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Enzymes are proteins that accelerate, or catalyze, chemical reactions. They are biological catalysts. In these reactions, the molecules at the beginning of the process are called substrates and the enzyme converts these into different molecules: the products. Almost all processes in the cell need enzymes in order to occur at significant rates. Since enzymes are extremely selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell.

Enzymes are known to catalyze about 4,000 reactions.^[1] However, not all biological catalysts are proteins, since some RNA molecules called ribozymes can also catalyze reactions. Enzymes are usually named according to the reaction they carry out. Typically the suffix -ase is added to the name of the substrate (e.g., lactase is the enzyme that cleaves lactose) or the type of reaction (e.g., DNA polymerase forms DNA polymers).

Like all catalysts, enzymes work by providing an alternative path of lower activation energy for a reaction and dramatically accelerating its rate. Some enzymes can make their conversion of substrate to product occur many millions of times faster. For example, the reaction catalysed by orotidine 5'-phosphate decarboxylase will consume half of its substrate in 78 million years if no enzyme is present. However, when the decarboxylase is added, the same process takes just 25 milliseconds.^[2] Chemically, enzymes are like any catalyst and are not consumed in chemical reactions, nor do they alter the equilibrium of a reaction. However, enzymes do differ from most other catalysts by being much more specific.

Enzyme activity can be affected by other molecules. Inhibitors are molecules that decrease enzyme activity, and activators are molecules that increase activity. Drugs and poisons are often enzyme inhibitors.

Some enzymes are used commercially, for example, in the synthesis of antibiotics. In addition, some household cleaning products use enzymes to speed up chemical reactions (e.g., enzymes in biological washing powders break down protein or fat stains on clothes).

Etymology and history

As early as the late 1700s and early 1800s, the digestion of meat by stomach secretions^[3] and the conversion of starch to sugars by plant extracts and saliva were known. However, the mechanism by which this occurred had not been identified.^[4]

In the 19th century, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur came to the conclusion that this fermentation was catalyzed by a vital force contained within the yeast cells called "ferments", which were thought to function only within living organisms. He wrote that "alcoholic fermentation is an act correlated with the life and organisation of the yeast cells, not with the death or putrefaction of the cells."^[5]

In 1878 German physiologist Wilhelm Kühne (1837–1900) coined the term enzyme, which comes from Greek ενζυμον "in leaven", to describe this process. The word enzyme was used later to refer to nonliving substances such as pepsin, and the word ferment used to refer to chemical activity produced by living organisms.

In 1897 Eduard Buchner began to study the ability of yeast extracts to ferment sugar despite the absence of living yeast cells. In a series of experiments at the University of Berlin, he found that the sugar was fermented even when there were no living yeast cells in the mixture.^[6] He named the enzyme that brought about the fermentation of sucrose "zymase".^[7] In 1907 he received the Nobel Prize in Chemistry "for his biochemical research and his discovery of cell-free fermentation".

Having shown that enzymes could function outside a living cell, the next step was to determine their chemical nature. Many early workers noted that enzymatic activity was associated with proteins, but several scientists (such as Nobel laureate Richard Willstätter) argued that proteins were merely carriers for the true enzymes and that proteins per se were incapable of catalysis. However, in 1926, James B. Sumner showed that the enzyme urease was a pure protein and crystallized it; Sumner did likewise for the enzyme catalase in 1937. The conclusion that pure proteins can be enzymes was definitively proved by Northrop and Stanley, who worked on the digestive enzymes pepsin (1930), trypsin and chymotrypsin. These three scientists were awarded the 1946 Nobel prize in Chemistry.^[8]

This discovery that enzymes could be crystalised eventually allowed their structures to be solved by x-ray crystallography. This was first done for lysozyme, an enzyme found in tears, saliva and egg whites that digests the coating of some bacteria; the structure was solved by a group led by David Chilton Phillips and published in 1965.^[9] This high-resolution structure of lysozyme marked the beginning of the field of structural biology and the effort to understand how enzymes work at an atomic level of detail.

