Enzymes

Enzyme: a specific protein that speeds up a specific chemical reaction by lowering the activation energy required for it, and remains unaltered itself in the process (a biocatalyst). Co-factor: an additional non-protein chemical component that aids an enzyme in catalysis. There are three kinds: metal ions (e.g. Zn⁺², Mg⁺²), prosthetic groups (e.g. hematin) and coenzymes (e.g. NAD⁺).

Coenzyme: a non-protein ORGANIC molecule that is LOOSELY attached to the enzyme; it takes part in catalysis, often by carrying electrons (hydrogen atoms) from one enzyme to another — e.g. NAD⁺, and vitamins such as niacin and riboflavin (or parts of coenzymes). Prosthetic group: a cofactor that forms a COVALENT (tight, permanent) bond with the enzyme; it may be an organic compound, e.g. hematin. So the difference is the type of attachment: loose (coenzyme) versus covalent (prosthetic group).

Hydrolases are enzymes that catalyse hydrolysis reactions: they break their substrates into monomers by adding water. Two examples: lipase — hydrolyses lipids into fatty acids and glycerol; amylase — hydrolyses starch into maltose. (Peptidase, which hydrolyses proteins, is another example.)

Activation energy is the energy required to initiate a chemical reaction. It works to destabilise the existing chemical bonds of the reactants so that new bonds can form. Enzymes lower the activation energy: they bring reactants together in the correct orientation or stress particular bonds of the reactants, so the reaction proceeds much faster than it would on its own.

Feedback inhibition is the control of a metabolic pathway by its own final product: the final product acts as an inhibitor of an initial enzyme of the pathway. It reacts with that enzyme and changes its conformation, so the enzyme can no longer bind its substrate, the pathway closes, and no more product is prepared. Example: when a cell has more ATP than it requires, ATP itself acts as a non-competitive inhibitor and blocks the enzyme that catalyses ATP synthesis.

Competitive: malonic acid — it resembles succinic acid and competes with it for the active site of succinic dehydrogenase, blocking the enzyme; antibiotic inhibitors that resemble bacterial substrates also compete for bacterial enzymes. Non-competitive: succinyl-CoA — the product of succinyl-CoA synthetase binds the enzyme elsewhere and stops further production; cyanide — binds copper and iron in the active site of cytochrome oxidase and blocks cellular respiration; excess ATP in feedback inhibition.

Optimum pH is the specific pH at which an enzyme works at its maximum rate; every enzyme has its own narrow optimum pH range, and a slight change retards or blocks its activity. Three human enzymes: pepsin — pH 1.5–1.6 (acidic, stomach); salivary amylase — pH 4.6–5.2; trypsin — pH 7.8–8.7 (alkaline, small intestine). (Pancreatic lipase, pH 8.0, is another example.)

Enzymes are three-dimensional globular proteins made of polypeptide chains coiled upon themselves. They are synthesized by ribosomes inside cells and then either work inside the cell or are secreted out. On the surface of the globular enzyme molecule there is a small cleft or depression made of only a few amino acids — this is the active site, the location at which catalysis occurs. Its shape is very specific and three-dimensional, and it bears a specific charge, so only a certain substrate molecule can fit into it. The active site has two distinct regions: the binding site, where the substrate fits by weak chemical forces such as hydrogen bonds, and the catalytic site, which catalyses the reaction and transforms the substrate into products. Without the precise shape and charge of the active site, the enzyme could not recognise its substrate or speed up its reaction.

Cofactors are additional non-protein components that aid enzymes in catalysis. (1) Metal ions: inorganic ions such as Ca⁺², Mg⁺², Mn⁺², Cu⁺² and Zn⁺². They change non-functional active sites into functional sites and change the enzyme's shape so it can combine with its substrate. (2) Prosthetic groups: cofactors that form covalent bonds with the enzyme, so they are permanently attached; a prosthetic group may be an organic compound, e.g. hematin. (3) Coenzymes: non-protein organic molecules that are loosely attached to the enzyme and take part in catalysis, often by transporting electrons (hydrogen atoms) from one enzyme to another; many vitamins (niacin, riboflavin) function as coenzymes, and the most important coenzyme in the cell is NAD⁺, the hydrogen acceptor that reduces to NADH. The protein part alone is the apoenzyme; apoenzyme + cofactor = holoenzyme (the complete, active enzyme).

