Enzyme

Enzymes /ˈɛnzaɪmz/ are macromolecular biological catalysts. Enzymes accelerate chemical reactions. The molecules upon which enzymes may act are called substrates and the enzyme converts the substrates into different molecules known as products. Almost all metabolic processes in the cell need enzyme catalysis in order to occur at rates fast enough to sustain life.[1]:8.1 Metabolic pathways depend upon enzymes to catalyze individual steps. The study of enzymes is called enzymology and a new field of pseudoenzyme analysis has recently grown up, recognising that during evolution, some enzymes have lost the ability to carry out biological catalysis, which is often reflected in their amino acid sequences and unusual 'pseudocatalytic' properties.[2][3]

Enzymes are known to catalyze more than 5,000 biochemical reaction types.[4] Most enzymes are proteins, although a few are catalytic RNA molecules. The latter are called ribozymes. Enzymes' specificity comes from their unique three-dimensional structures.

Like all catalysts, enzymes increase the reaction rate by lowering its activation energy. Some enzymes can make their conversion of substrate to product occur many millions of times faster. An extreme example is orotidine 5'-phosphate decarboxylase, which allows a reaction that would otherwise take millions of years to occur in milliseconds.[5][6] Chemically, enzymes are like any catalyst and are not consumed in chemical reactions, nor do they alter the equilibrium of a reaction. Enzymes 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. Many therapeutic drugs and poisons are enzyme inhibitors. An enzyme's activity decreases markedly outside its optimal temperature and pH, and many enzymes are (permanently) denatured when exposed to excessive heat, losing their structure and catalytic properties.

Some enzymes are used commercially, for example, in the synthesis of antibiotics. Some household products use enzymes to speed up chemical reactions: enzymes in biological washing powders break down protein, starch or fat stains on clothes, and enzymes in meat tenderizer break down proteins into smaller molecules, making the meat easier to chew.

Glucosidase enzyme
The enzyme glucosidase converts the sugar maltose to two glucose sugars. Active site residues in red, maltose substrate in black, and NAD cofactor in yellow. (PDB: 1OBB​)

Etymology and history

Eduardbuchner
Eduard Buchner

By the late 17th and early 18th centuries, the digestion of meat by stomach secretions[7] and the conversion of starch to sugars by plant extracts and saliva were known but the mechanisms by which these occurred had not been identified.[8]

French chemist Anselme Payen was the first to discover an enzyme, diastase, in 1833.[9] A few decades later, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur concluded that this fermentation was caused 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 organization of the yeast cells, not with the death or putrefaction of the cells."[10]

In 1877, German physiologist Wilhelm Kühne (1837–1900) first used the term enzyme, which comes from Greek ἔνζυμον, "leavened" or "in yeast", to describe this process.[11] The word enzyme was used later to refer to nonliving substances such as pepsin, and the word ferment was used to refer to chemical activity produced by living organisms.[12]

Eduard Buchner submitted his first paper on the study of yeast extracts in 1897. In a series of experiments at the University of Berlin, he found that sugar was fermented by yeast extracts even when there were no living yeast cells in the mixture.[13] He named the enzyme that brought about the fermentation of sucrose "zymase".[14] In 1907, he received the Nobel Prize in Chemistry for "his discovery of cell-free fermentation". Following Buchner's example, enzymes are usually named according to the reaction they carry out: the suffix -ase is combined with the name of the substrate (e.g., lactase is the enzyme that cleaves lactose) or to the type of reaction (e.g., DNA polymerase forms DNA polymers).[15]

The biochemical identity of enzymes was still unknown in the early 1900s. Many scientists observed that enzymatic activity was associated with proteins, but others (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.[16] In 1926, James B. Sumner showed that the enzyme urease was a pure protein and crystallized it; he did likewise for the enzyme catalase in 1937. The conclusion that pure proteins can be enzymes was definitively demonstrated by John Howard Northrop and Wendell Meredith Stanley, who worked on the digestive enzymes pepsin (1930), trypsin and chymotrypsin. These three scientists were awarded the 1946 Nobel Prize in Chemistry.[17]

The discovery that enzymes could be crystallized 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.[18] 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.[19]

Naming conventions

An enzyme's name is often derived from its substrate or the chemical reaction it catalyzes, with the word ending in -ase.[1]:8.1.3 Examples are lactase, alcohol dehydrogenase and DNA polymerase. Different enzymes that catalyze the same chemical reaction are called isozymes.[1]:10.3

The International Union of Biochemistry and Molecular Biology have developed a nomenclature for enzymes, the EC numbers; each enzyme is described by a sequence of four numbers preceded by "EC", which stands for "Enzyme Commission". The first number broadly classifies the enzyme based on its mechanism.[20]

The top-level classification is:

These sections are subdivided by other features such as the substrate, products, and chemical mechanism. An enzyme is fully specified by four numerical designations. For example, hexokinase (EC 2.7.1.1) is a transferase (EC 2) that adds a phosphate group (EC 2.7) to a hexose sugar, a molecule containing an alcohol group (EC 2.7.1).[21]

Structure

Q10 graph c
Enzyme activity initially increases with temperature (Q10 coefficient) until the enzyme's structure unfolds (denaturation), leading to an optimal rate of reaction at an intermediate temperature.

Enzymes are generally globular proteins, acting alone or in larger complexes. The sequence of the amino acids specifies the structure which in turn determines the catalytic activity of the enzyme.[22] Although structure determines function, a novel enzymatic activity cannot yet be predicted from structure alone.[23] Enzyme structures unfold (denature) when heated or exposed to chemical denaturants and this disruption to the structure typically causes a loss of activity.[24] Enzyme denaturation is normally linked to temperatures above a species' normal level; as a result, enzymes from bacteria living in volcanic environments such as hot springs are prized by industrial users for their ability to function at high temperatures, allowing enzyme-catalysed reactions to be operated at a very high rate.

