makes the acid/base catalysis even more efficient (or makes it possible). For example, a class of enzymes (see Section 6.5.6) catalyze their reaction using a pair of aspartic acid side chains (Scheme 6.3). The exact protonation state is controlled so that one may act as an acid and the other as a base at multiple stages of the reaction.

Scheme 6.3 – Example of General Acid and Base Catalysis in Enzymes: Steps from Aspartyl Protease Mechanism.

6.3.2.3. Catalysis by Covalent Bonding (Forming a Leaving Group)

One of the strategies is covalently bonding to the substrate to generate a leaving group, typically using the side chain(s) of amino acids nearby.

Many of the common amino acids possess side chains with functional groups that can act directly as nucleophiles or may become (more) nucleophilic (Figure 6.9). These catalyze reactions in a similar way as other “activating groups” by improving the electrophilicity and/or or leaving group ability of the substrate.

Figure 6.9 – Examples of Amino Acid Side Chains That Could Act as Nucleophiles for Covalent Bonding Catalysis.

In many cases the exact protonation state of all of the amino acid side chains in the enzyme is highly controlled and the side chains near the substrates affect their nucleophilicity/leaving group ability (see Section 6.4). This makes the covalent bonding catalysis even more efficient (or makes it possible). For example, a class of enzymes (see Section 6.5.3) catalyze their reaction using a serine side chain as a nucleophile/leaving group (Scheme 6.4). The alcohol of serine on its own is not nearly nucleophilic enough to attack amide functional groups. The nearby amino acid side chains work together with serine so that it may act as a nucleophile and later as a leaving group in the reaction.

Scheme 6.4 – Example of Covalent Bonding Catalysis in Enzymes: Steps from Serine Protease Mechanism.

6.3.2.4. Catalysis by Metal Ion Interactions

One of the strategies is metal ion catalysis. This involves a metal ion being bound inside the enzyme and then used to assist one or more steps in the reaction. Depending on the exact metal (the element) and the charge (anion/cation) there are several different ways catalysis can occur.

Cationic metals typically act as Lewis acids and accept electron density from the substrate(s) (and the enzyme). In general, these catalyze reactions by increasing the acidity or electrophilicity of a compound. Anionic metals typically act as Lewis bases and donate electron density to the substrate(s) (and the enzyme). In general, these catalyze reactions by increasing the basicity or nucleophilicity of a compound. In either case the availability of large d orbitals on the metal atom tends to allow otherwise-inaccessible geometries through coordination; the metal ion often also assists by helping to organize the substrates and enzyme in specific orientations. For example, a class of enzymes (see Section 6.5.5) catalyze their reaction using a bound zinc cation (Scheme 6.5). The metal ion both organizes the substrates and acts as a Lewis acid to increase the basicity and/or nucleophilicity of the catalyst and/or substrates.

Scheme 6.5 – Example of Metal Ion Catalysis in Enzymes: Steps from Zinc Metallo Protease Mechanism.