Enzymes

1. What are Enzymes?

• Biological catalyst = speeds up a reaction but remains unchanged at the end of it.
• Lowers the activation energy required by providing an alternate pathway.
• All enzymes are globular proteins with a tertiary structure (3^rd level of folding ⇒ 3D shape).
• Almost all metabolic reactions in living organisms are catalysed by enzymes ⇒ enzymes are essential for life.

Metabolism = sum of all chemical reactions going on in the body

Intracellular vs Extracellular enzymes

All enzymes are produced within a cell, but not all work inside it — some are secreted outside.

2. Properties of Enzymes

1. Globular proteins (soluble).
2. All have a tertiary structure ⇒ that is why they have an active site.
3. Sensitive to changes in external factors (e.g. temperature & pH) — because they are globular proteins.
4. Specific (to their substrate) because of the unique & complementary shape of the active site.
5. Efficient — a small amount of enzyme can catalyse a large number of reactions.
6. Lower the activation energy of a reaction by providing an alternate pathway.

Why “efficient”? Enzymes remain unchanged. One substrate binds → leaves as product → the next one binds → … so one enzyme molecule handles many substrates.

Enzyme activity & the factors affecting it

Enzyme activity = how well the enzyme catalyses its metabolic reaction (= rate of reaction).

#	Factor	Notation / note
1	Temperature	—
2	Amount / concentration of substrate	[S]
3	pH	power of H⁺
4	Amount / concentration of enzyme	[E]
5	Concentration of inhibitor	anything that holds back / halts the enzyme
6	Coenzymes / cofactors	(own sub-topic — see §9)

3. Mode of Action of Enzymes

Two models: (1) Lock & Key hypothesis and (2) Induced Fit hypothesis — both say the substrate binds the enzyme's active site.

Because of the tertiary structure (protein folding) an enzyme has a 3D shape. The active site is a hollow depression in the enzyme with a specific shape where another molecule (the substrate) can bind.

① Lock & Key Hypothesis

• Substrate = KEY, fits perfectly in the complementary active site ⇒ enzyme (active site) = LOCK. MCQ trap: which is lock, which is key?
• Held by weak hydrogen bonds ⇒ forms the enzyme–substrate (E–S) complex.
• Enzyme catalyses by lowering the activation energy ⇒ enzyme–product complex ⇒ products leave the active site.
• Like keys & locks: every key can’t open every lock ⇒ that is why enzymes are specific. (Old idea, 1800s.)

② Induced Fit Hypothesis

• Change in the active site shape = conformational change — induced by the substrate (the enzyme is “forced” to adjust).
• Held by weak hydrogen bonds ⇒ E–S complex ⇒ catalysis by lowering E_A ⇒ products leave, enzyme unchanged.

Dates (sir's recollection): lock & key came first (1800s); induced fit later (~1949). Induced fit is why a slightly changed active site can still bind the substrate.

	Lock & Key	Induced Fit
Active site vs substrate	complementary (exact)	NOT complementary at first
Active site	rigid	moulds around substrate
Common to both	weak H-bonds hold E–S complex · EA lowered via alternate pathway · enzyme unchanged at end

4. Activation Energy & How Enzymes Lower It

• Frequency of collisions = number of collisions per unit time.
• Frequency of EFFECTIVE collisions ★ = collisions that result in a chemical reaction (product formation).

Why do we give heat? Heat is given as E_A so reactant molecules gain kinetic energy, come close to one another and collide effectively & frequently.

Bound in the active site, substrates are aligned ⇒ effective collisions are much more likely ⇒ an alternate pathway with lower E_A ⇒ a relatively faster reaction (less energy and less time needed).

Forming products: the substrate sitting in the active site is put under strain (pressure) ⇒ it breaks off into products.

5. Affinity, V_max and K_m

• Greater affinity for substrate ⇒ faster enzymatic activity.
• Higher affinity ⇒ more likely that product will form in the presence of substrate.