Enzymes are usually much larger than their substrates. Sizes range from just 62 amino acid residues, for the monomer of 4-oxalocrotonate tautomerase,[25] to over 2,500 residues in the animal fatty acid synthase.[26] Only a small portion of their structure (around 2–4 amino acids) is directly involved in catalysis: the catalytic site.[27] This catalytic site is located next to one or more binding sites where residues orient the substrates. The catalytic site and binding site together comprise the enzyme's active site. The remaining majority of the enzyme structure serves to maintain the precise orientation and dynamics of the active site.[28]

In some enzymes, no amino acids are directly involved in catalysis; instead, the enzyme contains sites to bind and orient catalytic cofactors.[28] Enzyme structures may also contain allosteric sites where the binding of a small molecule causes a conformational change that increases or decreases activity.[29]

A small number of RNA-based biological catalysts called ribozymes exist, which again can act alone or in complex with proteins. The most common of these is the ribosome which is a complex of protein and catalytic RNA components.[1]:2.2

Mechanism

Enzyme structure
Organisation of enzyme structure and lysozyme example. Binding sites in blue, catalytic site in red and peptidoglycan substrate in black. (PDB: 9LYZ​)

Substrate binding

Enzymes must bind their substrates before they can catalyse any chemical reaction. Enzymes are usually very specific as to what substrates they bind and then the chemical reaction catalysed. Specificity is achieved by binding pockets with complementary shape, charge and hydrophilic/hydrophobic characteristics to the substrates. Enzymes can therefore distinguish between very similar substrate molecules to be chemoselective, regioselective and stereospecific.[30]

Some of the enzymes showing the highest specificity and accuracy are involved in the copying and expression of the genome. Some of these enzymes have "proof-reading" mechanisms. Here, an enzyme such as DNA polymerase catalyzes a reaction in a first step and then checks that the product is correct in a second step.[31] This two-step process results in average error rates of less than 1 error in 100 million reactions in high-fidelity mammalian polymerases.[1]:5.3.1 Similar proofreading mechanisms are also found in RNA polymerase,[32] aminoacyl tRNA synthetases[33] and ribosomes.[34]

Conversely, some enzymes display enzyme promiscuity, having broad specificity and acting on a range of different physiologically relevant substrates. Many enzymes possess small side activities which arose fortuitously (i.e. neutrally), which may be the starting point for the evolutionary selection of a new function.[35][36]

Hexokinase induced fit
Enzyme changes shape by induced fit upon substrate binding to form enzyme-substrate complex. Hexokinase has a large induced fit motion that closes over the substrates adenosine triphosphate and xylose. Binding sites in blue, substrates in black and Mg2+ cofactor in yellow. (PDB: 2E2N​, 2E2Q​)

"Lock and key" model

To explain the observed specificity of enzymes, in 1894 Emil Fischer proposed that both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another.[37] This is often referred to as "the lock and key" model.[1]:8.3.2 This early model explains enzyme specificity, but fails to explain the stabilization of the transition state that enzymes achieve.[38]

Induced fit model

In 1958, Daniel Koshland suggested a modification to the lock and key model: since enzymes are rather flexible structures, the active site is continuously reshaped by interactions with the substrate as the substrate interacts with the enzyme.[39] As a result, the substrate does not simply bind to a rigid active site; the amino acid side-chains that make up the active site are molded into the precise positions that enable the enzyme to perform its catalytic function. In some cases, such as glycosidases, the substrate molecule also changes shape slightly as it enters the active site.[40] The active site continues to change until the substrate is completely bound, at which point the final shape and charge distribution is determined.[41] Induced fit may enhance the fidelity of molecular recognition in the presence of competition and noise via the conformational proofreading mechanism.[42]

Catalysis

Enzymes can accelerate reactions in several ways, all of which lower the activation energy (ΔG, Gibbs free energy)[43]

  1. By stabilizing the transition state:
    • Creating an environment with a charge distribution complementary to that of the transition state to lower its energy[44]
  2. By providing an alternative reaction pathway:
    • Temporarily reacting with the substrate, forming a covalent intermediate to provide a lower energy transition state[45]
  3. By destabilising the substrate ground state:
    • Distorting bound substrate(s) into their transition state form to reduce the energy required to reach the transition state[46]
    • By orienting the substrates into a productive arrangement to reduce the reaction entropy change[47] (the contribution of this mechanism to catalysis is relatively small)[48]

Enzymes may use several of these mechanisms simultaneously. For example, proteases such as trypsin perform covalent catalysis using a catalytic triad, stabilise charge build-up on the transition states using an oxyanion hole, complete hydrolysis using an oriented water substrate.[49]

Dynamics

Enzymes are not rigid, static structures; instead they have complex internal dynamic motions – that is, movements of parts of the enzyme's structure such as individual amino acid residues, groups of residues forming a protein loop or unit of secondary structure, or even an entire protein domain. These motions give rise to a conformational ensemble of slightly different structures that interconvert with one another at equilibrium. Different states within this ensemble may be associated with different aspects of an enzyme's function. For example, different conformations of the enzyme dihydrofolate reductase are associated with the substrate binding, catalysis, cofactor release, and product release steps of the catalytic cycle.[50]

Allosteric modulation

Allosteric sites are pockets on the enzyme, distinct from the active site, that bind to molecules in the cellular environment. These molecules then cause a change in the conformation or dynamics of the enzyme that is transduced to the active site and thus affects the reaction rate of the enzyme.[51] In this way, allosteric interactions can either inhibit or activate enzymes. Allosteric interactions with metabolites upstream or downstream in an enzyme's metabolic pathway cause feedback regulation, altering the activity of the enzyme according to the flux through the rest of the pathway.[52]