Both models explain how an enzyme's active site recognises its substrate, but they differ on flexibility. The Lock-and-Key Model (Emil Fischer, 1894) says that, just as a specific key opens only a specific lock, a specific enzyme transforms only a specific substrate into products. The active site is a rigid structure: it does not change shape before, during or after enzyme action, and the substrate fits it exactly. The Induced Fit Model (Daniel Koshland, 1958) came from later evidence that did not support a rigid site in all reactions. It says that when a substrate combines with the binding site, it induces changes in the enzyme's structure; these changes enable the enzyme to perform its catalytic activity more effectively. So the active site is NOT rigid — it is capable of modification before catalysis starts, moulding itself around the substrate. In both models the outcome is the same: the enzyme-substrate complex (ES complex) forms, the catalytic site stresses and destabilises the substrate's bonds, activation energy is lowered, the substrate becomes products, and the enzyme detaches unaltered (E + S → ES → E + P). The induced fit model is the more complete picture because it explains how enzymes can be so precise and yet flexible.

Activation energy is the energy required to initiate a chemical reaction — the energy that works to destabilise the existing chemical bonds of the reactants so new bonds can form. On an energy-versus-reaction-time graph, the reactants start at a certain energy level and the products end at a lower level, but between them there is a high "hill" — the activation energy barrier that must be crossed. Without an enzyme, this hill is high (high Ea), so very few reactant molecules have enough energy to cross it and the reaction is slow. With an enzyme, the hill is much lower (low Ea): the enzyme brings reactants together in the correct orientation and stresses particular bonds of the reactants, so far more molecules can cross the barrier in the same time and the reaction runs much faster. The enzyme does not change the energy levels of the reactants or the products — sucrose still becomes glucose + fructose — it only lowers the barrier between them. That is why an enzyme-catalysed reaction can be 10³ to 10⁸ times faster than the uncatalysed reaction.

An enzyme's shape is held by hydrogen bonds and hydrophobic interactions between its polypeptide chains, and both are easily disrupted by temperature changes. Every enzyme has an optimum temperature at which it works at its maximum rate — for human enzymes, 37 °C. Below the optimum, the bonds that determine the enzyme's shape become less flexible, so the induced change in the active site that catalysis needs cannot happen properly, and the reaction rate is slow. As temperature rises up to a limit, heat adds activation energy and gives kinetic energy to enzyme and substrate molecules, so they move faster, collide more frequently, and the rate increases. But well above the optimum, atomic vibrations become too violent: bonds can no longer hold the polypeptide chains in position, the globular structure is lost, and the enzyme is denatured. The rate then falls rapidly and may be blocked completely. So the temperature graph is a bell curve: rising to the optimum, then falling steeply with denaturation.

Human enzymes have an optimum temperature of 37 °C, the normal temperature of the human body. Below 37 °C their activity falls because their shape becomes less flexible; above 37 °C their hydrogen bonds and hydrophobic interactions begin to break, and at much higher temperatures they denature and stop working. Thermophilic bacteria, which live in hot springs, have enzymes with an optimum temperature of 70 °C or higher. Their proteins have stronger bonding between their polypeptide arms, so their three-dimensional shape — and therefore their active sites — remains stable at temperatures that would denature human enzymes completely. In short: the same principle (an optimum, with denaturation above it) applies to both, but the thermophile's optimum is shifted far higher because its enzymes are built with stronger bonds.

Enzymes are very efficient: a small number of enzyme molecules can catalyse reactions of a large amount of substrate. If substrate concentration is unlimited, the overall rate of an enzyme-controlled reaction depends directly on the amount of enzyme present at a specific time. When enzyme concentration increases, there are more enzyme molecules and therefore more active sites; more substrate molecules bind with these new active sites and are transformed into products, so the rate rises. However, this rise has a limit: if enzyme concentration goes on increasing while substrate concentration remains the same, eventually there are no more free substrate molecules left to attach to the extra enzymes. The rate then stays constant and does not increase further. On a graph of rate versus enzyme concentration, the line rises steadily and then levels off into a plateau when substrate becomes the limiting factor.

