chapter 7 Enzymes: catalytic proteins

KEY POINTS

Enzymes are generally proteins used by cells as catalysts.

Most enzymes have at least two names.

Enzymes have a specific set of properties.

Chemical reactions involve the breaking and making of bonds.

For spontaneous reactions to occur some energy is taken from the environment.

Reactions involve the formation of unstable intermediate forms.

The active site contains the catalytic apparatus of an enzyme.

The main factors that influence the rate of an enzyme-catalysed reaction are temperature, pH and substrate concentration.

The behaviour of simple enzymes can be modelled using Michaelis-Menten kinetics.

Inhibitors lower the rate of reaction by several different types of mechanisms.

Many clinically important drugs are enzyme inhibitors.

Cells control the activity of various enzymes using a series of different mechanisms.

Many of the reactions that make up the metabolic profile of a cell are spontaneous. This means that they can occur without any external energy input. However, the spontaneity of these reactions does not guarantee rapidity. One way to increase the rate of a chemical reaction is to use a catalyst, a substance that speeds up a chemical reaction but which is not consumed in it. Cells use protein-based catalysts called enzymes to speed up cellular reactions that would normally occur spontaneously, but at a much slower rate. Most mammalian cells actually contain many hundreds to several thousand enzymes to give them diverse functionality.

Naming enzymes

Each enzyme has at least two names:

• An accepted name which is short and convenient for everyday use. These are often derived by taking the name of their substrate and adding the suffix ‘ase’. For example, amylase is an enzyme which hydrolyses amylose. Other enzyme names use a description of the reaction carried out by the enzyme and the same suffix. An example of this type of naming would be alanine transaminase, an enzyme that catalyses the removal of the amino group from alanine and its transfer to another molecule. Others such as trypsin, pepsin and chymotrypsin have trivial names that were derived in many different ways.

• A systematic name that is derived using rules developed by the International Union of Biochemistry and Molecular Biology. This nomenclature classifies enzymes into six major groups, with the name of each group derived by taking a complete description of the reaction catalysed followed by the suffix ‘ase’ (Table 7-1). However, for complex reactions these names can be very cumbersome and not appropriate for general use. For example, the systematic name for alanine transaminase is L-alanine:2-oxoglutarate aminotransferase which, while it gives more information, is much harder to remember and use.

TABLE 7-1 Classification of enzymes by type of reaction

General properties of an enzyme

The general properties of an enzyme are that it:

• contains an active site—the protein component of the enzyme contains a special pocket or cleft that contains the amino acid side chains that are involved in catalysis. The active site binds the substrate in such a manner that facilitates the chemical reaction, then releases the product.

• has specificity—most enzymes are highly specific, with their active sites able to bind and carry out catalysis on only one or a few closely related (structurally) substrates.

• may require a cofactor—some enzymes do not carry all the functional groups required for catalysis as the side chains of the amino acids that constitute the protein. Proteins can associate with other substances to complete their catalytic machinery. Common interactions are with metal ions such as Fe²⁺ and Zn²⁺, or with complex organic molecules that are often derived from dietary vitamins. If an enzyme requires a cofactor then, if it is present, it is referred to as the holoenzyme, but if the cofactor is absent the term apoenzyme is used.

• expresses catalytic efficiency—enzyme-catalysed reactions proceed much more rapidly than when uncatalysed. Enzymes are often able to carry out the reaction in the range of a hundred to a thousand times per second, making them 10³ to 10⁸ times quicker. The number of substrate molecules converted to product per second is termed the turnover number.

• has the ability to be controlled—not all the enzymes in a cell need to be active at the same time. Thus, enzymes have the ability to be ‘switched on’ (activated), and ‘switched off’ (inhibited) when not required. This is often referred to as regulation, and allows the cell to tailor the reactions occurring in the cell to the conditions that it is meeting.

• has controlled intracellular distribution—many enzymes catalyse highly specific reactions which are not required throughout the total cellular volume. Such enzymes are often compartmentalised into cellular organelles. This is particularly useful in separating enzymes which carry out competing reactions (Fig 7-1).

FIGURE 7-1 Subcellular compartmentalisation of enzymes. A shows that E₁ and E₂ catalyse competing reactions and if present at the same time the reaction would pass forwards and backwards setting up a ‘futile cycle’. However, if E₂ is in a different part of the cell to E₁ then the products produced by the action of E₁ can accumulate (B).

