Enzymes are macromolecular biological catalysts. Enzymes accelerate chemical reactions. The molecules upon which enzymes may act are called substrates and the enzyme converts the substrates into different molecules known as products. Almost all metabolic processes in the cell need enzymes in order to occur at rates fast enough to sustain life. Metabolic pathways depend upon enzymes to catalyze individual steps. The study of enzymes is called enzymology and a new field of pseudoenzyme analysis has recently grown up, recognizing that during evolution, some enzymes have lost the ability to carry out biological catalysis, which is often reflected in their amino acid sequences and unusual ‘pseudocatalytic’ properties.

Enzymes are known to catalyze more than 5,000 biochemical reaction types. Most enzymes are proteins, although a few are catalytic RNA molecules. The latter are called ribozymes. Enzymes’ specificity comes from their unique three-dimensional structures.

Like all catalysts, enzymes increase the reaction rate by lowering its activation energy. Some enzymes can make their conversion of substrate to product occur many millions of times faster. An extreme example is orotidine 5′-phosphate decarboxylase, which allows a reaction that would otherwise take millions of years to occur in milliseconds. Chemically, enzymes are like any catalyst and are not consumed in chemical reactions, nor do they alter the equilibrium of a reaction. Enzymes differ from most other catalysts by being much more specific. Enzyme activity can be affected by other molecules: inhibitors are molecules that decrease enzyme activity, and activators are molecules that increase activity. Many therapeutic drugs and poisons are enzyme inhibitors. An enzyme’s activity decreases markedly outside its optimal temperature and pH.

Some enzymes are used commercially, for example, in the synthesis of antibiotics. Some household products use enzymes to speed up chemical reactions: enzymes in biological washing powders break down protein, starch or fat stains on clothes, and enzymes in meat tenderizer break down proteins into smaller molecules, making the meat easier to chew.


An enzyme’s name is often derived from its substrate or the chemical reaction it catalyzes, with the word ending in –ase. Examples are lactase, alcohol dehydrogenase and DNA polymerase. Different enzymes that catalyze the same chemical reaction are called isozymes.

The International Union of Biochemistry and Molecular Biology have developed a nomenclature for enzymes called the EC numbers. Each enzyme is described by a sequence of four numbers preceded by “EC”, which stands for Enzyme Commission. The first number broadly classifies the enzyme based on its mechanism.  The top-level classification is;

  • EC 1, Oxidoreductases: catalyze oxidation/reduction reactions
  • EC 2, Transferases: transfer a functional group (e.g. a methyl or phosphate group)
  • EC 3, Hydrolases: catalyze the hydrolysis of various bonds
  • EC 4, Lyases: cleave various bonds by means other than hydrolysis and oxidation
  • EC 5, Isomerases: catalyze isomerization changes within a single molecule
  • EC 6, Ligases: join two molecules with covalent bonds.

These sections are subdivided by other features such as the substrate, products, and chemical mechanism. An enzyme is fully specified by four numerical designations. For example, hexokinase (EC is a transferase (EC 2) that adds a phosphate group (EC 2.7) to a hexose sugar, a molecule containing an alcohol group (EC 2.7.1).


A graph below shows that reaction rate increases exponentially with temperature until denaturation causes it to decrease again.

Figure 1; graph showing the activity of an enzyme as temperature increases

Enzyme activity initially increases with temperature until the enzyme’s structure unfolds (denaturation), leading to an optimal rate of reaction at an intermediate temperature

Enzymes are generally globular proteins, acting alone or in larger complexes. The sequence of the amino acids specifies the structure which in turn determines the catalytic activity of the enzyme. Although structure determines function, a novel enzymatic activity cannot yet be predicted from structure alone. Enzyme structures unfold (denature) when heated or exposed to chemical denaturants and this disruption to the structure typically causes a loss of activity. Enzyme denaturation is normally linked to temperatures above a species’ normal level; as a result, enzymes from bacteria living in volcanic environments such as hot springs are prized by industrial users for their ability to function at high temperatures, allowing enzyme-catalyzed reactions to be operated at a very high rate.

Enzymes are usually much larger than their substrates. Sizes range from just 62 amino acid residues, for the monomer of 4-oxalocrotonate tautomerase, to over 2,500 residues in the animal fatty acid synthase. Only a small portion of their structure (around 2–4 amino acids) is directly involved in catalysis at the catalytic site. This catalytic site is located next to one or more binding sites where residues orient the substrates. The catalytic site and binding site together comprise the enzyme’s active site. The remaining majority of the enzyme structure serves to maintain the precise orientation and dynamics of the active site.


In some enzymes, no amino acids are directly involved in catalysis instead, the enzyme contains sites to bind and orient catalytic cofactors. Enzyme structures may also contain allosteric sites where the binding of a small molecule causes a conformational change that increases or decreases activity.

A small number of RNA-based biological catalysts called ribozymes exist, which again can act alone or in complex with proteins. The most common of these is the ribosome which is a complex of protein and catalytic RNA components.


Substrate binding

Enzymes must bind their substrates before they can catalyze any chemical reaction. Enzymes are usually very specific as to what substrates they bind and then the chemical reaction catalysed. Specificity is achieved by binding pockets with complementary shape, charge and hydrophilic/hydrophobic characteristics to the substrates. Enzymes can therefore distinguish between very similar substrate molecules to be chemo selective, regioselective and stereospecific.

Some of the enzymes showing the highest specificity and accuracy are involved in the copying and expression of the genome. Some of these enzymes have “proof-reading” mechanisms. Here, an enzyme such as DNA polymerase catalyzes a reaction in a first step and then checks that the product is correct in a second step. This two-step process results in average error rates of less than 1 error in 100 million reactions in high-fidelity mammalian polymerases.