Cofactors

Transketolase + TPP
Chemical structure for thiamine pyrophosphate and protein structure of transketolase. Thiamine pyrophosphate cofactor in yellow and xylulose 5-phosphate substrate in black. (PDB: 4KXV​)

Some enzymes do not need additional components to show full activity. Others require non-protein molecules called cofactors to be bound for activity.[53] Cofactors can be either inorganic (e.g., metal ions and iron-sulfur clusters) or organic compounds (e.g., flavin and heme). These cofactors serve many purposes; for instance, metal ions can help in stabilizing nucleophilic species within the active site.[54] Organic cofactors can be either coenzymes, which are released from the enzyme's active site during the reaction, or prosthetic groups, which are tightly bound to an enzyme. Organic prosthetic groups can be covalently bound (e.g., biotin in enzymes such as pyruvate carboxylase).[55]

An example of an enzyme that contains a cofactor is carbonic anhydrase, which is shown in the ribbon diagram above with a zinc cofactor bound as part of its active site.[56] These tightly bound ions or molecules are usually found in the active site and are involved in catalysis.[1]:8.1.1 For example, flavin and heme cofactors are often involved in redox reactions.[1]:17

Enzymes that require a cofactor but do not have one bound are called apoenzymes or apoproteins. An enzyme together with the cofactor(s) required for activity is called a holoenzyme (or haloenzyme). The term holoenzyme can also be applied to enzymes that contain multiple protein subunits, such as the DNA polymerases; here the holoenzyme is the complete complex containing all the subunits needed for activity.[1]:8.1.1

Coenzymes

Coenzymes are small organic molecules that can be loosely or tightly bound to an enzyme. Coenzymes transport chemical groups from one enzyme to another.[57] Examples include NADH, NADPH and adenosine triphosphate (ATP). Some coenzymes, such as flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), thiamine pyrophosphate (TPP), and tetrahydrofolate (THF), are derived from vitamins. These coenzymes cannot be synthesized by the body de novo and closely related compounds (vitamins) must be acquired from the diet. The chemical groups carried include:

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 1000 enzymes are known to use the coenzyme NADH.[58]

Coenzymes are usually continuously 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. This continuous regeneration means that small amounts of coenzymes can be used very intensively. For example, the human body turns over its own weight in ATP each day.[59]

Thermodynamics

Enzyme catalysis energy levels 2
The energies of the stages of a chemical reaction. Uncatalysed (dashed line), substrates need a lot of activation energy to reach a transition state, which then decays into lower-energy products. When enzyme catalysed (solid line), the enzyme binds the substrates (ES), then stabilizes the transition state (ES) to reduce the activation energy required to produce products (EP) which are finally released.

As with all catalysts, enzymes do not alter the position of the chemical equilibrium of the reaction. In the presence of an enzyme, the reaction runs in the same direction as it would without the enzyme, just more quickly.[1]:8.2.3 For example, carbonic anhydrase catalyzes its reaction in either direction depending on the concentration of its reactants:[60]

(in tissues; high CO2 concentration)

(1)

(in lungs; low CO2 concentration)

(2)

The rate of a reaction is dependent on the activation energy needed to form the transition state which then decays into products. Enzymes increase reaction rates by lowering the energy of the transition state. First, binding forms a low energy enzyme-substrate complex (ES). Secondly the enzyme stabilises the transition state such that it requires less energy to achieve compared to the uncatalyzed reaction (ES). Finally the enzyme-product complex (EP) dissociates to release the products.[1]:8.3

Enzymes can couple two or more reactions, so that a thermodynamically favorable reaction can be used to "drive" a thermodynamically unfavourable one so that the combined energy of the products is lower than the substrates. For example, the hydrolysis of ATP is often used to drive other chemical reactions.[61]

Kinetics

Enzyme mechanism 2
A chemical reaction mechanism with or without enzyme catalysis. The enzyme (E) binds substrate (S) to produce product (P).
Michaelis Menten curve 2
Saturation curve for an enzyme reaction showing the relation between the substrate concentration and reaction rate.

Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products.[62] The rate data used in kinetic analyses are commonly obtained from enzyme assays. In 1913 Leonor Michaelis and Maud Leonora Menten proposed a quantitative theory of enzyme kinetics, which is referred to as Michaelis–Menten kinetics.[63] 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. This work was further developed by G. E. Briggs and J. B. S. Haldane, who derived kinetic equations that are still widely used today.[64]

Enzyme rates depend on solution conditions and substrate concentration. 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 on the right. Saturation happens because, as substrate concentration increases, more and more of the free enzyme is converted into the substrate-bound ES complex. At the maximum reaction rate (Vmax) of the enzyme, all the enzyme active sites are bound to substrate, and the amount of ES complex is the same as the total amount of enzyme.[1]:8.4

Vmax is only one of several important kinetic parameters. The amount of substrate needed to achieve a given rate of reaction is also important. This is given by the Michaelis-Menten constant (Km), which is the substrate concentration required for an enzyme to reach one-half its maximum reaction rate; generally, each enzyme has a characteristic KM for a given substrate. Another useful constant is kcat, also called the turnover number, which is the number of substrate molecules handled by one active site per second.[1]:8.4

The efficiency of an enzyme can be expressed in terms of kcat/Km. This is also called the specificity constant and incorporates the rate constants for all steps in the reaction up to and including the first irreversible step. 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, β-lactamase, and superoxide dismutase.[1]:8.4.2 The turnover of such enzymes can reach several million reactions per second.[1]:9.2 But most enzymes are far from perfect: the average values of and are about and , respectively.[65]

Michaelis–Menten kinetics relies on the law of mass action, which is derived from the assumptions of free diffusion and thermodynamically driven random collision. Many biochemical or cellular processes deviate significantly from these conditions, because of macromolecular crowding and constrained molecular movement.[66] More recent, complex extensions of the model attempt to correct for these effects.[67]

Inhibition

DHFR methotrexate inhibitor
An enzyme binding site that would normally bind substrate can alternatively bind a competitive inhibitor, preventing substrate access. Dihydrofolate reductase is inhibited by methotrexate which prevents binding of its substrate, folic acid.[68] Binding site in blue, inhibitor in green, and substrate in black. (PDB: 4QI9​)
Methotrexate vs folate 2
The coenzyme folic acid (left) and the anti-cancer drug methotrexate (right) are very similar in structure (differences show in green). As a result, methotrexate is a competitive inhibitor of many enzymes that use folates.