Structures and mechanisms

The activities of enzymes are determined by their three-dimensional structure.^[10]

Most enzymes are much larger than the substrates they act on, and only a very small portion of the enzyme (around 3–4 amino acids) is directly involved in catalysis.^[11] The region that contains these catalytic residues, binds the substrate and then carries out the reaction is known as the active site. Enzymes can also contain sites that bind cofactors, which are needed for catalysis. Some enzymes also have binding sites for small molecules, which are often direct or indirect products or substrates of the reaction catalyzed. This binding can serve to increase or decrease the enzyme's activity, providing a means for feedback regulation.

Like all proteins, enzymes are made as long, linear chains of amino acids that fold to produce a three-dimensional product. Each unique amino acid sequence produces a unique structure, which has unique properties. Individual protein chains may sometimes group together to form a protein complex. Most enzymes can be denatured—that is, unfolded and inactivated—by heating, which destroys the three-dimensional structure of the protein. Depending on the enzyme, denaturation may be reversible or irreversible.

Specificity

Enzymes are usually specific as to which reactions they catalyze and the substrates that are involved in these reactions. Complementary shape, charge and hydrophilic/hydrophobic characteristics of enzymes and substrates are responsible for this specificity. Enzymes can also show impressive levels of stereospecificity, regioselectivity and chemoselectivity.^[12]

Some of the enzymes showing the highest specificity and accuracy are involved in the copying and expression of the genome. These enzymes have "proof-reading" mechanisms. Here, an enzyme such as DNA polymerase catalyses a reaction in a first step and then checks the product is correct in a second step.^[13] This two-step process results in average error rates of less than one error 1 in 100 million reactions in high-fidelity mammalian polymerases.^[14] Similar proofreading mechanisms are also found in aminoacyl tRNA synthetases^[15] and ribosomes.^[16]

"Lock and key" model

Enzymes are very specific, and it was suggested by Emil Fischer in 1894 that this was because both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another.^[17] This is often referred to as "the lock and key" model. However, while this model explains enzyme specificity, it fails to explain the stabilization of the transition state that enzymes achieve.

Induced fit model

In 1958 Daniel Koshland suggested a modification to the lock and key model.^[18] Since enzymes are rather flexible structures, the active site can be modified as the substrate interacts with the enzyme. As a result, the amino acid side chains which make up the active site are molded into a precise shape which enables the enzyme to perform its catalytic function. In some cases the substrate molecule also changes shape slightly as it enters the active site.

Dynamics and function

Recent investigations have provided new insights into the connection between internal dynamics of enzymes and their mechanism of catalysis.^[19]^[20]^[21] An enzyme's internal dynamics are described as the movement of internal parts (e.g. amino acids, a group of amino acids, a loop region, an alpha helix, neighboring beta-sheets or even entire domain) of these biomolecules, which can occur at various time-scales ranging from femtoseconds to seconds. Networks of protein residues throughout an enzyme's structure can contribute to catalysis through dynamic motions.^[22]^[23]^[24] Protein motions are vital to many enzymes, but whether small and fast vibrations or larger and slower conformational movements are more important depends on the type of reaction involved. These new insights also have implications in understanding allosteric effects, producing designer enzymes and developing new drugs.

Allosteric modulation

Allosteric enzymes change their structure in response to binding of effectors. Modulation can be direct, where the effector binds directly to binding sites in the enzyme, or indirect, where the effector binds to other proteins or protein subunits that interact with the allosteric enzyme and thus influence catalytic activity.

Cofactors and coenzymes

Cofactors

Some enzymes do not need any additional components to show full activity. However, others require non-protein molecules to be bound for activity. Cofactors can be either inorganic (e.g., metal ions and iron-sulfur clusters) or organic compounds, (e.g., flavin and heme). Organic cofactors are usually called prosthetic groups. Tightly-bound cofactors are distinguished from coenzymes, such as NADH, since cofactors are regenerated as part of the catalytic mechanism and are not released from the active site during the reaction.

