Catalysts speed reactions without being used up

A catalyst is a substance that increases the rate of a chemical reaction without being permanently changed by that reaction. The key point is that it isn’t consumed like a reactant. A single catalyst molecule can work again and again, so cells only need small amounts compared with the quantities of substrates being converted.

An enzyme is a biological catalyst made by living cells that increases the rate of a biochemical reaction. A substrate is a reactant molecule that binds to an enzyme and is converted during the reaction. A product is a molecule formed from a substrate during a chemical reaction. In the simplest enzyme reaction: $substrate + enzyme \to enzyme$–$substrate complex \to product + enzyme$.

Why cells need faster reactions

Cells work at the temperatures, pressures and pH values compatible with life. Without enzymes, many essential reactions would still be chemically possible, but they would happen so slowly that the cell could not survive. Digestion, respiration, synthesis of macromolecules, detoxification and movement all rely on reaction rates being raised to useful levels.

So the benefit is not just that “faster is nice”. Enzymes make metabolism possible under cellular conditions. They let a cell obtain energy, build structures and respond to change quickly enough for life.

Metabolism is a network, not a list

Metabolism is the complex network of interdependent and interacting chemical reactions occurring in a living organism. Some reactions break molecules down. Others build them up. Many reactions also sit in pathways, where the product of one reaction becomes the substrate for the next.

Since these reactions are linked, a change in one step can alter the supply of substrates for later steps, the removal of products from earlier steps, or the balance of whole pathways. That is the interdependence in the topic title: enzymes don't work as isolated little machines; they operate inside a connected chemical system.

Enzyme specificity gives control

Enzyme specificity is the property of an enzyme by which its active site catalyses only one reaction or a narrow group of related reactions. Because enzymes are specific, organisms need many different enzymes. One enzyme can't simply run the whole of metabolism.

There is a big advantage to this specificity. When a cell controls the amount or activity of a particular enzyme, it controls the rate of a particular metabolic step. Producing more enzyme can increase flux through a pathway; inhibiting an enzyme can slow or stop it. That is why enzyme regulation is one of the main ways cells control their chemical composition and activities.

This also links to the idea of specificity and versatility. Specificity gives precise control and helps avoid unwanted side reactions. Versatility would reduce the number of enzymes needed, but it would also mean less fine control and more chemical confusion inside the cell.

Anabolism builds larger molecules

Anabolism is the set of metabolic reactions that uses energy to build larger molecules from smaller molecules. A common pattern is simple: monomers join to make macromolecules.

Many anabolic reactions are condensation reactions. In these chemical reactions, two molecules join together and a water molecule is released. Protein synthesis is anabolic because amino acid monomers join to form polypeptides. Glycogen formation is anabolic because glucose units join to form a storage polysaccharide. Photosynthesis is anabolic because small inorganic molecules are used to make larger organic molecules using energy from light.

Catabolism breaks larger molecules down

Catabolism is the set of metabolic reactions that breaks larger molecules into smaller molecules, often releasing energy that can be transferred to ATP.

In digestion, many catabolic reactions are hydrolysis reactions. These are chemical reactions in which water is used to break a covalent bond. During digestion, macromolecules such as polysaccharides, proteins and lipids are hydrolysed into smaller molecules that can be absorbed. In respiration, organic substrates are oxidized; for example, glucose and fatty acids can be broken down so that energy is transferred for cellular use.

Enzyme shape is part of enzyme function

A globular protein is a protein with a compact, roughly spherical three-dimensional structure that is soluble or functional in aqueous cellular environments. Most enzymes are globular proteins, and they work only if their folded shape is precise.

An active site is a specific region of an enzyme where the substrate binds and catalysis occurs. It usually takes up only a small part of the whole enzyme. A few amino acids may directly touch the substrate or take part in catalysis, but the rest of the protein still matters: it holds those amino acids in the correct positions.

A few amino acids, held in the right arrangement

The amino acids forming the active site do not have to sit next to each other in the primary structure of the polypeptide. When the protein folds, distant sections of the chain can be brought together. So the overall three-dimensional structure of an enzyme is not decoration. It gives the active site its shape, charge distribution, hydrophobic regions and ability to strain or stabilize bonds in the substrate.

