Cofactors, coenzymes and where enzymes work

What a coenzyme does

A coenzyme is a small organic molecule that an enzyme needs alongside its substrate. It binds to the enzyme, is chemically changed by the reaction that enzyme catalyses, and leaves carrying whatever the reaction removed: a pair of hydrogen atoms, a phosphate group, an acetyl group. It then binds a second enzyme elsewhere in the cell, gives that group up, and returns to the form it started in. A coenzyme is therefore a carrier between enzymes, recycled rather than used up, which is why a cell holds only a small quantity of one.

NAD is the example to work from. In glycolysis and the Krebs cycle a dehydrogenase removes two hydrogen atoms from its substrate; NAD accepts them and leaves the enzyme as reduced NAD. It carries them to the electron transport chain of the inner mitochondrial membrane, releases them there, and the oxidised form returns to accept another pair. Coenzyme A does the same with a different cargo, collecting the two-carbon acetate group made from pyruvate and delivering it into the Krebs cycle, and NADP carries hydrogen this way in photosynthesis. One molecule of a coenzyme therefore serves many reactions in succession.

A coenzyme is not a substrate, and the difference is worth stating precisely. A substrate is the molecule an enzyme exists to convert: it is complementary to that enzyme's active site, and it is consumed as product accumulates. A coenzyme is used by many different enzymes, is restored to its original form at one of them, and is not consumed, so the quantity a cell holds stays roughly constant while the pathway runs.

Four terms are used for the non-protein partners an enzyme may need, and the distinction to get right is whether the partner stays bound to the enzyme or binds, acts and leaves.

Apply the permanence test rather than the chemistry, because chemical type does not decide the category. The haem group of catalase is organic and permanently bound; the zinc ion of carbonic anhydrase is inorganic and permanently bound. Both are prosthetic groups.

Cofactors, coenzymes and prosthetic groups

Some enzymes require a non-protein component, called a cofactor, for catalytic activity. Chloride ions, for example, are required for full activity of salivary amylase. Organic cofactors are called coenzymes; NAD and coenzyme A are examples derived from B vitamins. A cofactor that remains tightly bound to a protein is called a prosthetic group; haem in catalase is an example.

Binding strength and chemical type are separate properties. A prosthetic group may be organic or inorganic, and some organic cofactors remain tightly bound. Where an examination specification defines coenzyme more narrowly as a temporary carrier, use that board's definition in the answer.

That grouping gives the three categories A-level questions ask for. Some cofactors are inorganic ions, such as the chloride required by amylase: the ion sits close to the active site and its charge helps the site take, or hold, the shape that is complementary to the substrate. Some are small organic molecules that bind and are released again, carrying a chemical group with them; these are the coenzymes of the usual convention. And some are non-protein groups bound tightly enough to be treated as part of the protein: prosthetic groups.

Prerequisite: haemoglobin as a conjugated protein. The haem group of haemoglobin, an iron-containing ring that is not made of amino acids, is a prosthetic group, and haemoglobin is called a conjugated protein because of it. Catalase also contains haem, and the iron in it catalyses the decomposition of hydrogen peroxide, a reaction no amino acid side chain catalyses on its own. Carbonic anhydrase, the enzyme in red blood cells that interconverts carbon dioxide and hydrogencarbonate, holds a single zinc ion in its active site permanently: an inorganic partner, but a prosthetic group rather than a visiting cofactor, because it never leaves.

Cofactor

A non-protein substance that an enzyme requires in order to function, such as the chloride ion required by amylase.

Coenzyme

An organic cofactor. In the usual A-level convention it binds to an enzyme, carries chemical groups or hydrogen between reactions and is released again; many are made from vitamins.

Prosthetic group

A cofactor tightly bound to an enzyme or other protein, and which may be organic, such as the haem group of catalase, or inorganic, such as the zinc ion of carbonic anhydrase.

Coenzymes and vitamins

In the usual A-level convention a coenzyme is a carrier. It binds to an enzyme alongside the substrate, accepts a group released by the reaction, such as a pair of hydrogen atoms or an acetate group, carries it to a different enzyme, and is returned to its original form when it gives that group up. It is recycled repeatedly, so a cell requires only a small quantity, but that quantity is not zero.

