Lecture 11 Summary

A catalyst is a substance that speeds up the rate of a chemical reaction without itself being consumed or generated by the reaction.. An enzyme is a protein that acts as a catalyst. The term substrate refers in enzymology to a reactant in an enzyme-catalyzed reaction. The rate enhancements achievable by an enzyme – speeding up a reaction by many orders of magnitude compared to its non-enzymatic rate - are accounted for by the direct interaction at the molecular level between enzyme and substrate. This interaction occurs in a part of the protein molecule (proteins are typically much larger than their substrates) called the active site. The features of active sites are described further below. Another characteristic of enzymes is their substrate specificity. In many instances enzymes show an exquisite ability to discriminate between closely related molecules. Often the existence of an enzyme-substrate complex is inferable or directly observable. Enzymes are involved in virtually every biological process. They are primarily and historically connected to metabolic reactions, but enzymes also participate in processes of regulation and energy transduction.

Enzyme classification

Enzymes are classified according to the type of reactions they catalyze.

1. Oxidoreductases (Redox reactions) Example: lactate dehydrogenase [EC 1.1.1.27]
2. Transferases (Transfer of functional groups) Example : aspartate transcarbamylase [EC 2.1.3.2]
3. Hydrolases (Hydrolysis reactions) Example : phosphodiesterases, proteases
4. Lyases (Group elimination to form double bonds) Example : carbonic anhydrase [EC 4.2.1.1]
5. Isomerases (Isomerization) Example : triose phosphate isomerase (TIM) [EC 5.3.1.1]
6. Ligases (Ligases) Example : T4 DNA ligase [EC 6.5.1.1]

Free energy and catalysis

In applying the principles of chemical thermodynamics to biological catalysis, we find that under the typical physiological conditions of constant temperature and pressure, the free energy function (G) is most useful. A chemical reaction's free energy change, ΔG, determines if a reaction is spontanaeous under specified conditions. Equilibrium conditions are governed by the standard free energy change ΔG°. By analogy to the methods of equilibrium thermodynamics (Big K), a kinetic rate constant (little k) can be related to the theoretical free energy required to reach the transition state (ΔG^‡). This approach to kinetics is refered to as transition state theory. Bearing in mind the distinction between thermodynamics and kinetics,

(1) Enzymes never alter the ΔG of reaction!
(2) Enzymes selectively lower ΔG^‡ for the reaction by forming an enzyme-substrate complex. This is what is referred to as transition state stabilization.

In studying enzymes and their complexes with substrates or inhibitors, we aim to learn how the enzyme active site is adapted to bind their substrates and selectively lower the energy of the transition state for the reaction.

Compare and contrast this with the key points listed in § 8.2 in BTS6 (p.208), which are concerned with chemical thermodynamics in general, and its application to enzymology.

Transition state theory and a simple mechanistic model

We can apply the formalism of thermodynamics to derive a general theoretical expression for the rate of a chemical reaction that quantitatively relates the velocity of a reaction to the free energy of activation, ΔG^‡.

This in turn, will allow us to quantify the rate enhancement that an enzyme can achieve by reducing the value of ΔG^‡ by a certain amount. This discussed in § 8.3 in BTS. By assuming the reactant is in equilibrium with the reaction transition state, an equation is derived that shows that the velocity (rate) of reaction is proportional to the concentration of the reactant and the exponential of the ratio (–ΔG^‡/RT). Using the appropriate form of this relation, it can be shown that at 25°C an enzyme can achieve a 10-fold rate enhancement for each 5.69 kJ/mol (1.36 kcal/mol) lowering of ΔG^‡ - this represents a factor of (ln10)RT with T in kelvins (see BTS6, p.212). Thus, the approach of transition state theory shows that the reduction of the free energy of activation for a reaction by an enzyme catalyst, which we can call ΔΔG^‡, is exponentially related to the rate enhancement, as represented by the ratio k_cat/k_uncat of the rate contants for the catalyzed and uncatalyzed reactions, respectively.

Enzymes and the chemistry of catalysis. Cofactors.