This is a classic structure–function relationship in a biological macromolecule: the protein’s folded structure creates a chemically suitable active site, and that active site allows the enzyme to catalyse a particular reaction.

Binding is not a rigid lock-and-key event

Induced-fit binding is enzyme–substrate binding where interactions between the substrate and the active site make both molecules change shape, which improves catalysis. The older lock-and-key model still helps as a first picture: the active site and substrate are complementary. But it’s too rigid for what really happens.

As a substrate enters the active site, weak interactions form between chemical groups on the substrate and amino acid side chains in the enzyme. These interactions can change bond angles, bond lengths and the positions of functional groups. So the enzyme shifts slightly, and the substrate shifts slightly too.

How induced fit helps catalysis

These shape changes help catalysis by stressing bonds that need to break, bringing reacting groups closer together, or orienting substrates correctly. In a reaction with two substrates, induced fit can hold them in the right relative position, making new bonds form more easily.

After the reaction, the products no longer fit the active site in the same way, so they are released. The enzyme then returns to a shape ready to bind more substrate. This return to its reusable form is why enzymes act as reusable catalysts rather than one-use reactants.

Collisions are needed before binding can happen

Molecular motion is the random movement of molecules caused by their kinetic energy. Inside a fluid cell environment, substrates and enzymes move, rotate, and bump into each other as molecules constantly jostle around.

A substrate–active site collision is an encounter where a substrate molecule contacts an enzyme’s active site. Many collisions go nowhere. For binding to occur, the substrate has to get close enough and line up in a suitable orientation. That’s why collision theory helps explain enzyme activity: reaction rate depends partly on how often successful collisions happen.

What affects collision frequency

Increasing substrate concentration puts more substrate molecules into a given volume, so collisions with active sites happen more often. Raising temperature, up to the point where denaturation becomes important, increases molecular motion and therefore collision frequency.

Movement isn’t always shared equally. Small soluble substrates usually move more than large enzyme molecules. Very large substrates, such as nucleic acids during copying processes, may be relatively immobilized while enzymes move along them. Some enzymes are immobilized because they are embedded in membranes; in those cases, the substrate must diffuse to the enzyme.

Specificity depends on active-site structure

Enzyme–substrate specificity is the ability of an enzyme to bind and catalyse a reaction for a particular substrate or limited group of substrates because of the active site’s shape and chemical properties. The active site has to be complementary enough for binding and catalysis, not just similar in outline.

An active site’s structure includes its three-dimensional shape, charges, polar groups, non-polar regions and the positions of amino acid side chains. When these features match the substrate, binding is likely. When they don’t, the molecule will usually fail to bind, or it may bind without being converted efficiently.

Denaturation disrupts catalysis

Denaturation is a structural change in a protein that disrupts its three-dimensional shape enough to reduce or stop its biological function. Enzymes are especially vulnerable here, because even a small change in the active site can prevent binding, induced fit or catalysis.

High temperature or extreme pH can cause denaturation because they disrupt the weak interactions that hold the enzyme in its folded shape, including hydrogen bonds, ionic interactions and hydrophobic interactions. The damage doesn’t have to happen directly at the active site. A change elsewhere in the protein can alter the folding pattern and therefore distort the active site.

The relationship is: amino acid sequence and folding produce active-site structure; active-site structure produces specificity; denaturation changes protein structure; changed structure reduces specificity and catalysis.

Temperature: faster collisions, then denaturation

At low temperatures, enzyme and substrate molecules have less kinetic energy. That means fewer successful substrate–active site collisions happen each second. As temperature rises, molecules move more, and enzyme activity usually increases.

Once the temperature goes above the enzyme’s optimum, bonds in the protein vibrate more strongly. This disrupts the enzyme’s three-dimensional structure. More enzyme molecules become denatured, leaving fewer active sites that still work. So the graph rises to an optimum and then falls, often sharply.

pH: active-site chemistry changes

pH is a logarithmic measure of hydrogen ion concentration in a solution. A lower pH means a higher concentration of hydrogen ions, so a drop of one pH unit represents a tenfold increase in hydrogen ion concentration.

