Lesson 3 of 5Biochemistry

Unit 1 · Lesson 3 of 5

Enzymes: Structure, Mechanism and Regulation

How enzymes lower activation energy, the active site and substrate-enzyme complex, lock-and-key versus induced fit, cofactors and coenzymes, the effects of temperature, pH and concentration, competitive, non-competitive and allosteric regulation, feedback inhibition, planning a catalase investigation, and enzymes in food, medicine and industry.

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Key things to remember

1Enzymes lower activation energy ( $E_{a}$ ); they do not change $Δ G$ and are not used up.
2 $E + S \to ES \to E + P$ . Induced fit: binding changes the active site's shape, straining the substrate's bonds.
3Rate rises with temperature up to an optimum, then falls as the enzyme denatures; each enzyme has an optimum pH.
4Increasing [S] raises the rate until the enzyme is saturated at $V_{ma x}$ ; increasing [E] raises $V_{ma x}$ when substrate is in excess.
5Competitive inhibitors block the active site (overcome by more substrate). Non-competitive inhibitors bind elsewhere and lower $V_{ma x}$ .
6Feedback inhibition: a pathway's end product allosterically inhibits its first enzyme.

Hydrogen peroxide ( $H_{2} O_{2}$ ) is a toxic by-product of normal metabolism. On its own it breaks down into water and oxygen so slowly that it would build up and poison your cells. Yet drop a piece of raw liver or potato into it and it fizzes instantly. The liver contains catalase, an enzyme that breaks down tens of millions of peroxide molecules per second. Almost every reaction in a cell is like this: possible, but far too slow without help. Enzymes are biological catalysts, mostly proteins, that speed up reactions enormously without being used up. Because each enzyme is shaped for one job, cells control their chemistry by controlling which enzymes are present and switched on. This topic explains how enzymes work, what slows them down, and how cells and industries put them to use.

Activation energy and catalysis

Fig 1A catalyst lowers the activation energy barrier but does not change the energy of the reactants or products, so the overall energy change is the same.

Even a reaction that releases energy must first get over a hump: bonds have to be strained or broken before new ones form. That energy barrier is the activation energy ( $E_{a}$ ). At body temperature only a tiny fraction of molecules collide with enough energy to get over it. An enzyme provides a different route with a lower $E_{a}$ , so far more molecules react each second. It does this by holding substrates in the correct orientation, straining the bonds that need to break, providing a favourable microenvironment (for example an acidic or nonpolar pocket), or briefly bonding to the substrate itself. Three things an enzyme does not do: it does not change the overall energy released or absorbed ( $Δ G$ ), it does not make an impossible reaction possible, and it is not consumed, so one enzyme molecule can be used over and over. Enzyme names usually end in -ase and often name the substrate: sucrase, lipase, DNA polymerase.

The active site, induced fit and helpers

Fig 2Induced fit: the substrate enters the active site, the enzyme changes shape to grip it and form the substrate-enzyme complex, then the products are released and the enzyme returns to its original shape.

Substrate: the molecule an enzyme acts on. Active site: the pocket or groove, formed by a few R groups of the folded protein, where the substrate binds and the reaction happens. Its shape and chemistry are why enzymes are specific.
Substrate-enzyme complex (enzyme-substrate complex): the temporary combination formed when substrate binds the active site. The overall cycle is $E + S \to ES \to E + P$ .
Lock-and-key model (Emil Fischer, 1894): the active site is a rigid shape that fits the substrate exactly, like a key in a lock. It explains specificity but not how bonds get strained.
Induced-fit model (Daniel Koshland, 1958): the active site is flexible. Binding causes the enzyme to change shape and close around the substrate, which strains its bonds and lowers $E_{a}$ . This is the model supported by modern evidence.
Cofactors: non-protein helpers that some enzymes need. Inorganic cofactors are metal ions such as $Zn^{2 +}$ , $Mg^{2 +}$ and $Fe^{2 +}$ .
Coenzymes: organic cofactors, many made from vitamins. $NAD^{+}$ comes from niacin (vitamin B3), FAD from riboflavin (B2) and coenzyme A from pantothenic acid (B5). Coenzymes often carry electrons or chemical groups between reactions, which is why vitamin deficiencies disrupt metabolism.

