(the enzymes catalyzing oxidation–reduction reactions), transferases (the enzymes catalyzing group transfers, namely to substitute one functional group for another), lyases (the enzymes catalyzing elimination of two groups from adjacent carbon atoms to form a C=C double bond), hydrolases (the enzymes catalyzing hydrolysis of biomolecules), isomerases (the enzymes catalyzing isomerization reactions), and ligases (the enzymes catalyzing bond formation coupled with ATP hydrolysis) [8].
An enzyme contains an active site which is the catalytic center where a biochemical reaction takes place. The active site is usually composed of functional groups from side chains of some α‐amino acid residues in the protein (enzyme) molecule. The biological reactant molecule(s) [often termed substrate(s), one or more] enters the structure of the enzyme and loosely binds to the enzyme usually by hydrogen bonding, hydrophobic interactions, and/or ionic bonding to form an intermediate enzyme–substrate complex (ES complex). Then various chemical processes take place in the ES complex to lead to the formation of the product. Finally, the product departs from structure of the enzyme to regenerate a free enzyme molecule. The enzyme catalyzed biochemical reactions are generalized as follows (Eq. 1.73):
In Equation 1.73, S and E represent the biological substrate and enzyme, respectively. ES is the intermediate enzyme–substrate complex. P is the reaction product. The formation of the ES complex is usually reversible, while the conversion of ES to the final product is irreversible.
An enzyme catalyzes a biochemical reaction by lowering activation energy of the reaction. On the basis of the types of interactions between the enzyme and the biological substrate in the transition state of a biochemical reaction, there are in general three types of catalytic mechanisms.
1 The acid–base catalysis. Hydrogen bonds are formed in the transition state, which stabilize the transition state (decrease the level of its free energy) to lower the activation energy (ΔGǂ). As a result, the reactivity of the substrate is enhanced and the reaction goes faster.
2 The metal–ion catalysis. The catalytic center is a metal ion which is attached to functional groups of some side chains of α‐amino acid residues of the enzyme. A coordination bond between the substrate and the metal ion is formed (or partially formed) in the transition state to stabilize the transition state and activate the substrate.
3 The covalent catalysis. A covalent bond between the substrate and the enzyme is formed (or partially formed) in the active site in the transition state to stabilize the transition state and activate the substrate.
A remarkable feature for enzymatic reactions is that the enzyme and substrate are both structurally and electronically complementary (also called key‐to‐lock model). As a result, the catalytic efficiency is extremely high. An enzymatic reaction is often 106–1012 times as fast as the uncatalyzed reaction [8]. In addition, most of the enzymatic reactions are highly selective and stereospecific due to the enzyme–substrate complementarity.
Many common enzymatic reactions follow mechanisms of the acid–base catalysis. The catalysis is usually concerted and achieved by proton transfer between the enzyme and substrate. The general principle for this type of catalysis can be illustrated using a generalized keto–enol tautomerization (Fig. 1.21).
Figure 1.21a shows an uncatalyzed, concerted keto–enol tautomerization taking place via a single transition state. The transition state is greatly destabilized (with a high level in free energy) by the formation of partial electric charges in different atoms. Figure 1.21b and c show acid‐ and base‐catalyzed tautomerization, respectively. The acid H–A and base :B− represent acidic and basic functional groups, respectively, in the active sites of enzymes. In the acid catalysis (Fig. 1.21b), a hydrogen bond is formed in the transition state on the carbonyl of the keto substrate to stabilize the partial negative charge on oxygen (via the electrostatic attraction to the partial positive charge on the HA proton). This stabilization leads to decrease in the energy level (in the free energy term) of the transition state and the activation energy (ΔGǂ) is lowered. In the base catalysis (Fig. 1.21c), a partial deprotonation on α‐carbon by the basic group occurs in the transition state. As a result, the partial positive charge developed on the α‐hydrogen of the keto substrate is stabilized (via the electrostatic attraction to the negative charge in the basic group B−). This stabilization leads to decrease in the free energy level of the transition state and the activation energy is lowered. In both acid‐ and base‐catalyzed tautomerization (Fig. 1.21b and c), the formations of the enol and regeneration of the free enzymes from the transition states (TS1 and TS2) proceed via additional proton transfer steps. However, these steps are spontaneous and fast and do not affect the activation energies. The comparison of energetics for the uncatalyzed tautomerization and acid‐ and base‐catalyzed tautomerization is demonstrated in Figure 1.21d.
Very often, enzymatic catalysis can also be achieved by splitting an uncatalyzed mechanistic step with a high activation energy into multiple catalyzed microscopic steps with lower activation energies. This way of catalysis can be demonstrated by using carbonic anhydrase, the enzyme that catalyzes the reaction of carbon dioxide (CO2) with water (H2O) giving bicarbonate (HCO3−) in human blood [8]. When CO2 produced from respiration is transferred into the venous blood, it combines with water in the blood and this reaction happens. By virtue, the reaction of CO2 with H2O is a simple inorganic chemical reaction. When it happens in the venous blood and is catalyzed by carbonic anhydrase, it becomes a biochemical reaction.
FIGURE 1.21 Acid–base catalysis for enzymatic reactions. (a) Uncatalyzed concerted keto–enol tautomerization; (b) The acid‐catalyzed mechanism for the keto–enol tautomerization; (c) The base‐catalyzed mechanism for the keto–enol tautomerization; and (d) Comparison of energetics for uncatalyzed and acid‐ or base‐catalyzed keto–enol tautomerization.
The catalytic center of carbonic anhydrase is a sp3‐hybridized zinc ion (Zn2+) connecting to three imidazole rings (Im) of histidine residues in the enzyme's polypeptide chain [8]. The unoccupied (vacant) sp3 orbital in Zn2+ is strongly electrophilic. The transition state (TS) of the uncatalyzed reaction of CO2 with H2O is highly destabilized (with a high level of free energy) due to the formation of partial electric charges (Fig. 1.22a). In the presence of carbonic anhydrase, the reaction pathway is altered such that the first step of the reaction is that H2O (hydrogen