Each enzyme has an optimum pH where its rate is highest. If conditions move away from that optimum, the ionization of amino acid side chains changes. This can disrupt ionic interactions and change the active site. At very high or very low pH, denaturation may occur. Different enzymes can have different pH optima because they work in different chemical environments.

Substrate concentration: more collisions, then active-site saturation

At low substrate concentration, adding more substrate increases the rate because more substrate molecules collide with active sites. The process depends on concentration, so it connects well with the wider idea that biological processes can depend on concentration differences or changes.

At high substrate concentration, many active sites are already occupied at any one time. The enzyme molecules are working close to their maximum capacity, so extra substrate has a smaller effect. The curve rises quickly at first, then levels off as active sites become saturated.

Interpreting enzyme graphs as models

A model is a simplified representation of a system that helps explain or predict behaviour. The smooth sketch graphs used for temperature, pH and substrate concentration are models. They show the general relationship, not every experimental detail.

When reading graphs, first identify the independent variable on the x-axis and the dependent variable on the y-axis. Then describe the relationship in words: for example, “rate increases to an optimum and then decreases” is much better than “it goes up then down”. Experimental results can then be compared with the model. If the data do not fit the model, the model or hypothesis may need refining.

Variables in enzyme experiments

An independent variable is the factor you deliberately change in an investigation to test its effect. In enzyme experiments, this could be temperature, pH, substrate concentration or enzyme concentration.

A dependent variable is the outcome you measure, which changes in response to the independent variable. For this topic, it’s usually a measurement used to calculate enzyme activity, such as the volume of oxygen produced, mass of substrate used, absorbance change, pH change or time taken to reach a visible endpoint.

A control variable is a factor kept constant, so that any change in the dependent variable can be linked to the independent variable. If substrate concentration is being tested, enzyme concentration, pH, temperature, total volume and reaction time should normally be controlled.

Calculating reaction rate

Reaction rate is the change in amount of substrate or product per unit time. One useful form is

If gas volume is measured instead, use

In school enzyme practicals, you may also see $\mathrm{cm^3\,s^{-1}}$, $\mathrm{g\,s^{-1}}$, $\mathrm{mol\,dm^{-3}\,s^{-1}}$ or $\mathrm{absorbance\,s^{-1}}$, depending on the measurement taken.

There are two common approaches. You can let the reaction run for a fixed short time and measure how much product forms or how much substrate disappears. Or you can start with a known amount of substrate and record the time taken to reach an endpoint. In both cases, divide the change measured by the time taken.

Practical examples of measurement

Catalase activity can be tracked by measuring the oxygen produced from hydrogen peroxide. The dependent variable can come from a gas syringe, an inverted measuring cylinder or an oxygen sensor. To test substrate concentration, prepare different hydrogen peroxide concentrations, while keeping the catalase source, temperature, pH and total volume constant. Eye protection is needed because hydrogen peroxide can irritate or damage tissues.

Other enzyme systems use different endpoints. Amylase digestion of starch can be timed with iodine tests until starch is no longer detected. Lipase activity can be monitored through pH change as fatty acids form. Protease digestion of gelatin can be measured by loss of gel structure or change in mass/size. Oxidase reactions that produce coloured products can be followed using a colorimeter.

The same logic applies when you use secondary data. Choose the steepest reliable part of a product-time graph, calculate the gradient, and include units. A straight line through early points often gives a better initial rate than late points, because substrate may be depleted and products may accumulate.

Reactions must pass through a transition state

Activation energy is the minimum energy reactants need to reach the transition state of a chemical reaction. The transition state is a high-energy, unstable arrangement of atoms that appears while bonds are breaking and new bonds are forming.

Bonds within the substrate need energy to break. When new bonds form in the products, energy is released. The overall reaction may release or absorb energy, but the activation energy still acts as a barrier that must be crossed before products can form.

Enzymes lower the barrier

Enzymes lower activation energy by binding substrates in ways that help them reach the transition state more easily. They may strain bonds, line up reacting groups correctly, bring substrates together or stabilize charged transition-state structures.