If enzyme molecules with vacant active sites are available, increasing the substrate concentration increases the rate of reaction, because new substrate molecules bind with the available active sites and more products are formed in the given time. But if the enzyme concentration is kept constant and the substrate amount keeps increasing, a point is reached where any further increase in substrate does not increase the rate any more. This happens because, at high substrate concentration, all the active sites of the enzymes are occupied: extra substrate molecules cannot find free active sites, so no additional product can be formed. On a graph of rate versus substrate concentration, the curve rises at first and then flattens into a plateau at the point where the enzymes are saturated with substrate. This is the mirror image of the enzyme-concentration effect: here the enzyme, not the substrate, becomes the limiting factor.

Enzyme inhibition is the blocking of an enzyme's activity by a chemical called an inhibitor. Inhibitors attach to enzymes but are not transformed into products, and they block active sites temporarily or permanently. Types: (a) Competitive inhibitors resemble the substrate and compete for the same binding site (e.g. malonic acid competing with succinic acid for succinic dehydrogenase). (b) Non-competitive inhibitors have no structural similarity to the substrate; they bind elsewhere on the enzyme and change its shape so the active site no longer fits the substrate (e.g. succinyl-CoA inhibiting succinyl-CoA synthetase; cyanide blocking cytochrome oxidase). Inhibitors may also be reversible (weak bonds, e.g. malonate — the effect is removed when the inhibitor is removed or substrate is increased) or irreversible (covalent bonds, e.g. penicillin permanently disabling the bacterial cell-wall enzyme). A special form is feedback inhibition, where the final product of a pathway inhibits an initial enzyme of that pathway. Significance: inhibition regulates metabolic pathways; many drugs work as inhibitors (antibiotics against bacterial enzymes, cancer drugs against cell-division enzymes, anticoagulants against clotting enzymes); understanding poisons (like cyanide) helps treat poisoning; and inhibitors are valuable tools in pharmaceutical research and in studying enzyme kinetics.

Competitive inhibitors: (i) They resemble the enzyme's substrate in structure. (ii) They compete with the substrate for the SAME binding site (active site) on the enzyme. (iii) When selected by the binding site, they block it, so the substrate cannot attach and the enzyme cannot act. (iv) Their effect can be overcome by increasing the substrate concentration, because more substrate molecules then out-compete the inhibitor. (v) Examples: malonic acid (versus succinic acid for succinic dehydrogenase); antibiotic inhibitors that mimic bacterial substrates. Non-competitive inhibitors: (i) They have NO real structural similarity to the substrate. (ii) They do NOT enter the active site; they bind the enzyme at some other place. (iii) Their binding alters the shape of the enzyme, so the active site no longer fits the substrate. (iv) Increasing substrate concentration cannot overcome their effect, because they do not compete for the active site. (v) Examples: succinyl-CoA (inhibits its own producing enzyme, succinyl-CoA synthetase); cyanide (binds copper and iron in cytochrome oxidase); excess ATP in feedback inhibition.

In a metabolic pathway, the product of one reaction becomes the substrate for the next reaction, and at the end of the pathway a desired product is synthesized. To regulate the concentration of that product, the pathway must be shut down when enough product has been made. This is done through feedback inhibition: the final product of the pathway acts as an inhibitor. It reacts with some initial enzyme of the pathway and changes its conformation, so that enzyme can no longer bind to its substrate. The pathway therefore closes and no more product is prepared. When the product is used up and its concentration falls, the inhibition is lifted and the pathway starts again. A clear example: when a cell has more ATP than it requires, ATP itself acts as a non-competitive inhibitor and blocks the enzyme that catalyses ATP synthesis. Feedback inhibition is thus the phenomenon where the product of a process controls the process itself, usually limiting the production of more product — an efficient built-in control system of the cell.