Enzyme reaction rates can be decreased by various types of enzyme inhibitors.[69]:73–74

Types of inhibition

Competitive

A competitive inhibitor and substrate cannot bind to the enzyme at the same time.[70] Often competitive inhibitors strongly resemble the real substrate of the enzyme. For example, the drug methotrexate is a competitive inhibitor of the enzyme dihydrofolate reductase, which catalyzes the reduction of dihydrofolate to tetrahydrofolate.[68] The similarity between the structures of dihydrofolate and this drug are shown in the accompanying figure. This type of inhibition can be overcome with high substrate concentration. In some cases, the inhibitor can bind to a site other than the binding-site of the usual substrate and exert an allosteric effect to change the shape of the usual binding-site.[71]

Non-competitive

A non-competitive inhibitor binds to a site other than where the substrate binds. The substrate still binds with its usual affinity and hence Km remains the same. However the inhibitor reduces the catalytic efficiency of the enzyme so that Vmax is reduced. In contrast to competitive inhibition, non-competitive inhibition cannot be overcome with high substrate concentration.[69]:76–78

Uncompetitive

An uncompetitive inhibitor cannot bind to the free enzyme, only to the enzyme-substrate complex; hence, these types of inhibitors are most effective at high substrate concentration. In the presence of the inhibitor, the enzyme-substrate complex is inactive.[69]:78 This type of inhibition is rare.[72]

Mixed

A mixed inhibitor binds to an allosteric site and the binding of the substrate and the inhibitor affect each other. The enzyme's function is reduced but not eliminated when bound to the inhibitor. This type of inhibitor does not follow the Michaelis-Menten equation.[69]:76–78

Irreversible

An irreversible inhibitor permanently inactivates the enzyme, usually by forming a covalent bond to the protein.[73] Penicillin[74] and aspirin[75] are common drugs that act in this manner.

Functions of inhibitors

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 at the beginning of the pathway 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. Major metabolic pathways such as the citric acid cycle make use of this mechanism.[1]:17.2.2

Since inhibitors modulate the function of enzymes they are often used as drugs. Many such drugs are reversible competitive inhibitors that resemble the enzyme's native substrate, similar to methotrexate above; other well-known examples include statins used to treat high cholesterol,[76] and protease inhibitors used to treat retroviral infections such as HIV.[77] A common example of an irreversible inhibitor that is used as a drug is aspirin, which inhibits the COX-1 and COX-2 enzymes that produce the inflammation messenger prostaglandin.[75] Other enzyme inhibitors are poisons. For example, the poison cyanide is an irreversible enzyme inhibitor that combines with the copper and iron in the active site of the enzyme cytochrome c oxidase and blocks cellular respiration.[78]

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.[79] They also generate movement, with myosin hydrolyzing ATP to generate muscle contraction, and also transport cargo around the cell as part of the cytoskeleton.[80] 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.[81] Viruses can also contain enzymes for infecting cells, such as the HIV integrase and reverse transcriptase, or for viral release from cells, like the influenza virus neuraminidase.[82]

An important function of enzymes is in the digestive systems of animals. Enzymes such as amylases and proteases break down large molecules (starch or proteins, respectively) into smaller ones, so they can be absorbed by the intestines. Starch molecules, for example, are too large to be absorbed from the intestine, but enzymes hydrolyze the starch chains into smaller molecules such as maltose and eventually glucose, which can then be absorbed. Different enzymes digest different food substances. In ruminants, which have herbivorous diets, microorganisms in the gut produce another enzyme, cellulase, to break down the cellulose cell walls of plant fiber.[83]

Metabolism

Glycolysis metabolic pathway
The metabolic pathway of glycolysis releases energy by converting glucose to pyruvate via a series of intermediate metabolites. Each chemical modification (red box) is performed by a different enzyme.

Several enzymes can work together in a specific order, creating metabolic pathways.[1]:30.1 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 catalyze the same reaction in parallel; this can allow more complex regulation: with, for example, a low constant activity provided by one enzyme but an inducible high activity from a second enzyme.[84]

Enzymes determine what steps occur in these pathways. Without enzymes, metabolism would neither progress through the same steps and could not be regulated to serve the needs of the cell. Most central metabolic pathways are regulated at a few key steps, typically through enzymes whose activity involves the hydrolysis of ATP. Because this reaction releases so much energy, other reactions that are thermodynamically unfavorable can be coupled to ATP hydrolysis, driving the overall series of linked metabolic reactions.[1]:30.1

Control of activity

There are five main ways that enzyme activity is controlled in the cell.[1]:30.1.1

Regulation

Enzymes can be either activated or inhibited by other molecules. For example, the end product(s) of a metabolic pathway are often inhibitors for one of the first enzymes of the pathway (usually the first irreversible step, called committed step), thus regulating the amount of end product made by the pathways. Such a regulatory mechanism is called a negative feedback mechanism, because the amount of the end product produced is regulated by its own concentration.[85]:141–48 Negative feedback mechanism can effectively adjust the rate of synthesis of intermediate metabolites according to the demands of the cells. This helps with effective allocations of materials and energy economy, and it prevents the excess manufacture of end products. Like other homeostatic devices, the control of enzymatic action helps to maintain a stable internal environment in living organisms.[85]:141

Post-translational modification

Examples of post-translational modification include phosphorylation, myristoylation and glycosylation.[85]:149–69 For example, in the response to insulin, the phosphorylation of multiple enzymes, including glycogen synthase, helps control the synthesis or degradation of glycogen and allows the cell to respond to changes in blood sugar.[86] Another example of post-translational modification is the cleavage of the polypeptide chain. Chymotrypsin, a digestive protease, is produced in inactive form as chymotrypsinogen in the pancreas and transported in this form to the stomach where it is activated. This stops the enzyme from digesting the pancreas or other tissues before it enters the gut. This type of inactive precursor to an enzyme is known as a zymogen[85]:149–53 or proenzyme.