Proteins make good enzymes because of their structural and chemical diversity. Catalytic power and specificity are both related to shape (or steric) complementarity and electrostatic complementarity. Complementarity to the transition state lowers the activation energy for a reaction, and complementarity to substrate has much to do with specificity. Because proteins can adopt a variety of shapes and create precisely adapted electrostatic environments, a process of natural selection can act, resulting in the evolution of proteins that are highly efficient enzymes.

The chemical diversity of the twenty amino acids makes proteins well-suited to catalyze reactions via a number of common mechanisms. Many reactions in organic chemistry proceed via nucleophilic attack, general acid, and general base mechanisms. The side chains of proteins include groups that can act as nucleophiles such as the -OH and -SH groups of Ser and Cys, respectively. Groups such as -COOH and ImH⁺ (protonated imidazole side chain of His) can act as general acids (i.e. proton donors), while groups such as -COO⁻, Im, and -NH₂ can act as general bases (proton acceptors) in catalytic mechanisms.

There are some catalytic chemistries for which protein groups alone are not so well-suited, for example electrophilic catalysis. To extend their catalytic capabilities, enzymes often make use of accessories, generally termed cofactors. Table 8.2 (BTS6, p.207) lists many examples of cofactors.

Cofactors can be defined in general as "accessories" upon which the catalytic activity of many enzymes depend. These cofactors are further subdivided into two groups: metals and "small" organic molecules. The term coenzyme is typically applied to the latter, and a coenzyme that is very tightly bound to an enzyme is often referred to as a prosthetic group. The roles that cofactors play in enzyme activity are varied. In some cases, they are clearly catalytic in nature, helping enzymes perform some chemistry that proteins in general are not particularly adept at. In other cases, the coenzymes act as modular carriers of chemical species such as electrons, one-carbon units, or acyl groups. In this role, the term cosubstrate is perhaps more apt, but their modular nature - being used by a range of enzymes - and their relationship to vitamins (from which they are derived) distinguishes them from typical substrates.

Some cofactors clearly play a catalytic role - such as Zn²⁺ in carbonic anhydrase. Others act more like substrates, except that they associate with many different enzymes, acting as carriers of electrons (NADH, FADH) or chemical groups (Coenzyme A is a carrier of acyl groups). The term "prosthetic group" refers to cofactors that remain fairly tightly associated with an enzyme - in many cases is actually covalently linked to the enzyme, such as the pyridoxal phosphate (PLP) prosthetic group of the β subunit of tryptophan synthase [EC 4.2.1.20].

Active sites of enzymes and the enzyme-substrate (ES) complex

The active site is a three-dimensional cleft or crevice or "pocket" that forms by the juxtaposition of different residues in the tertiary structure of the enzyme. The lysozyme example shows how different residues - sometimes quite far separated in the sequence - contribute to the formation of an enzyme active site.

(I) Saturation kinetics. Reaction velocity as a function of substrate concentration reaches a maximum, suggesting a saturation of catalytic sites on the enzyme where the enzyme and substrate interact. This evidence is indirect.

(II) Spectroscopic changes.

(III) X-ray crystallography of enzyme-substrate or enzyme-substrate analog complexes has provided explicit structural details of how many enzymes recognize and bind their substrates.

Binding energy and basic catalytic principles

Binding energy

Catalyic strategies

Covalent catalysis
General acid - general base mechanisms
Metal ion catalysis - electrophilic catalysis
Catalysis by approximation

Learning objectives

• Define or explain the following terms: catalyst, enzyme, substrate, active site.
• Describe the classification of enzymes.
• Discuss the implications of chemical thermodynamics and kinetics for enzyme catalysis.
• Use transition state theory to explain and predict rate enhancements .
• Define or explain the following terms: cofactor, coenzyme, prosthetic group.
• Distinguish between catalytic and cosubstrate functions of coenzymes.
• Explain or diagram binding energy, and how it contributes to catalysis.

Page updated 10-18-09

References

1. Fersht A. Structure and Mechanism in Protein Science (1999, WH Freeman and Co.)
2. Jencks WP. Catalysis in Chemistry and Enzymology (1987, Dover)
3. Walsh, C. Enzymatic Reaction Mechanisms (1979, WH Freeman and Co.)