The key graph point is simple: an enzyme lowers the activation energy, but it does not change the overall energy difference between substrates and products. The start and end energy levels stay the same with or without the enzyme; only the route and the height of the energy barrier change.

With a lower activation energy, a larger proportion of substrate molecules have enough energy to react at a given temperature. So the reaction rate increases without the cell needing damagingly high temperatures.

Where the enzyme works matters

An intracellular enzyme catalyses reactions inside a cell. Glycolysis counts as an intracellular pathway because its enzymes work in the cytoplasm. The Krebs cycle is intracellular too, since its enzymes act inside mitochondria in eukaryotic cells.

An extracellular enzyme is secreted by a cell and catalyses reactions outside that cell. The main example is chemical digestion in the gut: enzymes are released into the lumen of the digestive system, where they hydrolyse large food molecules into smaller molecules that can be absorbed.

Why extracellular digestion is useful

Large macromolecules often can’t cross membranes directly. Extracellular enzymes deal with this by breaking polymers into smaller products outside the cell, or outside the body tissue that secretes them. Those products can then be absorbed through membranes.

Intracellular and extracellular reactions are both part of metabolism. What changes is the location, not the importance. Glycolysis and the Krebs cycle help cells transfer energy from substrates; gut digestion provides the smaller molecules that cells later use in their internal metabolic pathways.

Metabolic energy transfers are not perfectly efficient

Heat is energy transferred from a warmer body or region to a cooler one because of a temperature difference. In metabolism, cells cannot avoid producing heat, because energy transfers in chemical reactions are not 100% efficient.

As cells move energy from substrates into useful forms such as ATP or concentration gradients, some of that energy spreads out as heat. Biology has not “failed” here; energy transformations work this way. So, while metabolic pathways run, they warm cells and tissues.

Heat helps some animals maintain body temperature

Mammals, birds and some other animals rely on metabolic heat production to keep a relatively constant body temperature. If heat production drops too low, they can raise metabolic activity. Shivering is a familiar example: muscle contraction uses ATP and releases heat.

Some tissues make heat especially well. Brown adipose tissue contains many mitochondria and can oxidize substrates in a way that releases more energy as heat rather than conserving it in ATP. Metabolism and temperature regulation are linked: chemical reactions produce heat, and body temperature affects the rates of those same reactions.

Linear pathways have a start and an end

A metabolic pathway is a sequence of enzyme-catalysed reactions in which the product of one reaction becomes the substrate for another. A linear pathway is a metabolic pathway with a distinct initial substrate, a sequence of intermediates and an end product.

Glycolysis is a linear pathway. It converts glucose through a sequence of intermediates into pyruvate. A specific enzyme catalyses each step, so the molecule changes gradually rather than in one huge chemical leap.

Cyclical pathways regenerate an intermediate

A cyclical pathway is a metabolic pathway in which intermediates are regenerated so that the pathway can turn repeatedly. In a cycle, each intermediate acts as the product of one step and the substrate for another.

The Krebs cycle and the Calvin cycle are examples. During cell respiration, the Krebs cycle processes carbon compounds in a cycle. During photosynthesis, carbon compounds cycle in the Calvin cycle. Don’t confuse a cycle with “going in circles and doing nothing”; cycles allow repeated processing while regenerating a key acceptor molecule.

Metabolism as a whole is more complex than a single line or circle. Pathways can branch, share intermediates and influence one another. Cells therefore need regulation: a change in one pathway can alter concentrations and reaction rates elsewhere.

Allosteric sites regulate enzyme shape

An allosteric site is a specific binding site on an enzyme. It sits away from the active site, and a regulatory molecule can bind there to change the enzyme’s shape. Only specific substances fit a particular allosteric site, just as only suitable substrates fit an active site.

When a molecule binds to an allosteric site, the interactions inside the enzyme shift. That shift causes a conformational change, which is a change in the three-dimensional shape of a protein. If this change affects the active site enough, the substrate may no longer bind properly, or catalysis may not happen.

Non-competitive inhibition

A non-competitive inhibitor is a substance that reduces enzyme activity by binding somewhere other than the active site. This changes the enzyme’s shape, so catalysis is prevented. The inhibitor does not compete with the substrate for the active site.