Factors that affect the rate of reaction

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Change temperature, pH and substrate concentration, then add a competitive or non-competitive inhibitor and compare the rate curves.

Factor	Effect on rate	Why
Temperature	Rises (roughly doubling every 10 °C) up to an optimum, then falls sharply	Warmer molecules collide more often and with more energy. Above the optimum, vibrations break the weak bonds holding the active site's shape and the enzyme denatures. Most human enzymes peak near 37 °C; Taq polymerase from hot-spring bacteria peaks near 75 °C.
pH	Highest at an optimum pH, lower on either side	$H^{+}$ and $OH^{-}$ change the charges on R groups, disrupting ionic and hydrogen bonds in the active site. Pepsin (stomach) works best near pH 2, salivary amylase near 7 and trypsin (small intestine) near 8.
Substrate concentration	Rises, then levels off at a maximum rate ( $V_{ma x}$ )	More substrate means more collisions with active sites, until every active site is busy. The enzyme is then saturated, and adding more substrate cannot help.
Enzyme concentration	Rises in proportion, as long as substrate is in excess	More enzyme means more active sites working at once, so doubling the enzyme doubles the maximum rate.

Low temperatures slow enzymes reversibly; high temperatures and extreme pH usually denature them permanently.

Inhibition and regulation

Fig 3A competitive inhibitor blocks the active site; a non-competitive inhibitor binds elsewhere and changes the active site's shape.

Type	How it works	Can more substrate overcome it?	Example
Competitive inhibition	An inhibitor shaped like the substrate occupies the active site, so substrate cannot bind while it is there	Yes: at high [S] the substrate outcompetes the inhibitor, so $V_{ma x}$ can still be reached	Statin drugs block the enzyme HMG-CoA reductase to lower cholesterol production
Non-competitive inhibition	The inhibitor binds to a different site on the enzyme, changing its shape so the active site works poorly or not at all	No: the inhibited enzymes are effectively removed, so $V_{ma x}$ drops	Heavy metal ions such as $Hg^{2 +}$ and $Pb^{2 +}$ bind sulfhydryl groups and distort enzymes
Allosteric regulation	A molecule binds to an allosteric site (a regulatory site away from the active site). An allosteric activator stabilizes the active shape; an allosteric inhibitor stabilizes the inactive shape	Not by itself; the regulator controls the enzyme like a switch	ATP is an allosteric inhibitor of phosphofructokinase, an early enzyme in glycolysis
Feedback inhibition	The end product of a pathway allosterically inhibits the pathway's first enzyme, so the product shuts down its own production when it is plentiful	Rate recovers when the end product is used up and leaves the allosteric site	Isoleucine inhibits threonine deaminase, the first enzyme of the pathway that makes isoleucine from threonine

Most inhibition is reversible. Irreversible inhibitors, such as penicillin (bacterial cell-wall enzyme) and aspirin (the COX enzyme), bond covalently to the enzyme.

Planning an investigation and using enzymes in technology

Question and hypothesis. How does temperature (or pH, or $H_{2} O_{2}$ concentration) affect the rate of catalase activity? Predict with reasons, e.g. rate rises to an optimum near body temperature, then falls as catalase denatures.
Variables. Independent: the factor you change, with at least five values (e.g. 0, 20, 37, 50, 70 °C). Dependent: the rate, measured as volume of $O_{2}$ per unit time. Controlled: mass and surface area of potato or liver, volume and concentration of $H_{2} O_{2}$ , pH, time allowed.
Method. Catalase runs $2 H_{2} O_{2} \to 2 H_{2} O + O_{2}$ . Collect the $O_{2}$ in a gas syringe or an inverted water-filled graduated cylinder, or time how long a catalase-soaked filter paper disc takes to rise in peroxide. Equilibrate the enzyme and substrate at each temperature before mixing.
Controls and reliability. Use boiled potato as a negative control (denatured catalase should give no gas). Run at least three trials per condition and average them.
Safety. Wear goggles: concentrated hydrogen peroxide irritates eyes and skin. Handle hot water baths and sharp cork borers carefully.
Analysis. Calculate rate = volume ÷ time, graph rate against the independent variable, and identify the optimum and any sources of error (such as gas escaping before the stopper is in).