What slows spontaneous reactions

Chemical reactions involve the breaking of existing bonds and the construction of new molecular bonds. To do this one molecule is often forced into an unstable intermediate before the reaction can occur. To reach this state energy is absorbed from the environment. When the reaction occurs the molecules in the unstable intermediate form rearrange their bonding to generate new products. These products have structures which require less energy to maintain than the unstable intermediates, and the excess energy is released into their surroundings.

The energy needed to form the unstable intermediate form is called the activation energy (E_A) of the reaction (Fig 7-2). A chemical reaction can be envisaged as moving a barrel from one side of a hill to the other. First we have to push the barrel to the top of the hill. In doing this we are putting energy into the system, and this is analogous to the activation energy required by a chemical reaction. At the top of the hill the barrel is now unstable and a small push is all that is required to roll it down to the bottom. This is analogous to the unstable intermediate in a chemical reaction. Once the barrel reaches the bottom of the hill and stops rolling, it has reached a stable state. A similar phenomenon occurs in a chemical reaction. The unstable intermediates transform into products that are more stable molecules.

FIGURE 7-2 Energy diagram for catalysed and uncatalysed reactions showing that the energy input to form the reaction intermediate, activation energy (E_A), is greater for an uncatalysed reaction than for an enzyme-catalysed one.

In a test-tube the easiest way to provide the energy to form the unstable intermediate state is to heat up the reaction mixture. Heat makes the molecules less stable by causing their atoms to vibrate to such an extent that their bonds are more likely to break. The molecules themselves also gain speed and collisions between them become more frequent and forceful. Unfortunately the application of heat is not the answer for living systems as cellular macromolecules such as proteins denature and become inactive when excessive heat is applied. This means cells must find another way of providing the activation energy for their reactions at relatively modest temperatures, for example, at about 37°C for humans.

Lowering activation energy

Cells overcome their prevailing conditions by providing alternative reaction pathways which lower the energy required to convert the substrate (lowered energy of activation) to the unstable intermediate prior to formation of the product. These alternative pathways are provided by enzymes whose active sites provide environments which lower the activation energy for reactions (Fig 7-2). These active sites have multiple functions in that they not only have to carry out catalysis, but also have to recognise specific substrate molecules, and be able to release the product. This means that active sites are actually highly complex molecular machines.

The mechanisms of catalysis used by enzymes are as diverse as the chemical reactions they promote. One of the main mechanisms used to increase catalytic efficiency is referred to as transition-state stabilisation. The active site can act as a three-dimensional template that interacts with the substrate to bind it in a form similar to its transition state. This then means that at any time more molecules of substrate are in their transition state, and are able to be readily converted to product, leading to an increased rate of reaction. As well as interacting with the substrate to promote the transition state, functional groups in the active site can also act as proton donors or acceptors. This is termed general acid–base catalysis.

The internal movement of parts of an enzyme’s structure, such as individual amino acid residues, a group of amino acids or even large portions of the protein, can also be related to catalysis. These movements can occur very quickly (10⁻¹⁵ s) or much more slowly (seconds). These movements are vital to many enzymes, but whether small and fast vibrations or larger and slower conformational movements are more important depends on the reaction catalysed. These movements are important in the binding and releasing of substrates and products. However, it is not clear if they accelerate the chemical steps involved in catalysis.

Kinetics of enzyme-catalysed reactions

Many of the conclusions needed to model the kinetics of enzyme-catalysed reactions were drawn by two researchers, Leonor Michaelis and Maud Menten. They proposed a simple model in which a single substrate transiently combines with the enzyme (the ES complex) which then breaks down to yield the product. It can be represented as:

where E = enzyme, S = substrate, ES = enzyme-substrate complex, P = product, and k₁, k_–1 and k₂ are rate constants for the respective reactions.

They then developed an equation to explain how the rate of reaction varies with substrate concentration. This became known as the Michaelis-Menten equation.

where V₀ = the initial reaction velocity, V_max = the maximal reaction velocity, K_m = the Michaelis constant ((k₁ + k₂)/k_–1) and [S] = the substrate concentration.

The derivation of the equation required some assumptions to be made. First, it was assumed that there was a large excess of substrate such that the amount of substrate bound to the enzyme at any time is always a small fraction of the total. Next it was assumed that [ES] does not change over time, that it achieves steady state. This means that the researchers assumed that the rate of formation (E + S → ES) was the same as the rate of breakdown (ES → E + P). Finally, they only used the initial velocity of the reaction which was determined in the laboratory as soon as the enzyme and substrate were mixed (Fig 7-3). This ensured that the concentration of product was negligible and that the reverse reaction (P → S) could be ignored.