On the basis of the general type of reaction, enzymes are classified into six classes: 1. Oxidoreductases — catalyse oxidation/reduction; add or remove H⁺ ions or electrons (e.g. cytochrome oxidase oxidises cytochrome). 2. Transferases — transfer a specific functional group (methyl, acyl, amino or phosphate) from one substrate to another (e.g. hexokinase transfers a phosphate group from ATP to glucose). 3. Hydrolases — catalyse hydrolysis; break substrates into monomers by adding water (e.g. lipase, amylase, peptidase). 4. Lyases — catalyse non-hydrolytic addition or removal of groups such as CO₂ or NH₃ (e.g. pyruvate decarboxylase removes CO₂ from pyruvic acid). 5. Isomerases — catalyse intra-molecular rearrangement, converting one isomer into another (e.g. hexose isomerase converts glucose to fructose). 6. Ligases — catalyse joining of two molecules by forming new C–C, C–N, C–O or C–S bonds, using energy from ATP (e.g. polymerase enzymes join monomers). Each class is further divided into subclasses, e.g. dehydrogenases/oxidases/reductases under oxidoreductases, and peptidases/lipases/glycosidases under hydrolases.

Enzymes are often named after the substrate they act upon, usually with the suffix "-ase". On this basis they fall into four groups: 1. Proteases — act on proteins: pepsin and trypsin break large polypeptides into smaller polypeptides; aminopeptidases break small polypeptides into dipeptides; erepsin breaks dipeptides into amino acids. 2. Lipases — act on lipids: pancreatic lipase hydrolyses lipids into fatty acids and glycerol. 3. Carbohydrases — act on carbohydrates: amylase acts on starch or glycogen and breaks them into maltose; cellulase breaks cellulose into cellobiose or glucose; maltase breaks maltose into glucose; sucrase breaks sucrose into glucose and fructose; lactase breaks lactose into glucose and galactose. 4. Nucleases — act on nucleic acids: RNase breaks down RNA, DNase breaks down DNA, and ATPase breaks down ATP. So the substrate in the name tells the student exactly what the enzyme works on.

Both. During exercise the body needs a lot of ATP quickly, so catabolic processes dominate at that time: glucose and glycogen are broken down (glycolysis and respiration) to release energy, and fats may also be broken down. Evidence: breathing and heart rate rise to supply oxygen for this breakdown, and muscles use up their glycogen stores. After exercise, anabolic processes take over: the body rebuilds glycogen stores and repairs and builds muscle protein — this is why rest and protein-rich food after exercise help muscles grow stronger. So exercise is catabolic while it is happening and anabolic during recovery.

Because speed matters for life. A reaction that an enzyme completes in 30 minutes could take a whole year without the enzyme. Cells must carry out thousands of reactions every second — digestion, energy release, growth, repair — and if each reaction ran at its uncatalysed speed, the organism could not survive: food would not be digested in time, energy would not be released when needed, and waste would pile up. Enzymes also give control: because each enzyme is specific and its activity can be changed by temperature, pH, concentration and inhibitors, the cell can switch pathways on and off exactly when needed. So enzymes are important not because reactions are impossible without them, but because life needs them fast and controlled.

Temperature graph: a bell-shaped curve. Rate rises as temperature increases towards the optimum (37 °C for human enzymes), peaks at the optimum, then falls steeply as the enzyme denatures. Interpretation: moderate heat speeds molecules up; excess heat destroys the enzyme's shape. Enzyme concentration graph: a straight rising line that levels off into a plateau. Interpretation: more enzyme means more active sites and a faster rate, until substrate runs out and becomes limiting. Substrate concentration graph: a curve that rises steeply at first and then flattens into a plateau. Interpretation: at low substrate levels, adding substrate fills empty active sites and raises the rate; at high levels all active sites are occupied (saturation), so extra substrate cannot raise the rate further.

Use these tests. A chemical is a competitive inhibitor if its molecule resembles the substrate: it competes for the same active site, its effect can be overcome by adding more substrate, and it is often a drug designed to mimic a natural substrate (e.g. many antibiotics; malonic acid versus succinic acid). A chemical is a non-competitive inhibitor if it does not resemble the substrate: it binds somewhere else on the enzyme, changes the enzyme's shape so the substrate no longer fits, and adding more substrate cannot overcome it (e.g. cyanide in rat poison; heavy metals like lead and mercury that bind enzymes; excess product in feedback inhibition). So: look at the shape (does it mimic the substrate?), the binding place (active site or elsewhere?), and whether extra substrate reverses the effect.