Quantity

Enzyme production (transcription and translation of enzyme genes) can be enhanced or diminished by a cell in response to changes in the cell's environment. This form of gene regulation is called enzyme induction. For example, bacteria may become resistant to antibiotics such as penicillin because enzymes called beta-lactamases are induced that hydrolyse the crucial beta-lactam ring within the penicillin molecule.[87] Another example comes from enzymes in the liver called cytochrome P450 oxidases, which are important in drug metabolism. Induction or inhibition of these enzymes can cause drug interactions.[88] Enzyme levels can also be regulated by changing the rate of enzyme degradation.[1]:30.1.1 The opposite of enzyme induction is enzyme repression.

Subcellular distribution

Enzymes can be compartmentalized, with different metabolic pathways occurring in different cellular compartments. For example, fatty acids are synthesized by one set of enzymes in the cytosol, endoplasmic reticulum and Golgi and used by a different set of enzymes as a source of energy in the mitochondrion, through β-oxidation.[89] In addition, trafficking of the enzyme to different compartments may change the degree of protonation (e.g., the neutral cytoplasm and the acidic lysosome) or oxidative state (e.g., oxidizing periplasm or reducing cytoplasm) which in turn affects enzyme activity.[90] In contrast to partitioning into membrane bound organelles, enzyme subcellular localisation may also be altered through polymerisation of enzymes into macromolecular cytoplasmic filaments.[91][92]

Organ specialization

In multicellular eukaryotes, cells in different organs and tissues have different patterns of gene expression and therefore have different sets of enzymes (known as isozymes) available for metabolic reactions. This provides a mechanism for regulating the overall metabolism of the organism. For example, hexokinase, the first enzyme in the glycolysis pathway, has a specialized form called glucokinase expressed in the liver and pancreas that has a lower affinity for glucose yet is more sensitive to glucose concentration.[93] This enzyme is involved in sensing blood sugar and regulating insulin production.[94]

Involvement in disease

Phenylalanine hydroxylase mutations
In phenylalanine hydroxylase over 300 different mutations throughout the structure cause phenylketonuria. Phenylalanine substrate and tetrahydrobiopterin coenzyme in black, and Fe2+ cofactor in yellow. (PDB: 1KW0​)

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 malfunction of just one type of enzyme out of the thousands of types present in the human body can be fatal. An example of a fatal genetic disease due to enzyme insufficiency is Tay–Sachs disease, in which patients lack the enzyme hexosaminidase.[95][96]

One example of enzyme deficiency is the most common type of phenylketonuria. Many different single amino acid mutations in the enzyme phenylalanine hydroxylase, which catalyzes the first step in the degradation of phenylalanine, result in build-up of phenylalanine and related products. Some mutations are in the active site, directly disrupting binding and catalysis, but many are far from the active site and reduce activity by destabilising the protein structure, or affecting correct oligomerisation.[97][98] This can lead to intellectual disability if the disease is untreated.[99] Another example is pseudocholinesterase deficiency, in which the body's ability to break down choline ester drugs is impaired.[100] Oral administration of enzymes can be used to treat some functional enzyme deficiencies, such as pancreatic insufficiency[101] and lactose intolerance.[102]

Another way enzyme malfunctions can cause disease comes from germline mutations in genes coding for DNA repair enzymes. Defects in these enzymes cause cancer because cells are less able to repair mutations in their genomes. This causes a slow accumulation of mutations and results in the development of cancers. An example of such a hereditary cancer syndrome is xeroderma pigmentosum, which causes the development of skin cancers in response to even minimal exposure to ultraviolet light.[103][104]

Industrial applications

Enzymes are used in the chemical industry and other industrial applications when extremely specific catalysts are required. Enzymes in general are limited in the number of reactions they have evolved to catalyze and also by their lack of stability in organic solvents and at high temperatures. As a consequence, 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.[105][106] These efforts have begun to be successful, and a few enzymes have now been designed "from scratch" to catalyze reactions that do not occur in nature.[107]

Application Enzymes used Uses
Biofuel industry Cellulases Break down cellulose into sugars that can be fermented to produce cellulosic ethanol.[108]
Ligninases Pretreatment of biomass for biofuel production.[108]
Biological detergent Proteases, amylases, lipases Remove protein, starch, and fat or oil stains from laundry and dishware.[109]
Mannanases Remove food stains from the common food additive guar gum.[109]
Brewing industry Amylase, glucanases, proteases Split polysaccharides and proteins in the malt.[110]:150–9
Betaglucanases Improve the wort and beer filtration characteristics.[110]:545
Amyloglucosidase and pullulanases Make low-calorie beer and adjust fermentability.[110]:575
Acetolactate decarboxylase (ALDC) Increase fermentation efficiency by reducing diacetyl formation.[111]
Culinary uses Papain Tenderize meat for cooking.[112]
Dairy industry Rennin Hydrolyze protein in the manufacture of cheese.[113]
Lipases Produce Camembert cheese and blue cheeses such as Roquefort.[114]
Food processing Amylases Produce sugars from starch, such as in making high-fructose corn syrup.[115]
Proteases Lower the protein level of flour, as in biscuit-making.[116]
Trypsin Manufacture hypoallergenic baby foods.[116]
Cellulases, pectinases Clarify fruit juices.[117]
Molecular biology Nucleases, DNA ligase and polymerases Use restriction digestion and the polymerase chain reaction to create recombinant DNA.[1]:6.2
Paper industry Xylanases, hemicellulases and lignin peroxidases Remove lignin from kraft pulp.[118]
Personal care Proteases Remove proteins on contact lenses to prevent infections.[119]
Starch industry Amylases Convert starch into glucose and various syrups.[120]

