Proteins

In a nutshell

Proteins are the workhorses of the cell: they build structures, carry substances, defend the body and, as enzymes, catalyse almost every reaction in a living organism.

This subtopic is about how amino acids join into polypeptides, how a polypeptide folds into a working 3-D shape, and how that shape lets an enzyme speed up a reaction. Everything an enzyme does comes back to its structure.

Assumed knowledge: Monomers and polymers.

Core content

Amino acids: the monomers of proteins

Amino acids are the monomers from which proteins are made. All twenty amino acids that are common in all organisms share the same general structure and differ only in their R group (their side chain).

Every amino acid has a central carbon atom bonded to four things:

• an amine group, NH₂
• a carboxyl group, COOH
• a hydrogen atom, H
• a variable R group (the side chain that differs between amino acids)

Amino acids contain the elements carbon, hydrogen, oxygen and nitrogen; two of the twenty also contain sulfur in their R group, which matters later for disulfide bridges.

The peptide bond

Two amino acids join by a condensation reaction: a bond forms and a molecule of water is released.

The bond formed between them is a peptide bond, and it forms between the carboxyl group of one amino acid and the amine group of the next.

• Two amino acids joined this way form a dipeptide.
• Many amino acids joined this way form a polypeptide.

Adding water back breaks the peptide bond: this is hydrolysis. So proteins are built by condensation and digested by hydrolysis.

A functional protein may contain one or more polypeptides.

The four levels of protein structure

A protein's shape is described at four levels. Each higher level depends on the one below it, and the final shape is what gives the protein its function.

The key exam idea: the primary structure (sequence of amino acids) determines the final 3-D shape, because the sequence decides where the R groups are and therefore where the bonds that hold the tertiary structure can form. Change one amino acid and you can change the whole shape, and so the function.

Because their shapes differ, proteins have a huge variety of functions: enzymes, structural proteins (collagen), transport proteins (haemoglobin), antibodies and hormones (insulin).

The bonds that hold a protein together

Three kinds of bond hold the tertiary and quaternary structure, and you must be able to name them:

• Hydrogen bonds between R groups: many but individually weak.
• Ionic bonds between oppositely charged R groups: stronger than hydrogen bonds.
• Disulfide bridges between the sulfur-containing R groups of two cysteines: strong covalent bonds that do not break easily.

The peptide bonds of the primary structure are separate from these: they are not what holds the tertiary structure together, and they are not the bonds that break during denaturation.

Required practical skill: the biuret test for proteins

The biuret test detects the presence of protein (specifically, peptide bonds).

• Add biuret reagent (sodium hydroxide solution followed by copper(II) sulfate solution) to the sample.
• A positive result is a colour change from blue to purple (lilac/violet).
• A negative result stays blue.
• No heating is needed for this test.

Many proteins are enzymes

An enzyme is a biological catalyst: it speeds up the rate of a reaction without being used up, and it does so by lowering the activation energy of the reaction it catalyses.

Enzymes are globular proteins. Each has an active site, a region whose shape comes from the enzyme's tertiary structure. Only a substrate with a complementary shape can bind to the active site to form an enzyme-substrate complex. This is why enzymes are specific: one enzyme's active site fits one substrate (or a small group of similar substrates).

The induced-fit model

The modern model of how an enzyme works is the induced-fit model:

• The active site is not an exact fit for the substrate to begin with.
• As the substrate binds, the active site changes shape to mould around it, becoming complementary.
• This change puts a strain on the bonds in the substrate, which is what lowers the activation energy.

An earlier idea, the lock-and-key model, treated the active site as a fixed, exact fit. Induced fit replaced it because it better explains how binding actually lowers activation energy. (You should appreciate that models of enzyme action have changed over time.)

Still don't get it? · how an enzyme lowers activation energy

Think of activation energy as a wall a reaction has to get over before it can happen. A reaction that would eventually go on its own is stuck behind a high wall, so it happens very slowly. Lowering the wall does not change what is on the other side, it just lets far more molecules get over, so the reaction goes faster.