Conversely, some enzymes display enzyme promiscuity, having broad specificity and acting on a range of different physiologically relevant substrates. Many enzymes possess small side activities which arose fortuitously (i.e. neutrally), which may be the starting point for the evolutionary selection of a new function.


For two molecules to react they must collide with one another. They must collide in the right direction (orientation) and with sufficient energy. Sufficient energy means that between them they have enough energy to overcome the energy barrier to reaction. This is called the activation energy.

Enzymes have an active site. This is part of the molecule that has just the right shape and functional groups to bind to one of the reacting molecules. The reacting molecule that binds to the enzyme is called the substrate.

An enzyme-catalyzed reaction takes a different ‘route’. The enzyme and substrate form a reaction intermediate. Its formation has a lower activation energy than the reaction between reactants without a catalyst.

Route A; reactant 1 + reactant 2  –> product


Route B; reactant 1 + enzyme  –>  intermediate

intermediate + reactant 2  –>  product + enzyme

So the enzyme is used to form a reaction intermediate, but when this reacts with another reactant the enzyme reforms

“Lock and key” model

To explain the observed specificity of enzymes, in 1894 Emil Fischer proposed that both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This is often referred to as “the lock and key” model. This early model explains enzyme specificity, but fails to explain the stabilization of the transition state that enzymes achieve.

Figure 2;lock and key mechanism

In this analogy, the lock is the enzyme and the key is the substrate. Only the correctly sized key (substrate) fits into the key hole (active site) of the lock (enzyme). Smaller keys, larger keys, or incorrectly positioned teeth on keys (incorrectly shaped or sized substrate molecules) do not fit into the lock (enzyme). Only the correctly shaped key opens a particular lock. This is illustrated in graphic on the left.

Nature of Active Site and Substrate Interaction

Enzymes have varying degrees of specificity. Some enzymes have absolute specificity for one substrate and no others, while other enzymes react with substrates with similar functional groups, side chains, or positions on a chain. The least specific enzymes catalyze a reaction at a particular chemical bond regardless of other structural features.

Much experimental work is devoted to gaining an understanding of the nature of the active site in an enzyme. Since enzymes are proteins, the nature of amino acid side chains in the vicinity of the active site is important. The specific amino acid side chains have been determined for many enzymes. The active site for carboxypeptidase A will be used to illustrate the principles involved as shown in the graphic on the left.

The substrate can interact with the active site through opposite charges, hydrogen bonding (shown in yellow), hydrophobic non-polar interaction, and coordinate covalent bonding to the metal ion activator as shown in magenta. The numbers behind the amino acids indicate the sequence position of the amino acid in the protein. The white lines represent the wire frames of the other amino acids in the enzyme. The carbonyl bond is activated by interaction with the Zn ions. This leads to the addition of -OH from water to the carbonyl to produce an acid and the ultimate rupture of the C-N bond.


  • Temperature

Figure3; effect of temperature on enzyme activity

As the temperature rises, reacting molecules have more and more kinetic energy. This increases the chances of a successful collision and so the rate increases. There is a certain temperature at which an enzyme’s catalytic activity is at its greatest (see graph). This optimal temperature is usually around human body temperature (37.5 oC) for the enzymes in human cells.

Above this temperature the enzyme structure begins to break down (denature) since at higher temperatures intra- and intermolecular bonds are broken as the enzyme molecules gain even more kinetic energy.


  • pH

Each enzyme works within quite a small pH range. There is a pH at which its activity is greatest (the optimal pH). This is because changes in pH can make and break intra- and intermolecular bonds, changing the shape of the enzyme and, therefore, its effectiveness.


  • Concentration of enzyme and substrate

The rate of an enzyme-catalyzed reaction depends on the concentrations of enzyme and substrate. As the concentration of either is increased the rate of reaction increases (see graphs).

For a given enzyme concentration, the rate of reaction increases with increasing substrate concentration up to a point, above which any further increase in substrate concentration produces no significant change in reaction rate. This is because the active sites of the enzyme molecules at any given moment are virtually saturated with substrate. The enzyme/substrate complex has to dissociate before the active sites are free to accommodate more substrate. (See graph)


Provided that the substrate concentration is high and that temperature and pH are kept constant, the rate of reaction is proportional to the enzyme concentration. (See graph).

Inhibition of enzyme activity

Some substances reduce or even stop the catalytic activity of enzymes in biochemical reactions. They block or distort the active site. These chemicals are called inhibitors, because they inhibit reaction. Inhibitors that occupy the active site and prevent a substrate molecule from binding to the enzyme are said to be active site-directed (or competitive, as they ‘compete’ with the substrate for the active site). Inhibitors that attach to other parts of the enzyme molecule, perhaps distorting its shape, are said to be non-active site-directed (or non-competitive).

Immobilized enzymes

Enzymes are widely used commercially, for example in the detergent, food and brewing industries. Protease enzymes are used in ‘biological’ washing powders to speed up the breakdown of proteins in stains like blood and egg. Pectinase is used to produce and clarify fruit juices. Problems using enzymes commercially include;

  • they are water soluble which makes them hard to recover
  • some products can inhibit the enzyme activity (feedback inhibition)

Enzymes can be immobilized by fixing them to a solid surface. This has a number of commercial advantages

  • the enzyme is easily removed
  • the enzyme can be packed into columns and used over a long period
  • speedy separation of products reduces feedback inhibition
  • thermal stability is increased allowing higher temperatures to be used
  • higher operating temperatures increase rate of reaction

There are four principal methods of immobilization currently in use:

  • covalent bonding to a solid support
  • adsorption onto an insoluble substance
  • entrapment within a gel
  • encapsulation behind a selectively permeable membrane




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