Enzymes

• Intracellular and extracellular enzymes; active site, enzyme–substrate complex, activation energy.
• Lock-and-key and induced-fit hypotheses of specificity.
• Following reactions with catalase (product formed) and amylase (substrate lost); using a colorimeter.
• Effects of temperature, pH, enzyme, substrate and inhibitor concentration.
• V_max and K_m as a measure of affinity.
• Competitive and non-competitive reversible inhibition.
• Immobilised enzymes in alginate and their advantages.

Globular proteins that catalyse reactions

Enzymes are globular proteins that act as biological catalysts: they speed up reactions and are not used up.

• Intracellular enzymes catalyse reactions inside cells, e.g. catalase (breaks down hydrogen peroxide), DNA polymerase.
• Extracellular enzymes are secreted to catalyse reactions outside the cell, e.g. salivary amylase, trypsin in the small intestine.

How enzymes work

• The active site is a small region of the enzyme, formed by a few amino acids, with a specific 3-D shape determined by the tertiary structure.
• The substrate has a shape complementary to the active site. It binds to form an enzyme–substrate complex, then an enzyme–product complex; the products are released and the enzyme is free to be used again.
• Binding holds substrates close together in the right orientation or strains bonds within them, so the enzyme lowers the activation energy of the reaction.
• Each enzyme is specific: only substrates of the right shape fit the active site.

Two models of specificity

• Lock-and-key hypothesis: the active site has a fixed, rigid shape that exactly fits the substrate, like a key in a lock.
• Induced-fit hypothesis: the active site is flexible; as the substrate binds, the enzyme changes shape slightly to mould more tightly around it, putting strain on the substrate’s bonds. This better explains how some enzymes bind several similar substrates.

Following the progress of a reaction

Catalase: rate of formation of product

2H₂O₂ → 2H₂O + O₂. Add a catalase source (e.g. potato or yeast suspension) to hydrogen peroxide in a flask connected to a gas syringe (or an upturned measuring cylinder over water). Record the volume of oxygen at regular intervals. The initial rate is the gradient of the curve at time zero (draw a tangent), when substrate concentration is highest and not yet limiting.

Amylase: rate of disappearance of substrate

Mix amylase with starch solution. At regular intervals take a drop and add it to iodine on a spotting tile. While starch remains, the iodine turns blue-black; the time taken for the iodine to stay orange-brown is the time for all the starch to be hydrolysed. Rate = 1 ÷ time.