Now build it up properly. Molecules only react when they collide with enough energy and in the right way. The enzyme's active site grabs the substrate and, as it moulds around it (induced fit), it bends and strains the substrate's own bonds. Those bonds are now closer to breaking, so less extra energy is needed to make the reaction happen. It also holds the reacting molecules together in the right orientation.

Back to the exam wording: the enzyme forms an enzyme-substrate complex and, by straining the bonds in the substrate, it lowers the activation energy of the reaction, so the rate increases. The examiner's mark is not for saying "it lowers activation energy" alone; it is for saying how, via the strain the induced fit puts on the substrate.

Factors affecting the rate of enzyme-controlled reactions

Five factors change the rate. For each, the story comes back to how many enzyme-substrate complexes form.

Enzyme concentration. More enzyme means more active sites available, so more enzyme-substrate complexes form and the rate rises. If substrate is limited, adding more enzyme eventually has no effect, because substrate concentration becomes the limiting factor.

Substrate concentration. More substrate means more collisions with active sites, so more complexes form and the rate rises. Once every active site is occupied, the enzyme is saturated: adding more substrate cannot increase the rate, which levels off at its maximum (V_max).

Temperature. As temperature rises, enzyme and substrate molecules gain kinetic energy, move faster and collide more often, so the rate rises up to the optimum temperature. Above the optimum, the extra vibration breaks the hydrogen and ionic bonds holding the tertiary structure; the enzyme denatures, its active site changes shape and is no longer complementary to the substrate, so fewer (then no) enzyme-substrate complexes form and the rate falls.

pH. Each enzyme has an optimum pH. Moving away from it, the excess H⁺ or OH^- ions disrupt the hydrogen and ionic bonds in the tertiary structure. The active site changes shape and is no longer complementary, so the rate falls; at extreme pH the enzyme denatures.

Inhibitor concentration. Inhibitors reduce the rate by lowering the number of enzyme-substrate complexes that form. There are two types, covered next.

Competitive and non-competitive inhibitors

Still don't get it? · why more substrate beats a competitive but not a non-competitive inhibitor

Picture the active site as a single parking space and the substrate and a competitive inhibitor as two cars fighting for it. If you flood the car park with substrate cars, they win most of the space just by outnumbering the inhibitor, so almost every enzyme gets used. That is why adding substrate lets a competitively-inhibited reaction climb back up to its normal maximum rate.

A non-competitive inhibitor is not fighting for the parking space at all. It parks somewhere else on the enzyme and bends the whole structure, so the parking space itself is now the wrong shape. Adding more substrate cars does not help, because the space is broken no matter how many cars arrive.

In exam terms: a competitive inhibitor binds to the active site, so raising substrate concentration means substrate outcompetes it and the rate returns towards Vmax. A non-competitive inhibitor binds away from the active site and changes the tertiary structure (and so the shape of the active site), so more substrate cannot restore the rate and Vmax is not reached.

Worked examples

Example 1 - finding the initial rate from a tangent (a data skill).

The graph shows the volume of oxygen given off as catalase breaks down hydrogen peroxide. The rate is fastest at the start and slows as substrate is used up, so we measure the initial rate by drawing a tangent at t = 0 and finding its gradient.

Read two points on the tangent: (0 s, 0 cm³) and (20 s, 44 cm³).

$\text{initial rate} = \frac{\Delta y}{\Delta x} = \frac{44 - 0}{20 - 0} = 2.2 \ \text{cm}^3\,\text{s}^{-1}$

The final answer must carry its units (cm³ s^-1); the units are worth their own mark.

Example 2 - a model 5-mark answer: "Explain why the rate of an enzyme-controlled reaction decreases above the optimum temperature."

A full-mark answer is a causal chain, each link a separate marking point, in order:

1. Above the optimum, the enzyme molecules vibrate more (they have more kinetic energy).
2. This breaks the hydrogen bonds and ionic bonds holding the tertiary structure.
3. The tertiary structure changes, so the shape of the active site changes (the enzyme is denatured).
4. The active site is no longer complementary to the substrate.
5. So fewer enzyme-substrate complexes form, and the rate decreases.

