Enzymes & kinetics

What a catalyst does, and what it doesn't

Almost every reaction in your cells is run by an enzyme, a biological catalyst. A catalyst speeds up a reaction without being used up itself. Most enzymes are proteins, although a few are made of RNA.

Even a reaction that releases energy has to get over a hump first. Old bonds must be stretched and partly broken before new ones can form, and that costs energy. This hump is the activation energy (Eₐ). At body temperature, only a tiny fraction of molecules collide with enough energy to get over it, so many reactions are painfully slow on their own.

An enzyme lowers the activation energy by giving the reaction an easier route. It does not change where the reaction starts or ends. The energy of the reactants and products stays the same, so the overall free-energy change, ΔG, stays the same too. An enzyme can't make an uphill (endergonic) reaction go on its own.

The active site: where the work happens

The molecule an enzyme acts on is its substrate. It binds in a pocket or groove called the active site, formed where the folded protein chain brings particular amino acids close together. The shape and chemistry of the active site mean that each enzyme binds only one substrate, or a few similar ones. That's enzyme specificity.

The old "lock and key" picture suggests a rigid fit. In reality the active site changes shape slightly as the substrate binds, gripping it more tightly. This is the induced fit model. Once bound, the enzyme lowers the activation energy in several ways:

• Holding two substrates close together and in the right orientation, so they don't have to meet by chance.
• Straining or bending the substrate's bonds toward the shape they have at the top of the hump (the transition state).
• Providing a small chemical environment, for example acidic or charged side chains, that the reaction needs.
• Briefly forming a bond with the substrate, which is undone before the product leaves.

Many enzymes also need a helper. Inorganic ions such as Mg²⁺ or Zn²⁺ are called cofactors; organic helpers, often made from vitamins, are called coenzymes. When the product leaves, the enzyme is back in its original form, ready for the next substrate.

More substrate, faster reaction, up to a limit

Imagine a fixed amount of enzyme and you keep adding substrate. At first, the rate rises almost in step with the substrate, because most active sites are empty and each new substrate molecule finds one easily.

Eventually, nearly every active site is busy all the time. Adding more substrate can't help, because the enzymes are working as fast as they can. The curve levels off at the maximum rate, Vmax. The enzyme is said to be saturated. The only way to go faster now is to add more enzyme.

Temperature and pH: why the curves have peaks

Plot rate against temperature and you get a lopsided hill. On the left, warming speeds things up, because molecules move faster and collide more often and more forcefully. On the right, the rate crashes. Too much heat makes the protein chain vibrate so hard that the hydrogen bonds and other weak interactions holding its shape break. The active site loses its shape: the enzyme is denatured. The peak is the optimum temperature.

Cold is different. A cold enzyme is still correctly folded; its molecules just collide less often, so it works slowly. Warm it up and the activity comes back. Heat denaturation, on the other hand, usually can't be undone.

The optimum reflects where an organism lives. Most human enzymes work best near 37 °C. The DNA polymerase of Thermus aquaticus, a bacterium from hot springs, works fastest at about 75–80 °C, which is why it's used in PCR.

pH gives a similar hill, but for a different reason. Changing the concentration of H⁺ adds or removes protons from charged side chains in and around the active site. That changes the charges the substrate relies on and, at extremes, disrupts the ionic bonds that hold the fold. Pepsin, which digests proteins in your acidic stomach, works best at a pH of about 1.5–2, while the digestive enzymes released into your slightly alkaline small intestine work best at a higher pH.

Inhibitors: competing for the site, or changing its shape

An inhibitor is a molecule that slows an enzyme down. Two main kinds behave very differently, and the easiest way to tell them apart is to flood the enzyme with substrate.

A competitive inhibitor looks enough like the substrate to sit in the active site, but it can't be turned into product. While it's there, the substrate can't bind. It's a contest for the same seat. Add lots of substrate and the substrate wins most of the contests, so the enzyme can still reach nearly the same Vmax. You just need more substrate to get there.

A noncompetitive inhibitor binds somewhere else on the enzyme and changes its shape so that the active site works poorly, whether or not the substrate is bound. Adding more substrate doesn't help, because the substrate isn't competing with anything. It effectively takes some enzyme molecules out of action, so Vmax falls.

Switching enzymes on and off

Cells don't just put up with inhibitors; they use them as switches. Many key enzymes have a second binding site, away from the active site, called an allosteric site. A molecule binding there changes the enzyme's shape and makes it more or less active. That's allosteric regulation. Allosteric enzymes often have several subunits and give an S-shaped (sigmoidal) rate curve rather than a smooth hill.

A common pattern is feedback inhibition: the final product of a pathway binds an allosteric site on an enzyme near the start and slows it down. When the product piles up, the pathway slows; when it's used up, the brake comes off. The cell makes only what it needs.

An example you'll meet in glycolysis is phosphofructokinase-1 (PFK-1). ATP is one of its substrates, but ATP also binds an allosteric site that slows the enzyme. AMP, which builds up when the cell is short of energy, speeds it up. So glycolysis runs fast when energy is low and slows when ATP is plentiful.

Key terms

Enzyme

A biological catalyst, usually a protein, that speeds up a reaction without being used up.

Activation energy (Eₐ)

The energy needed to reach the transition state, the hump every reaction must get over.

Active site

The pocket on an enzyme where the substrate binds and the reaction is catalysed.

Induced fit

The slight change in an enzyme's shape as its substrate binds, which tightens the fit.

Vmax / saturation

The maximum rate, reached when nearly every active site is occupied all the time.

Denaturation

Loss of a protein's folded shape, for example from high temperature or extreme pH, which destroys the active site.

Competitive inhibitor

A molecule that competes with the substrate for the active site; overcome by adding more substrate.

Noncompetitive inhibitor

A molecule that binds elsewhere on the enzyme and lowers Vmax, whatever the substrate concentration.

Allosteric regulation / feedback inhibition

Control of an enzyme by a molecule binding away from the active site / a pathway's end product inhibiting an early enzyme.

Check yourself

Try answering in your head before you open each answer.

• 1.A reaction mixture has already reached equilibrium. You add a large amount of the enzyme that catalyses the reaction. What happens to the amounts of reactant and product?Show answerHide

  Nothing changes. The enzyme speeds up the forward and reverse reactions equally, so it can't shift the equilibrium. It only helps a mixture get to equilibrium faster, and this one is already there.

• 2.Sample A of an enzyme is kept at 4 °C for an hour; sample B is heated to 90 °C for an hour. Both are then tested at 37 °C. Predict the results.Show answerHide

  Sample A should work normally: cold only slowed its molecules, and its fold was intact. Sample B will probably show little or no activity: the heat unfolded (denatured) the protein, and that usually isn't reversed by cooling.

• 3.Methotrexate, a drug used against some cancers, closely resembles the normal substrate of the enzyme it blocks. What kind of inhibitor is it likely to be, and what would happen if the substrate concentration in the cell rose sharply?Show answerHide

  Because it mimics the substrate, it most likely competes for the active site, so it's a competitive inhibitor. A big rise in substrate would out-compete it and restore much of the enzyme's activity, weakening the drug's effect.