An example of an enzyme that contains a cofactor is carbonic anhydrase, and is shown in the diagram above with four zinc cofactors bound in its active sites.^[25] These tightly-bound molecules are usually found in the active site and are involved in catalysis. For example, flavin and heme cofactors are often involved in redox reactions.

Enzymes that require a cofactor but do not have one bound are called apoenzymes. An apoenzyme together with its cofactor(s) is called a holoenzyme (i.e., the active form). Most cofactors are not covalently attached to an enzyme, but are very tightly bound. However, organic prosthetic groups can be covalently bound (e.g., thiamine pyrophosphate in the enzyme pyruvate dehydrogenase).

Coenzymes

Coenzymes are small molecules that transport chemical groups from one enzyme to another.^[26] Some of these chemicals such as riboflavin, thiamine and folic acid are vitamins, this is when these compounds cannot be made in the body and must be acquired from the diet. The chemical groups carried include the hydride ion (H+ + 2e-) carried by NAD or NADP⁺, the acetyl group carried by coenzyme A, formyl, methenyl or methyl groups carried by folic acid and the methyl group carried by S-adenosylmethionine.

Since coenzymes are chemically changed as a consequence of enzyme action, it is useful to consider coenzymes to be a special class of substrates, or second substrates, which are common to many different enzymes. For example, about 700 enzymes are known to use the cofactor NADH.^[27]

Coenzymes are usually regenerated and their concentrations maintained at a steady level inside the cell: for example, NADPH is regenerated through the pentose phosphate pathway and S-adenosylmethionine by methionine adenosyltransferase.

Thermodynamics

As with all catalysts, all reactions catalyzed by enzymes must be "spontaneous" (containing a net negative Gibbs free energy). In the presence of an enzyme, a reaction runs in the same direction as it would without the enzyme, just more quickly. However, the uncatalyzed, "spontaneous" reaction might lead to different products than the catalyzed reaction. Furthermore, enzymes can couple two or more reactions, so that a thermodynamically favorable reaction can be used to "drive" a thermodynamically unfavorable one. For example, the cleavage of the high-energy compound ATP is often used to drive other energetically unfavorable chemical reactions.

Enzymes catalyze the forward and backward reactions equally. They do not alter the equilibrium itself, but only the speed at which it is reached. For example, carbonic anhydrase catalyzes its reaction in either direction depending on the concentration of its reactants.

Nevertheless, if the physiological concentrations of the substrates and products have a large negative Gibbs free energy (exergonic), then the reaction is effectively irreversible. Under these conditions it is possible that the enzyme will only catalyze the reaction in one direction.

Kinetics

Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products. The rate data used in kinetic analyses are obtained from enzyme assays. In 1913 Leonor Michaelis and Maud Menten proposed a quantitative theory of enzyme kinetics, which is referred to as Michaelis-Menten kinetics.^[28] Their work was further developed by G. E. Briggs and J. B. S. Haldane, who derived kinetic equations that are still widely used today.^[29]

The major contribution of Michaelis and Menten was to think of enzyme reactions in two stages. In the first, the substrate binds reversibly to the enzyme, forming the enzyme-substrate complex. This is sometimes called the Michaelis-Menten complex in their honor. The enzyme then catalyzes the chemical step in the reaction and releases the product.

Enzymes can catalyze up to several million reactions per second. To find the maximum speed of an enzymatic reaction, the substrate concentration is increased until a constant rate of product formation is seen. This is shown in the saturation curve, shown on the right. Saturation happens because, as substrate concentration increases, more and more of the free enzyme is converted into the substrate-bound ES form. At the maximum velocity (V_max) of the enzyme, all enzyme active sites are saturated with substrate, and the amount of ES complex is the same as the total amount of enzyme.

However, V_max is only one kinetic constant of enzymes. The amount of substrate needed to achieve a given rate of reaction is also important. This is given by the Michaelis-Menten constant (K_m), which is the substrate concentration required for an enzyme to reach one-half its maximum velocity. Each enzyme has a characteristic K_m for a given substrate, and this can show how tight the binding of the substrate is to the enzyme. Another useful constant is k_cat, which is the number of substrate molecules handled by one active site per second.