Notice this is five linked points, not one: a "5-mark explain" wants five distinct ideas in a sequence.

Common exam mistakes

• Writing that a change (heat or pH) "breaks the bonds" without saying which bonds: the mark needs hydrogen and ionic bonds. Do not say peptide or disulfide bonds break during denaturation, they do not.
• Saying denaturation "changes the shape of the enzyme": too vague. The mark needs the tertiary structure changes, so the shape of the active site changes and it is no longer complementary to the substrate.
• Giving a non-enzyme protein (a receptor, an antibody, a channel protein) an "active site". Not all proteins are enzymes; only enzymes have active sites. This loses the mark.
• Describing induced fit as the substrate having "the same shape" as the active site. It must be complementary, and the active site changes shape as the substrate binds.
• Thinking induced fit lets one enzyme change its active site to fit any substrate. Enzymes stay specific; the active site moulds around its substrate only.
• Saying an enzyme "lowers activation energy" but not how. For the full mark, say it forms an enzyme-substrate complex and strains the bonds in the substrate.
• Defining quaternary structure as "four polypeptide chains". It is two or more (more than one) polypeptide chains; four is only true for haemoglobin.
• Reading the biuret result backwards: purple means protein is present, blue means it is absent. Also, biuret needs no heating (unlike the Benedict's test).
• Confusing condensation (joins molecules, releases water) with hydrolysis (breaks the bond, uses water).
• Saying a mutation makes "different amino acids". A changed base sequence changes which amino acid is placed in the sequence, not the amino acids themselves.

Key definitions

• Condensation reaction: a reaction that joins two molecules together with the formation of a chemical bond and the elimination of a molecule of water.
• Hydrolysis: the breaking of a chemical bond between two molecules involving the use of a water molecule.
• Peptide bond: the bond formed between two amino acids in a condensation reaction (between the carboxyl group of one and the amine group of the next).
• Primary structure: the sequence of amino acids in a polypeptide chain.
• Secondary structure: the coiling of the polypeptide into an α-helix or folding into a β-pleated sheet, held by hydrogen bonds.
• Tertiary structure: the overall 3-D shape of a polypeptide, held by hydrogen bonds, ionic bonds and disulfide bridges between R groups.
• Quaternary structure: a protein made of more than one polypeptide chain.
• Enzyme: a biological catalyst.
• Activation energy: the minimum amount of energy needed for a reaction to take place.
• Induced fit: the active site changes shape as the substrate binds so that it becomes complementary to the substrate.
• Enzyme-substrate complex: the structure formed when a substrate binds to the active site of an enzyme.
• Competitive inhibitor: a molecule with a similar shape to the substrate that binds to the active site of the enzyme.
• Non-competitive inhibitor: a molecule that binds to the enzyme at a site other than the active site, changing the shape of the active site.

Specification

• I can draw and label the general structure of an amino acid (amine group, carboxyl group, R group) and state that the twenty common amino acids differ only in their R group.
• I can describe the condensation of two amino acids to form a dipeptide and a peptide bond, and the hydrolysis of that bond.
• I can state that a functional protein may contain one or more polypeptides.
• I can describe the role of hydrogen bonds, ionic bonds and disulfide bridges in protein structure.
• I can explain the relationship between the primary, secondary, tertiary and quaternary structure of a protein and its function.
• I can describe the biuret test for proteins and its positive result.
• I can state that each enzyme lowers the activation energy of the reaction it catalyses.
• I can describe the induced-fit model of enzyme action, including the enzyme-substrate complex and enzyme specificity.
• I can explain how enzyme concentration, substrate concentration, competitive and non-competitive inhibitors, pH and temperature affect the rate of an enzyme-controlled reaction.

Related notes

• Monomers and polymers
• Lipids
• Nucleic acids
• Transport across cell membranes