Application	Enzyme	What it does
Cheese making	Rennet (chymosin)	Cuts the milk protein casein so milk curdles into curds and whey. Once taken only from calves' stomachs, most chymosin today is made by genetically modified microbes.
Breadmaking	Amylase	Breaks flour starch into maltose that yeast can ferment, giving more $CO_{2}$ for a better rise and more sugar for a browner crust.
Lactose intolerance	Lactase supplements	Hydrolyze lactose into glucose and galactose. Without lactase, undigested lactose reaches the colon, where bacteria ferment it and cause gas, bloating and cramps.
Digestive disorders	Pancreatic enzyme supplements (lipase, protease, amylase)	Taken with meals by people with cystic fibrosis or pancreatic insufficiency, whose pancreas cannot deliver enough enzymes to digest food.
Laundry and dish detergents	Proteases, lipases, amylases	Digest protein, grease and starch stains; engineered versions work in cold water, which saves energy.
Pharmaceuticals	Enzymes as drugs and as drug targets	Clot-dissolving enzymes treat strokes and heart attacks; many drugs are enzyme inhibitors, such as statins and HIV protease inhibitors.

Enzymes suit industry because they are specific, work at mild temperatures and are biodegradable.

Worked examples

Reveal one step at a time and try to beat it to the next line.

Example 1

In a catalase investigation at 37 °C, three trials collected 27.0 mL, 25.5 mL and 26.1 mL of oxygen in 60.0 s. Calculate the average rate in mL/s to three significant figures, and explain why the investigator used a boiled potato core as well.

Example 2

An enzyme is tested with and without an inhibitor at increasing substrate concentrations. At low [S] both rates are low. At very high [S] the inhibited rate reaches the same maximum as the uninhibited rate. What type of inhibitor is it, and why?

Example 3

A bacterium makes the amino acid isoleucine from threonine in five enzyme-catalyzed steps. When isoleucine is added to the growth medium, the bacterium stops converting threonine. Explain the mechanism and why it benefits the cell.

Where marks get lost

Saying enzymes "die" at high temperatures. Enzymes are molecules, not living things; they denature, meaning they lose their 3D shape.
Saying an enzyme provides the activation energy. It lowers the activation energy by giving the reaction a different pathway; it does not supply energy or change $Δ G$ .
Thinking cold temperatures denature enzymes. Cold slows collisions, but the shape survives, so activity returns on warming; heat denaturation is usually permanent.
Mixing up the two inhibitors. Competitive inhibitors compete for the active site and can be outcompeted by more substrate; non-competitive inhibitors bind elsewhere and lower $V_{ma x}$ whatever the [S].
Explaining the plateau at high [S] as the enzyme being used up. The enzyme is saturated: every active site is busy, so the rate is limited by how fast each enzyme can turn substrate over.

Flashcards 9

Activation energy

Active site

Substrate-enzyme complex

Induced fit

+ 5 more

Practice

Which statement about an enzyme-catalyzed reaction is correct?

Put the steps of an induced-fit catalytic cycle in order.

1Products are released and the enzyme is ready again
2Substrate is converted into products
3Substrate-enzyme complex forms and bonds are strained
4Substrate collides with the active site
5Active site changes shape to grip the substrate

A catalase reaction produces 18.0 mL of oxygen in 45.0 s. What is the average rate of oxygen production in mL/s? Give three significant figures.

mL/s

Below its optimum, an enzyme's rate roughly doubles for every 10 °C rise. If the rate is 5.0 units at 15 °C, estimate the rate at 35 °C (still below the optimum).

units

Adding more substrate does not increase the rate of an inhibited reaction, and the maximum rate is lower than without the inhibitor. What kind of inhibitor is present?

Match each technological application to the enzyme it uses.

Curdling milk to make cheese

Helping yeast make bread rise

Letting lactose-intolerant people digest dairy

Removing grease stains in cold-water laundry

What term describes the process in which the end product of a metabolic pathway inhibits the first enzyme of that pathway?

Pepsin works in the stomach at pH 2. What happens to its activity when it moves into the small intestine at pH 8?

Name the regulatory site on an enzyme, away from the active site, where activators or inhibitors bind to change the enzyme's shape.

A student tests catalase at five temperatures but uses a different-sized piece of potato each time. What is the main problem with this design?

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