The efficiency of an enzyme can be expressed in terms of k_cat/K_m. This is also called the specificity constant and incorporates the rate constants for all steps in the reaction. Because the specificity constant reflects both affinity and catalytic ability, it is useful for comparing different enzymes against each other, or the same enzyme with different substrates. The theoretical maximum for the specificity constant is called the diffusion limit and is about 10⁸ to 10⁹ (M^-1 s^-1). At this point every collision of the enzyme with its substrate will result in catalysis, and the rate of product formation is not limited by the reaction rate but by the diffusion rate. Enzymes with this property are called catalytically perfect or kinetically perfect. Example of such enzymes are triose-phosphate isomerase, carbonic anhydrase, acetylcholinesterase, catalase, fumarase, ß-lactamase, and superoxide dismutase.

Some enzymes operate with kinetics which are faster than diffusion rates, which would seem to be impossible. Several mechanisms have been invoked to explain this phenomenon. Some proteins are believed to accelerate catalysis by drawing their substrate in and pre-orienting them by using dipolar electric fields. Other models invoke a quantum-mechanical tunneling explanation, whereby a proton or an electron can tunnel through activation barriers, although for proton tunneling this model remains somewhat controversial.^[30]^[31] Quantum tunneling for protons has been observed in tryptamine.^[32] This suggests that enzyme catalysis may be more accurately characterized as "through the barrier" rather than the traditional model, which requires substrates to go "over" a lowered energy barrier.

Inhibition

Reversible inhibitors

In competitive inhibition the inhibitor binds to the substrate binding site as shown (right top), thus preventing substrate from binding (EI complex). Often competitive inhibitors strongly resemble the real substrate of the enzyme. For example, methotrexate is a competitive inhibitor of the enzyme dihydrofolate reductase, which catalyzes the reduction of dihydrofolate to tetrahydrofolate. The similarity between the structures of folic acid and this drug are shown on the right.

Non-competitive inhibitors never bind to the active site, but to other parts of the enzyme that can be far away from the substrate binding site (right, bottom). Moreover, non-competitive inhibitors only bind to the enzyme-substrate (ES) complex and not to free enzyme. Their binding to this site changes the shape of the enzyme and stops the active site binding substrate(s). Consequently, since there is no direct competition between the substrate and inhibitor for the enzyme, the extent of inhibition depends only on the inhibitor concentration and will not be affected by the substrate concentration.

Irreversible inhibitors

Some enzyme inhibitors react with the enzyme and form a covalent adduct with the protein. The inactivation produced by this type of inhibitor cannot be reversed. A class of these compounds called Suicide inhibitors includes eflornithine a drug used to treat the parasitic disease sleeping sickness.

Uses of inhibitors

Inhibitors are often used as drugs, but they can also act as poisons. However, the difference between a drug and a poison is usually only a matter of amount, since most drugs are toxic at some level, as Paracelsus wrote, "In all things there is a poison, and there is nothing without a poison."^[33] Equally, antibiotics and other anti-infective drugs are just specific poisons that can kill a pathogen but not its host.

An example of an inhibitor being used as a drug is aspirin, which inhibits the COX-1 and COX-2 enzymes that produce the inflammation messenger prostaglandin, thus suppressing pain and inflammation. The poison cyanide is an irreversible enzyme inhibitor that combines with the copper prosthetic groups of the enzyme cytochrome c oxidase and blocks cellular respiration.

In many organisms inhibitors may act as part of a feedback mechanism. If an enzyme produces too much of one substance in the organism, that substance may act as an inhibitor for the enzyme that produces it, causing production of the substance to slow down or stop when there is sufficient amount. This is a form of negative feedback.

