Proteins: enzymes

enzymes as proteins

• Enzymes are globular proteins

• They act as biological catalysts

  • ‘Biological’ because they function in living systems

  • ‘Catalysts’ because they speed up the rate of chemical reactions without being used up or changed

• Critical to the enzyme's function is the active site where the substrate binds

• Metabolic pathways are controlled by enzymes in a biochemical cascade of reactions

  • Virtually every metabolic reaction within living organisms is catalysed by an enzyme; enzymes are therefore essential for life to exist

• Enzymes can be intracellular or extracellular, and these determine the structures and functions from the cellular to the whole-organism level

  • Intracellular enzymes are produced and function inside the cell

  • Extracellular enzymes are secreted by cells and catalyse reactions outside cells (e.g. digestive enzymes in the gut)

mode of enzymes action

• Enzymes have an active site where specific substrates bind, forming an enzyme-substrate complex

  • Substrates collide with the enzyme's active site, and this must happen at the correct orientation and speed for a reaction to occur

specificity

• The active site of an enzyme has a specific shape to fit a specific substrate

  • Extremes of heat or pH can change the shape of the active site, preventing substrate binding – this is called denaturation

• The specificity of an enzyme is a result of the complementary nature between the shape of the active site on the enzyme and its substrate(s)

• The shape of the active site is determined by the complex tertiary structure of the protein that makes up the enzyme:

  • Proteins are formed from chains of amino acids held together by peptide bonds

  • The order of amino acids determines the shape of an enzyme

  • If the order is altered, the resulting three-dimensional shape changes

enzyme - substrate complex

• An enzyme-substrate complex forms when an enzyme and its substrate join together

• The enzyme-substrate complex is only formed temporarily, before the enzyme catalyses the reaction and the product(s) are released

  • This way, enzymes are free to be recycled for future reactions

activation energy of a reaction

• All chemical reactions are associated with energy changes

• For a reaction to proceed, there must be enough activation energy

• Activation energy is the amount of energy needed by the substrate to become unstable enough for a reaction to occur and for products to be formed

  • Enzymes speed up chemical reactions because they influence the stability of bonds in the reactants

  • The destabilisation of bonds in the substrate makes it more reactive

• Enzymes work by lowering the activation energy of a reaction by providing an alternative energy pathway

the induced fit model

• Also known as the ‘induced-fit hypothesis’

• In this model, the enzyme and substrate interact with each other in the following way:

  • The enzyme and its active site (and sometimes the substrate) can change shape slightly as the substrate molecule enters the enzyme

  • These changes in shape are known as conformational changes

  • This ensures that an ideal binding arrangement between the enzyme and substrate is achieved

  • This maximises the ability of the enzyme to catalyse the reaction

development of the induced-fit enzyme model

• Scientists often use models to explain their observations from experiments

• As technology and research advances within a field, new models can be developed and old ones disproven

• The lock and key model covered at GCSE was originally thought to be an accurate model of enzyme action

  • It was suggested that the rigid shape of the active site of the enzyme is a precise fit for the specific shape of the substrate

• New techniques have allowed scientists to discover that proteins are not rigid structures

  • Experiments showed that multiple regions of an enzyme molecule moved in response to the environment

  • Many of these movements were minimal, but some of them were more significant

  • The larger movements occurred when the substrate bound to the enzyme

• These findings led to the now widely accepted induced fit model

• There is evidence to support the induced fit model:

  • X-ray diffraction techniques allow for 3D pictures of molecules to be formed

  • This technique was used to produce pictures of the enzyme hexokinase before and after it bound to its substrate glucose

  • The images confirmed that the active site of the enzyme changed shape after the substrate bound

effect of temperatures - lower temperature

• Lower temperatures either prevent reactions from proceeding or slow them down:

  • Molecules move relatively slowly

  • There is a lower frequency of successful collisions between substrate molecules and the active site of the enzyme

  • Therefore, less frequent enzyme-substrate complex formation

  • Substrate(s) and enzyme collide with less energy, making it less likely for bonds to be formed or broken (stopping the reaction from occurring)

effect of temperatures - higher temperature

• Molecules move more quickly

• There is a higher frequency of successful collisions between substrate molecules and the active site of the enzyme

• Therefore, more frequent enzyme-substrate complex formation

• Substrate(s) and enzyme collide with more energy, making it more likely for bonds to be formed or broken (allowing the reaction to occur)

effect of temperatures - too high of a temperature

• However, as temperatures continue to increase, the rate at which an enzyme catalyses a reaction drops sharply, as the enzyme begins to denature:

  • Bonds (e.g. hydrogen bonds) holding the enzyme molecule in its precise shape start to break

  • This causes the tertiary structure of the protein (i.e. the enzyme) to change

  • This permanently damages the active site, preventing the substrate from binding

  • Denaturation has occurred if the substrate can no longer bind

  • Very few human enzymes can function at temperatures above 50°C

    • This is because humans maintain a body temperature of about 37°C, therefore, even temperatures exceeding 40°C will cause the denaturation of enzymes

    • High temperatures cause the hydrogen bonds between amino acids to break, changing the conformation of the enzyme

effect of pH

• All enzymes have an optimum pH

• Enzymes are denatured at extremes of pH

  • Hydrogen and ionic bonds hold the tertiary structure of the protein (i.e. the enzyme) together

  • Below and above the optimum pH of an enzyme, solutions with an excess of H⁺ ions (acidic solutions) and OH^- ions (alkaline solutions) can cause these bonds to break

  • This alters the shape of the active site, which means enzyme-substrate complexes form less easily

  • Eventually, enzyme-substrate complexes can no longer form at all

  • At this point, complete denaturation of the enzyme has occurred

• Where an enzyme functions can be an indicator of its optimal environment:

  • E.g. pepsin is found in the stomach, an acidic environment at pH 2

  • Pepsin’s optimum pH is therefore pH 2

buffer solutions

• When investigating the effect of pH on the rate of an enzyme-catalysed reaction, you can use buffer solutions to measure the rate of reaction at different pH values:

  • Buffer solutions each have a specific pH

  • Buffer solutions maintain this specific pH, even if the reaction taking place would otherwise cause the pH of the reaction mixture to change

calculating pH

If the hydrogen ion (H⁺) concentration of a solution is known, the pH can be calculated using the equation:

pH = -log₁₀ [H⁺]

the effect of enzyme concentration

• Enzyme concentration affects the rate of reaction

  • The higher the enzyme concentration in a reaction mixture, the greater the number of active sites available and the greater the likelihood of enzyme-substrate complex formation

  • As long as there is sufficient substrate available, the initial rate of reaction increases linearly with enzyme concentration

  • If the amount of substrate is limited, further increases in enzyme concentration will not increase the reaction rate as the amount of substrate becomes a limiting factor

the effect of substrate concentration

• The greater the substrate concentration, the higher the rate of reaction:

  • As the number of substrate molecules increases, the likelihood of enzyme-substrate complex formation increases

  • If the enzyme concentration remains fixed but the amount of substrate is increased past a certain point, however, all available active sites eventually become saturated, and any further increase in substrate concentration will not increase the reaction rate

  • When the active sites of the enzymes are all full, any substrate molecules that are added have nowhere to bind to form an enzyme-substrate complex

• In the graph below, there is a linear increase in reaction rate as substrate is added, which then plateaus when all active sites become occupied

enzyme inhibitors

• An enzyme's activity can be reduced or stopped, temporarily, by a reversible inhibitor

• There are two types of reversible inhibitors:

  • Competitive inhibitors have a similar shape to that of the substrate molecules and therefore compete with the substrate for the active site

  • Non-competitive inhibitors bind to the enzyme at an alternative site, which alters the shape of the active site and therefore prevents the substrate from binding to it

end product inhibitors

• Reversible inhibitors can act as regulators in metabolic pathways

• Metabolic reactions must be very tightly controlled and balanced, so that no single enzyme can continuously and uncontrollably generate more and more of a particular product

• Metabolic reactions can be controlled by using the end-product of a specific sequence of metabolic reactions as a non-competitive, reversible inhibitor:

  • As the enzyme converts substrate to product, the process is itself slowed down as the end-product of the reaction binds to an alternative site on the original enzyme, changing the shape of the active site and preventing the formation of further enzyme-substrate complexes

  • The end-product can then detach from the enzyme, allowing the active site to reform and the enzyme to return to an active state

  • This means that as product levels fall, the enzyme begins catalysing the reaction once again, in a continuous feedback loop

  • This process is known as end-product inhibition

the effect of inhibitor concentration

• Increasing the concentration of an inhibitor reduces the rate of reaction, and eventually, if the inhibitor concentration continues to be increased, the reaction will stop completely

• For competitive inhibitors, countering the increase in inhibitor concentration by increasing the substrate concentration can increase the rate of reaction once more

  • More substrate molecules mean they are more likely to collide with enzymes and form enzyme-substrate complexes

• For non-competitive inhibitors, increasing the substrate concentration cannot increase the rate of reaction once more, as the shape of the active site of the enzyme remains changed and enzyme-substrate complexes are still unable to form