See also

References

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Further reading

General

  • Berg JM, Tymoczko JL, Stryer L (2002). Biochemistry (5th ed.). New York, NY: W. H. Freeman. ISBN 0-7167-3051-0., A biochemistry textbook available free online through NCBI Bookshelf.open access publication – free to read

Etymology and history

Enzyme structure and mechanism

  • Suzuki H (2015). How Enzymes Work: From Structure to Function. Boca Raton, FL: CRC Press. ISBN 978-981-4463-92-8.

Kinetics and inhibition

  • Cornish-Bowden A (2012). Fundamentals of Enzyme Kinetics (4th ed.). Weinheim: Wiley-VCH. ISBN 978-3527330744.
ACE inhibitor

An angiotensin-converting-enzyme inhibitor (ACE inhibitor) is a pharmaceutical drug used primarily for the treatment of hypertension (elevated blood pressure) and congestive heart failure.

This group of drugs causes relaxation of blood vessels as well as a decrease in blood volume, which leads to lower blood pressure and decreased oxygen demand from the heart. They inhibit the angiotensin-converting enzyme, an important component of the renin–angiotensin system.

Frequently prescribed ACE inhibitors include benazepril, zofenopril, perindopril, trandolapril, captopril, enalapril, lisinopril, and ramipril.

Active site

In biology, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of residues that form temporary bonds with the substrate (binding site) and residues that catalyse a reaction of that substrate (catalytic site). Although the active site is small relative to the whole volume of the enzyme (it only occupies 10~20% of the total volume), it is the most important part of the enzyme as it directly catalyzes the chemical reaction. It usually consists of three to four amino acids, while other amino acids within the protein are required to maintain the protein tertiary structure of the enzyme.Each active site is specially designed in response to their substrates, as a result, most enzymes have specificity and can only react with particular substrates. This specificity is determined by the arrangement of amino acids within the active site and the structure of the substrates. Sometimes enzymes also need to bind with some cofactors to fulfil their function. The active site is usually a groove or pocket of the enzyme which can be located in a deep tunnel within the enzyme, or between the interfaces of multimeric enzymes. An active site can catalyse a reaction repeatedly as residues are not altered at the end of the reaction (they may change during the reaction, but are regenerated by the end). This process is achieved by lowering the activation energy of the reaction, so more substrates have enough energy to undergo reaction.

BRENDA

BRENDA (The Comprehensive Enzyme Information System) is an information system representing one of the most comprehensive enzyme repositories. It is an electronic resource that comprises molecular and biochemical information on enzymes that have been classified by the IUBMB. Every classified enzyme is characterized with respect to its catalyzed biochemical reaction. Kinetic properties of the corresponding reactants (that is, substrates and products) are described in detail. BRENDA contains enzyme-specific data manually extracted from primary scientific literature and additional data derived from automatic information retrieval methods such as text mining. It provides a web-based user interface that allows a convenient and sophisticated access to the data.

Cofactor (biochemistry)

A cofactor is a non-protein chemical compound or metallic ion that is required for an enzyme's activity. Cofactors can be considered "helper molecules" that assist in biochemical transformations. The rates at which these happen are characterized by enzyme kinetics.

Cofactors can be subclassified as either inorganic ions or complex organic molecules called coenzymes, the latter of which is mostly derived from vitamins and other organic essential nutrients in small amounts. A coenzyme that is tightly or even covalently bound is termed a prosthetic group. Cosubstrates are transiently bound to the protein and will be released at some point, then get back in. The prosthetic groups, on the other hand, are bound permanently to the protein. Both of them have the same function, which is to facilitate the reaction of enzymes and protein. Additionally, some sources also limit the use of the term "cofactor" to inorganic substances. An inactive enzyme without the cofactor is called an apoenzyme, while the complete enzyme with cofactor is called a holoenzyme.Some enzymes or enzyme complexes require several cofactors. For example, the multienzyme complex pyruvate dehydrogenase at the junction of glycolysis and the citric acid cycle requires five organic cofactors and one metal ion: loosely bound thiamine pyrophosphate (TPP), covalently bound lipoamide and flavin adenine dinucleotide (FAD), and the cosubstrates nicotinamide adenine dinucleotide (NAD+) and coenzyme A (CoA), and a metal ion (Mg2+).Organic cofactors are often vitamins or made from vitamins. Many contain the nucleotide adenosine monophosphate (AMP) as part of their structures, such as ATP, coenzyme A, FAD, and NAD+. This common structure may reflect a common evolutionary origin as part of ribozymes in an ancient RNA world. It has been suggested that the AMP part of the molecule can be considered to be a kind of "handle" by which the enzyme can "grasp" the coenzyme to switch it between different catalytic centers.

Diffusion limited enzyme

A Diffusion limited enzyme is an enzyme which catalyses a reaction so efficiently that the rate limiting step is that of substrate diffusion into the active site, or product diffusion out. This is also known as kinetic perfection or catalytic perfection. Since the rate of catalysis of such enzymes is set by the diffusion-controlled reaction, it therefore represents an intrinsic, physical constraint on evolution (a maximum peak height in the fitness landscape). Diffusion limited perfect enzymes are very rare. Most enzymes catalyse their reactions to a rate that is 1,000-10,000 times slower than this limit. This is due to both the chemical limitations of difficult reactions, and the evolutionary limitations that such high reaction rates do not confer any extra fitness.