Biological function

Enzymes serve a wide variety of functions inside living organisms. They are indispensable for signal transduction and cell regulation, often via kinases and phosphatases. They also generate movement, with myosin hydrolysing ATP to generate muscle contraction and also moving cargo around the cell as part of the cytoskeleton. Other ATPases in the cell membrane are ion pumps involved in active transport. Enzymes are also involved in more exotic functions, such as luciferase generating light in fireflies.

Viruses can contain enzymes for infecting cells, such as the HIV integrase and reverse transcriptase, or for viral release from cells, like the influenza virus neuraminidase.

Metabolism

Several enzymes can work together in a specific order, creating metabolic pathways. In a metabolic pathway, one enzyme takes the product of another enzyme as a substrate. After the catalytic reaction, the product is then passed on to another enzyme. Sometimes more than one enzyme can catalyse the same reaction in parallel, this can allow more complex regulation: with for example a low contant activity being provided by one enzyme but an inducible high activity from a second enzyme.

Enzymes determine what steps occur in these pathways. Without enzymes, metabolism would neither progress through the same steps, nor be fast enough to serve the needs of the cell. Indeed, a metabolic pathway such as glycolysis could not exist independently of enzymes. Glucose, for example, can react directly with ATP to become phosphorylated at one or more of its carbons. However, if hexokinase is present, glucose-6-phosphate is the only product, as this reaction will occur most swiftly. Consequently, the network of metabolic pathways within each cell depends on the set of functional enzymes that are present.

Control of activity

Involvement in disease

Since the tight control of enzyme activity is essential for homeostasis, any malfunction (mutation, overproduction, underproduction or deletion) of a single critical enzyme can lead to a genetic disease. The importance of enzymes is shown by the fact that a lethal illness can be caused by the malfunction of just one type of enzyme out of the thousands of types present in our bodies.

One example is the most common type of phenylketonuria. Mutation of this gene causes a single amino acid change in the enzyme phenylalanine hydroxylase, which catalyzes the first step in the degradation of phenylalanine. The resulting build-up of phenylalanine and related products can lead to mental retardation if the disease is untreated.^[36]

Another example is when germline mutations in genes coding for DNA repair enzymes cause hereditary cancer syndromes such as xeroderma pigmentosum. Defects in these enzymes cause cancer since the body is less able to repair mutations in the genome. This causes a slow accumulation of mutations and results in the development of many types of cancer in the sufferer.

Naming conventions

An enzyme's name is a description of what it does, with the word ending in -ase. Examples are alcohol dehydrogenase and DNA polymerase. Kinases are enzymes that transfer phosphate groups. This results in different enzymes with the same function having the same basic name; they are therefore distinguished by other characteristics, such as their optimal pH (alkaline phosphatase) or their location (membrane ATPase). Furthermore, the reversibility of chemical reactions means that the normal physiological direction of an enzyme reaction may not be seen under laboratory conditions. This can result in the same enzyme being identified with two different names: one coming from the laboratory identification and the other from its behavior in the cell. For instance, the enzyme formally known as xylitol:NAD+ 2-oxidoreductase (D-xylulose-forming) is more commonly referred to from the cellular viewpoint as D-xylulose reductase, since the function of the enzyme in the cell is actually the reverse of what is often seen under laboratory conditions.

The International Union of Biochemistry and Molecular Biology and the International Union of Pure and Applied Chemistry have developed a nomenclature for enzymes, the EC numbers; each enzyme is described by a sequence of four numbers preceded by "EC". However, this is not a perfect solution, as enzymes from different species or even very similar enzymes in the same species may have identical EC numbers.

The complete nomenclature can be browsed at http://www.chem.qmul.ac.uk/iubmb/enzyme/.

Industrial applications

Enzymes are used in the chemical industry and other industrial applications when extremely specific catalysts are required. However, enzymes in general are limited in the number of reactions they have evolved to catalyse and also by their lack of stability in organic solvents and at high temperatures. Consequently, protein engineering is an active area of research and involves attempts to create new enzymes with novel properties, either through rational design or in vitro evolution.^[37]^[38]

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