V_max = rate of reaction at which the enzyme shows its highest turnover. Turnover value = rate at which an enzyme converts its substrate into products. (Max rate / max velocity of the reaction.)

• Look at the substrate amount at ½V_max — that [S] is K_m.
• K_m is used as a measure of the efficiency AND affinity of an enzyme for its substrate.

A has the lower K_m (4 < 6) ⇒ A needs less substrate to get going ⇒ higher affinity / more efficient than B.

6. Factors Affecting the Rate of Enzyme Activity

Always recall: enzymes = globular proteins ⇒ sensitive to changes in temperature & pH. Their 3D active-site shape is held by 4 tertiary bonds (H-bonds, hydrophobic, ionic, disulfide).

① Temperature

Describe: ① As temperature increases, the rate of reaction increases until the optimum temperature (V_max). ② Above the optimum temperature the rate of reaction decreases.

Explain:

1. As temperature ↑ ⇒ kinetic energy ↑ ⇒ frequency of effective collisions ↑ ⇒ E–S complexes formed more frequently ⇒ rate of reaction ↑ until V_max. (Same enzymes & substrates were already there — they just move/vibrate more and collide more.)
2. Beyond the optimum: the enzyme / active site begins to lose its 3D (tertiary) structure because the hydrogen bonds, hydrophobic interactions and ionic bonds break down ⇒ specific shape of the active site lost ⇒ fewer E–S complexes ⇒ rate ↓.

② pH (= power of H⁺)

Explain: enzymes are sensitive to pH changes: they lose their tertiary structure at pH values other than optimum ⇒ fewer E–S complexes ⇒ the reaction recedes to zero.

Why? (changed R groups): pH tells the amount of H⁺ ions. Low pH = many H⁺; high pH (alkaline) = few. H⁺ is positive ⇒ it is attracted to negatively charged R groups and bonds with them ⇒ the normal tertiary bonding between R groups changes ⇒ specific 3D structure lost.

③ Substrate concentration [S]

Describe: ① Initially the rate of enzyme activity increases as [S] increases. ② At V_max, increasing [S] is not affecting / not increasing the rate of reaction.

1. Explain ①: at low [S], [S] is the limiting factor; increasing it ⇒ frequency of effective collisions ↑ ⇒ more E–S complexes.
2. Explain ②: at a certain [S] activity reaches V_max. All enzymes are saturated (all active sites occupied), so [E] is now the limiting factor ⇒ more [S] no longer ↑ the rate.

④ Enzyme concentration [E]

No enzyme ⇒ no catalysis, however much substrate there is. Two graphs exist:

Fixed [S] — Describe: ① ↑[E] increases the rate until V_max is reached. ② Increasing [E] after V_max has no effect — the rate stays constant (say “constant”, NOT “stops” — it doesn't fall to zero).

1. Explain ①: at low [E], [E] is the limiting factor; ↑[E] ⇒ frequency of collisions ↑ ⇒ more E–S complexes formed.
2. Explain ②: at a certain [E] the rate becomes constant and now [S] is the limiting factor (substrate used up — fixed amount at the start).

7. Enzyme Inhibitors

	Competitive inhibitor	Non-competitive inhibitor
Shape	structurally similar to substrate (not identical)	not similar to the substrate
Binds to	the active site — competes with substrate for it	the allosteric site — no competition for the active site
Effect	substrate & inhibitor compete	binding changes (conformational change) the active-site shape ⇒ substrate can't attach ⇒ no E–S complex
Reversible?	Reversible: ↑[S] ⇒ substrate wins the site	Usually called non-reversible: ↑[S] does not help (reverses only if the inhibitor detaches)
Km / affinity	Km ↑ ⇒ affinity ↓	Km unchanged
Vmax	remains the same	decreases

Why does K_m ↑ (competitive)? The enzyme mistakes the inhibitor for the substrate, so its attraction (affinity) is divided between the two ⇒ affinity ↓ ⇒ more substrate is needed to reach ½V_max ⇒ K_m ↑. V_max same because enough substrate can still push every active site to work.