ELISA

The enzyme-linked immunosorbent assay (ELISA) (, ) is a commonly used analytical biochemistry assay, first described by Weiland in 1978. The assay uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a ligand (commonly a protein) in a liquid sample using antibodies directed against the protein to be measured. The ELISA has been used as a diagnostic tool in medicine, plant pathology, and biotechnology, as well as a quality control check in various industries.In the most simple form of an ELISA, antigens from the sample are attached to a surface. Then, a matching antibody is applied over the surface so it can bind to the antigen. This antibody is linked to an enzyme, and in the final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate.

Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation. Between each step, the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are non-specifically bound. After the final wash step, the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.

Of note, ELISA can perform other forms of ligand binding assays instead of strictly "immuno" assays, though the name carried the original "immuno" because of the common use and history of development of this method. The technique essentially requires any ligating reagent that can be immobilized on the solid phase along with a detection reagent that will bind specifically and use an enzyme to generate a signal that can be properly quantified. In between the washes, only the ligand and its specific binding counterparts remain specifically bound or "immunosorbed" by antigen-antibody interactions to the solid phase, while the nonspecific or unbound components are washed away. Unlike other spectrophotometric wet lab assay formats where the same reaction well (e.g., a cuvette) can be reused after washing, the ELISA plates have the reaction products immunosorbed on the solid phase, which is part of the plate, and so are not easily reusable.

Enzyme Commission number

The Enzyme Commission number (EC number) is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze.

As a system of enzyme nomenclature, every EC number is associated with a recommended name for the respective enzyme.

Strictly speaking, EC numbers do not specify enzymes, but enzyme-catalyzed reactions. If different enzymes (for instance from different organisms) catalyze the same reaction, then they receive the same EC number. Furthermore, through convergent evolution, completely different protein folds can catalyze an identical reaction and therefore would be assigned an identical EC number (these are called non-homologous isofunctional enzymes, or NISE). By contrast, UniProt identifiers uniquely specify a protein by its amino acid sequence.

Enzyme activator

Enzyme activators are molecules that bind to enzymes and increase their activity. They are the opposite of enzyme inhibitors. These molecules are often involved in the allosteric regulation of enzymes in the control of metabolism. An example of an enzyme activator working in this way is fructose 2,6-bisphosphate, which activates phosphofructokinase 1 and increases the rate of glycolysis in response to the hormone insulin. In some cases, when a substrate binds to one catalytic subunit of an enzyme, this can trigger an increase in the substrate affinity as well as catalytic activity in the enzyme's other subunits, and thus the substrate acts as an activator.

Enzyme catalysis

Enzyme catalysis is the increase in the rate of a chemical reaction by the active site of a protein. The protein catalyst (enzyme) may be part of a multi-subunit complex, and/or may transiently or permanently associate with a Cofactor (e.g. adenosine triphosphate). Catalysis of biochemical reactions in the cell is vital due to the very low reaction rates of the uncatalysed reactions at room temperature and pressure. A key driver of protein evolution is the optimization of such catalytic activities via protein dynamics.The mechanism of enzyme catalysis is similar in principle to other types of chemical catalysis. By providing an alternative reaction route the enzyme reduces the energy required to reach the highest energy transition state of the reaction. The reduction of activation energy (Ea) increases the amount of reactant molecules that achieve a sufficient level of energy, such that they reach the activation energy and form the product. As with other catalysts, the enzyme is not consumed during the reaction (as a substrate is) but is recycled such that a single enzyme performs many rounds of catalysis.

Enzyme inhibitor

An enzyme inhibitor is a molecule that binds to an enzyme and decreases its activity. Since blocking an enzyme's activity can kill a pathogen or correct a metabolic imbalance, many drugs are enzyme inhibitors. They are also used in pesticides. Not all molecules that bind to enzymes are inhibitors; enzyme activators bind to enzymes and increase their enzymatic activity, while enzyme substrates bind and are converted to products in the normal catalytic cycle of the enzyme.

The binding of an inhibitor can stop a substrate from entering the enzyme's active site and/or hinder the enzyme from catalyzing its reaction. Inhibitor binding is either reversible or irreversible. Irreversible inhibitors usually react with the enzyme and change it chemically (e.g. via covalent bond formation). These inhibitors modify key amino acid residues needed for enzymatic activity. In contrast, reversible inhibitors bind non-covalently and different types of inhibition are produced depending on whether these inhibitors bind to the enzyme, the enzyme-substrate complex, or both.

Many drug molecules are enzyme inhibitors, so their discovery and improvement is an active area of research in biochemistry and pharmacology. A medicinal enzyme inhibitor is often judged by its specificity (its lack of binding to other proteins) and its potency (its dissociation constant, which indicates the concentration needed to inhibit the enzyme). A high specificity and potency ensure that a drug will have few side effects and thus low toxicity.

Enzyme inhibitors also occur naturally and are involved in the regulation of metabolism. For example, enzymes in a metabolic pathway can be inhibited by downstream products. This type of negative feedback slows the production line when products begin to build up and is an important way to maintain homeostasis in a cell. Other cellular enzyme inhibitors are proteins that specifically bind to and inhibit an enzyme target. This can help control enzymes that may be damaging to a cell, like proteases or nucleases. A well-characterised example of this is the ribonuclease inhibitor, which binds to ribonucleases in one of the tightest known protein–protein interactions. Natural enzyme inhibitors can also be poisons and are used as defences against predators or as ways of killing prey.

Enzyme kinetics

Enzyme kinetics is the study of the chemical reactions that are catalysed by enzymes. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction are investigated. Studying an enzyme's kinetics in this way can reveal the catalytic mechanism of this enzyme, its role in metabolism, how its activity is controlled, and how a drug or an agonist might inhibit the enzyme.