In the cases required here, binding is reversible. If the inhibitor leaves the allosteric site, the enzyme can return to its active form. It’s a clear structure–function link: a small change in enzyme shape at one site can alter function at another site.

Competitive inhibitors occupy the active site

A competitive inhibitor reduces enzyme activity by binding reversibly to the active site. While it is there, the substrate cannot bind. Competitive inhibitors often resemble the substrate closely enough to fit into the active site, but the enzyme does not convert them into the normal product.

Substrate and inhibitor are competing for the same active site, so concentration makes a difference. A high inhibitor concentration gives stronger inhibition. If substrate concentration rises enough, substrate molecules are more likely to bind before inhibitor molecules, so a fixed low concentration of competitive inhibitor has less effect.

Competitive versus non-competitive inhibition on graphs

Without an inhibitor, the rate rises as substrate concentration increases, until the active sites are close to saturation. With a competitive inhibitor, the curve sits lower at low substrate concentration, but it can approach the same maximum rate when substrate concentration becomes very high. With a non-competitive inhibitor, extra substrate cannot remove the inhibitor from the allosteric site, so the maximum rate stays lower.

Statins as competitive inhibitors

Statins lower blood cholesterol by competitively inhibiting HMG-CoA reductase, an enzyme in the cholesterol synthesis pathway. The statin molecule binds reversibly to the active site and slows the pathway. Dose matters: too little causes insufficient inhibition, while too much inhibition can disrupt normal metabolism.

End products can switch pathways down

Feedback inhibition is regulation of a metabolic pathway where the end product inhibits an earlier enzyme in that pathway. The enzyme blocked is usually the one that catalyses the first committed step, so once enough product has built up, the whole pathway slows.

This is negative feedback. When the end product concentration rises, inhibition increases and production drops. When the end product concentration falls, less inhibitor stays bound, and the pathway becomes active again. The change in concentration acts as the signal that regulates the pathway.

Isoleucine production as the example

In the pathway that produces isoleucine, threonine is converted through a series of steps into isoleucine. Isoleucine is the end-product inhibitor. At high concentration, it binds to an allosteric site on the first enzyme in the pathway, threonine deaminase.

That binding changes the enzyme’s shape and stops catalysis, so less threonine enters the pathway. This prevents wasteful accumulation of intermediates and avoids making more isoleucine than the cell needs. When isoleucine concentration decreases, it dissociates from the allosteric site, and the pathway can run again.

Irreversible inhibition permanently inactivates enzyme molecules

An irreversible inhibitor is a substance that permanently reduces enzyme activity by forming a stable bond or causing a lasting chemical change in the enzyme. That’s different from the competitive and non-competitive inhibition discussed earlier, where the inhibitor can bind reversibly.

Mechanism-based inhibition is irreversible inhibition in which an inhibitor binds at the active site and triggers chemical changes that permanently inactivate the enzyme. Often, the inhibitor resembles the substrate closely enough that the enzyme starts to interact with it as if it were the normal substrate. But then a stable covalent bond forms, or the active site is chemically altered, and that enzyme molecule can no longer catalyse the reaction.

Penicillin as the required example

Penicillin is an antibiotic that kills susceptible bacteria by mechanism-based inhibition of transpeptidase enzymes involved in bacterial cell wall formation. Transpeptidase is an enzyme that cross-links peptidoglycan strands in bacterial cell walls.

Penicillin binds to the active site of transpeptidase and forms a permanent covalent bond. After that, the enzyme can’t cross-link the cell wall properly. As the bacterium grows, its wall weakens, water enters by osmosis, and the cell may burst.

Resistance through changed transpeptidase

Penicillin resistance can arise when bacteria have changed transpeptidases with altered active sites. If the active site changes so that penicillin binds less effectively, the inhibitor no longer inactivates the enzyme efficiently. The enzyme can keep cross-linking peptidoglycan, so penicillin has less effect on the bacterium.

This is another structure–function relationship: a small change in enzyme structure can change inhibitor binding, and that can change survival.