Why K_m same but V_max ↓ (non-competitive)? Inhibitor and substrate share no similarity, so the enzyme's attraction to the substrate is not divided ⇒ affinity & K_m unchanged. But enzymes with the inhibitor bound can never form E–S complexes, however much [S] ⇒ the maximum rate falls ⇒ V_max ↓.

Importance of enzyme inhibitors

1. Antibiotics inhibit bacterial enzymes.
2. They control the rate of metabolic reactions.
3. Pesticides & herbicides contain inhibitors.

End-product inhibition (= feedback inhibition)

A metabolic pathway is a chain: products of enzyme A are the substrate of enzyme B, whose products are the substrate of enzyme C …

• The final product binds an allosteric site on the early-stage enzyme ⇒ its active-site shape changes ⇒ substrate 1 can no longer bind ⇒ entire metabolic pathway is halted (all later steps depend on the first).
• Reduces the rate of the pathway; used to regulate a range of metabolic pathways.
• An example of negative feedback — homeostatic control: stimulus = end product made in more than required ⇒ feedback shuts production off (like a barrier closing a full street).
• Named “feedback” because the end product is giving a message back to the first enzyme: “stop — we don't need more”.

8. Immobilised Enzymes

• Inert ⇒ does not take part in the E–S reaction. Insoluble ⇒ the enzyme does not dissolve and can be reused.
• Free enzymes are soluble globular proteins and can move freely in the solution. Immobilising limits their mobility.
• Methods exist (adsorption, covalent cross-linking, entrapment) — the syllabus method is entrapment in calcium alginate beads.

Using alginate to immobilise enzymes

1. Mix the enzyme with sodium alginate solution and stir.
2. Draw the mixture into a syringe; introduce it drop by drop into a beaker of CaCl₂(aq).
3. Ca²⁺ ions displace Na⁺ from the sodium alginate ⇒ gel-like, insoluble calcium alginate beads with the enzyme trapped inside.
4. Rinse the beads with water before use.

Advantages of immobilising enzymes

1. Immobilised enzymes can be reused (they don't dissolve; beads are removed, rinsed, washed and used again).
2. Saves the cost of extracting enzymes (from micro-organisms / animals) again and again — extraction is expensive & time-consuming.
3. Enzymes become more stable (robust) to pH and temperature changes because it is harder to change their tertiary structure (protective inert coat).
4. The enzyme does not contaminate the product and is easily separated from it.
5. Immobilised enzymes have a larger shelf life.
6. Higher yield.

Real-life use: lactose-free milk

• Lactose-intolerant people can't consume dairy (milk, cheese, yogurt) because it contains lactose.
• Lactose is a disaccharide = glucose + galactose. The enzyme lactase hydrolyses lactose ⇒ milk becomes lactose-free.

Milk is allowed to run through a column of lactase-containing beads; the lactase hydrolyses the lactose to glucose & galactose. The collected milk contains no lactase — it is all trapped in the beads, which can be reused again and again.

9. Cofactors and Coenzymes

• Coenzymes = organic molecules required for carrying out catalysis (usually vitamin derivatives).
• Cofactors = inorganic materials, e.g. metals / metal ions.
• Not every enzyme needs one — but some cannot work without their cofactor/coenzyme (their active-site shape is incomplete until it binds).

Enzyme	Needs	Type
Amylase	Cl⁻ (chloride ion)	cofactor
Carbonic anhydrase (in RBCs)	Zn²⁺ (zinc ion)	cofactor
(respiration / photosynthesis, A2)	NAD — nicotinamide adenine dinucleotide	coenzyme
(respiration / photosynthesis, A2)	FAD — flavin adenine dinucleotide	coenzyme

Cofactors & coenzymes matter again in A2 (respiration & photosynthesis); for AS, know the definitions and the apoenzyme → holoenzyme idea.

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