Two coenzymes are named in this unit, and both act in respiration. NAD collects hydrogen from the oxidation reactions of glycolysis and the Krebs cycle and delivers it to the electron transport chain, where the energy it carries is used to make ATP. Coenzyme A picks up the two-carbon acetate made from pyruvate and delivers it into the Krebs cycle. Both are coenzymes and both are derived from B vitamins. Next topic for the reactions themselves: glycolysis, the link reaction and the Krebs cycle.

The 'made from' column explains why vitamins are examinable here. A cell cannot synthesise nicotinamide or pantothenic acid: both are obtained in food as vitamins and converted into NAD and coenzyme A inside the cell. A diet lacking vitamin B3 therefore limits NAD supply in every cell, and the deficiency disease pellagra, which affects the skin, digestive system and nervous system, results from that shortage in tissues with a high respiratory rate.

Several other B vitamins are coenzymes or precursors of coenzymes, which is why the group is treated together. Only a small dietary quantity is required, because coenzymes are recycled rather than consumed.

Intracellular and extracellular enzymes

Enzymes are also classified by where they act, and both terms below are examinable. An intracellular enzyme is made and used within the same cell. Catalase is the standard example: it sits inside peroxisomes, small organelles bounded by a single membrane, and breaks down the hydrogen peroxide that the cell's own metabolism produces, before that peroxide oxidises other cell components. The enzymes of glycolysis, dissolved in the cytoplasm, are intracellular too, and so is DNA polymerase in the nucleus.

An extracellular enzyme is secreted from the cell that made it, by exocytosis, and does its work outside. Digestion is the reason this category has to exist. Starch, proteins and triglycerides are far too large to cross a cell surface membrane, so no cell can take them in and hydrolyse them internally; the enzymes are secreted to act on the food. Salivary amylase is secreted into the mouth and begins starch on its way to maltose; trypsin is made in the pancreas and secreted into the small intestine, where it hydrolyses proteins it never shares a cell with. Only the small, soluble products of that external digestion, glucose, amino acids and the rest, are absorbed. Fungi take the same idea further: a mould secretes its digestive enzymes onto the food it grows across and absorbs what they release.

The classification has a practical consequence. An extracellular enzyme must remain active in conditions the secreting cell never experiences, which is why pepsin's optimum is at the pH of the stomach rather than the pH of the cell that secreted it. The pancreas secretes some proteases in an inactive form that is activated only in the gut, which prevents them hydrolysing pancreatic tissue.

Intracellular enzyme

An enzyme that catalyses reactions inside the cell that made it, such as catalase in peroxisomes.

Extracellular enzyme

An enzyme secreted from the cell that made it and catalysing reactions outside it, such as amylase and trypsin in digestion.

Km and apparent affinity

Prerequisite: Km as used to distinguish competitive from non-competitive inhibitors. Km, the substrate concentration at which an enzyme works at half its maximum rate, is also used to compare different enzymes acting on the same substrate.

In the simple Michaelis-Menten comparisons used at A level, an enzyme that reaches half of Vmax at a low substrate concentration is described as having a higher apparent affinity for its substrate. An enzyme requiring a much higher substrate concentration to reach half its maximum rate has a lower apparent affinity. The relationship is inverse: an enzyme with a very large Km works well below half its maximum rate at physiological substrate concentrations.

Hexokinase and glucokinase both catalyse the same reaction, the phosphorylation of glucose at the start of glycolysis. Hexokinase, found in nearly all tissues, has a Km far below the normal concentration of glucose in blood, so it operates near its maximum rate continuously, and tissues such as the brain maintain glycolysis at a steady rate regardless of the last meal. Glucokinase, the liver isoform, has a Km roughly a hundred times higher, in the range of glucose concentrations reached after a meal. The liver enzyme therefore works well below its maximum rate between meals and its rate rises steeply when blood glucose rises, which is when the liver stores glucose as glycogen.