How Enzymes Work: Active Sites, Activation Energy and Cofactors

A million times a second

Every time you breathe out, your red blood cells release CO₂ that was carried as bicarbonate ions. The reaction H₂CO₃ ⇌ CO₂ + H₂O happens on its own, but far too slowly for life. The enzyme carbonic anhydrase speeds it up enormously: one molecule of the enzyme can handle on the order of a million molecules per second.

Almost every reaction in a cell has its own enzyme. In Grade 11 you learned what enzymes do. This lesson is about how they do it, at the level of molecules and energy.

Enzymes are (mostly) folded proteins

An enzyme's active site is a small region, often a cleft or pocket, formed where the polypeptide folds bring particular R groups together. Those R groups do two jobs:

• some bind the substrate (by hydrogen bonds, ionic bonds and hydrophobic interactions), which gives the enzyme its specificity;
• others catalyze: they take part in the chemistry of breaking or forming bonds.

Because the active site depends on tertiary (and sometimes quaternary) structure, anything that denatures the enzyme destroys the site. (A few catalysts in cells are RNA molecules, called ribozymes; the part of the ribosome that forms peptide bonds is one.)

Enzymes are usually named after their substrate or reaction plus -ase: sucrase, lipase, DNA polymerase, lactate dehydrogenase (removes H, a redox reaction). The international system sorts enzymes into main classes by reaction type, including oxidoreductases (redox), transferases (move a group), hydrolases (hydrolysis), lyases, isomerases and ligases (join molecules using ATP).

Lowering activation energy

Even a reaction that releases energy (negative ΔG) needs an input of activation energy to reach the transition state, an unstable arrangement in which old bonds are partly broken and new ones partly formed. At body temperature, few molecules have enough energy, so the uncatalyzed reaction is slow.

An enzyme provides a different pathway with a lower activation energy. Look at the energy diagram:

• The enzyme lowers Eₐ, so many more molecules can react each second.
• It does not change the energies of the reactants or products, so ΔG is the same. An enzyme cannot make an energy-requiring reaction happen on its own (that needs coupling to ATP, which you'll meet in Metabolic Processes).
• It speeds up the forward and reverse reactions equally, so it does not change where the equilibrium lies; it only helps the reaction reach equilibrium faster.
• The enzyme comes out unchanged and can be used again and again.

Induced fit and the catalytic cycle

1. The substrate binds to the active site, forming the enzyme-substrate complex.
2. Binding causes the enzyme to change shape slightly (induced fit), wrapping the active site around the substrate.
3. The reaction happens; the complex becomes an enzyme-product complex.
4. The products are released, and the enzyme returns to its original shape, ready for the next substrate.

Four ways an active site lowers Eₐ

• Orientation: it holds two substrates close together in exactly the right position to react, instead of waiting for a lucky collision.
• Strain: induced fit stretches or bends bonds in the substrate toward the transition state, making them easier to break.
• Microenvironment: the active site can provide conditions different from the surrounding solution, such as a pocket of acidic R groups that donate H⁺ or a non-polar pocket.
• Direct participation: R groups (or a cofactor) may briefly form covalent bonds with the substrate, or pass electrons or protons to it, then return to their original state.

Cofactors and coenzymes

Many enzymes need a non-protein partner, a cofactor:

• Inorganic ions: carbonic anhydrase holds a zinc ion (Zn²⁺) in its active site; many enzymes that use ATP need Mg²⁺; catalase contains iron in a heme group.
• Coenzymes are organic cofactors, many made from vitamins: NAD⁺ from niacin (vitamin B₃), FAD from riboflavin (vitamin B₂), and coenzyme A from pantothenic acid (vitamin B₅). They often carry electrons or chemical groups from one enzyme to another. This is one reason a lack of certain vitamins harms so many body functions.
• A cofactor bound very tightly, even permanently, is called a prosthetic group (such as the heme in catalase).

An enzyme without its cofactor is an inactive apoenzyme; with it, it is a holoenzyme.

How fast? Rate and substrate concentration

If you keep the amount of enzyme constant and increase the substrate concentration, the rate rises at first, because enzymes and substrates meet more often. Eventually every active site is busy almost all the time: the enzyme is saturated and the rate levels off at a maximum, Vmax. The substrate concentration that gives half of Vmax is called Km. A low Km means the enzyme reaches half speed at low substrate concentration: it binds its substrate tightly (high affinity).

The turnover number tells how many substrate molecules one enzyme molecule handles per second at Vmax: from a few per second for some enzymes to around a million for carbonic anhydrase.