Enzymes are usually protein molecules that manipulate other molecules—the enzymes' substrates. These target molecules bind to an enzyme's active site and are transformed into products through a series of steps known as the enzymatic mechanism

E + S ⇄ ES ⇄ ES* ⇄ EP ⇄ E + PThese mechanisms can be divided into single-substrate and multiple-substrate mechanisms. Kinetic studies on enzymes that only bind one substrate, such as triosephosphate isomerase, aim to measure the affinity with which the enzyme binds this substrate and the turnover rate. Some other examples of enzymes are phosphofructokinase and hexokinase, both of which are important for cellular respiration (glycolysis).

When enzymes bind multiple substrates, such as dihydrofolate reductase (shown right), enzyme kinetics can also show the sequence in which these substrates bind and the sequence in which products are released. An example of enzymes that bind a single substrate and release multiple products are proteases, which cleave one protein substrate into two polypeptide products. Others join two substrates together, such as DNA polymerase linking a nucleotide to DNA. Although these mechanisms are often a complex series of steps, there is typically one rate-determining step that determines the overall kinetics. This rate-determining step may be a chemical reaction or a conformational change of the enzyme or substrates, such as those involved in the release of product(s) from the enzyme.

Knowledge of the enzyme's structure is helpful in interpreting kinetic data. For example, the structure can suggest how substrates and products bind during catalysis; what changes occur during the reaction; and even the role of particular amino acid residues in the mechanism. Some enzymes change shape significantly during the mechanism; in such cases, it is helpful to determine the enzyme structure with and without bound substrate analogues that do not undergo the enzymatic reaction.

Not all biological catalysts are protein enzymes; RNA-based catalysts such as ribozymes and ribosomes are essential to many cellular functions, such as RNA splicing and translation. The main difference between ribozymes and enzymes is that RNA catalysts are composed of nucleotides, whereas enzymes are composed of amino acids. Ribozymes also perform a more limited set of reactions, although their reaction mechanisms and kinetics can be analysed and classified by the same methods.

Hydrolase

Hydrolase is a class of enzyme that is commonly used as biochemical catalysts that use water to break a chemical bond. This results in a division of a larger molecule to smaller molecules. Some common examples of hydrolase enzymes are esterases including lipases, phosphatases, glycosidases, peptidases, and nucleosidases. Esterases cleave ester bonds in lipids and phosphatases cleave phosphate groups off molecules. An example of crucial esterase is the acetylcholine esterase, which assists in transforming the neuron impulse into acetic acid after it the hydrolase breaks the acetylcholine into choline and acetic acid. Acetic acid is an important metabolite in the body, which becomes a nice intermediate for other reactions such as glycolysis. Lipases hydrolyze glycerides. Glycosidases cleave sugar molecules off carbohydrates and peptidases hydrolyze peptide bonds. Nucleosidases hydrolyze the bonds of nucleotides.Hydrolase enzymes are important for the body because they have degradative properties. In lipids, lipases contribute to the breakdown of fats and lipoproteins and other larger molecules into smaller molecules like fatty acids and glycerol. Fatty acids and other small molecules are used for synthesis and as a source of energy.In biochemistry, a hydrolase is an enzyme that catalyzes the hydrolysis of a chemical bond. For example, an enzyme that catalyzes the following reaction is a hydrolase:

A–B + H2O → A–OH + B–H

Ligase

In biochemistry, a ligase is an enzyme that can catalyze the joining of two large molecules by forming a new chemical bond, usually with accompanying hydrolysis of a small pendant chemical group on one of the larger molecules or the enzyme catalyzing the linking together of two compounds, e.g., enzymes that catalyze joining of C-O, C-S, C-N, etc. In general, a ligase catalyzes the following reaction:

Ab + C → A–C + bor sometimes

Ab + cD → A–D + b + c + d + e + fwhere the lowercase letters can signify the small, dependent groups. Ligase can join two complementary fragments of nucleic acid and repair single stranded breaks that arise in double stranded DNA during replication.

Lineweaver–Burk plot

In biochemistry, the Lineweaver–Burk plot (or double reciprocal plot) is a graphical representation of the Lineweaver–Burk equation of enzyme kinetics, described by Hans Lineweaver and Dean Burk in 1934.

Lyase

In biochemistry, a lyase is an enzyme that catalyzes the breaking (an "elimination" reaction) of various chemical bonds by means other than hydrolysis (a "substitution" reaction) and oxidation, often forming a new double bond or a new ring structure. The reverse reaction is also possible (called a "Michael addition"). For example, an enzyme that catalyzed this reaction would be a lyase:

ATP → cAMP + PPiLyases differ from other enzymes in that they require only one substrate for the reaction in one direction, but two substrates for the reverse reaction.

Michaelis–Menten kinetics

In biochemistry, Michaelis–Menten kinetics is one of the best-known models of enzyme kinetics. It is named after German biochemist Leonor Michaelis and Canadian physician Maud Menten. The model takes the form of an equation describing the rate of enzymatic reactions, by relating reaction rate (rate of formation of product, ) to , the concentration of a substrate S. Its formula is given by

This equation is called the Michaelis–Menten equation. Here, represents the maximum rate achieved by the system, at saturating substrate concentration. The Michaelis constant is the substrate concentration at which the reaction rate is half of . Biochemical reactions involving a single substrate are often assumed to follow Michaelis–Menten kinetics, without regard to the model's underlying assumptions.

PRIAM enzyme-specific profiles

PRIAM enzyme-specific profiles (PRofils pour l'Identification Automatique du Métabolisme) is a method for the automatic detection of likely enzymes in protein sequences. PRIAM uses position-specific scoring matrices (also known as profiles) automatically generated for each enzyme entry.

Restriction enzyme

A restriction enzyme or restriction endonuclease is an enzyme that cleaves DNA into fragments at or near specific recognition sites within the molecule known as restriction sites. Restrictions enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix.

These enzymes are found in bacteria and archaea and provide a defence mechanism against invading viruses. Inside a prokaryote, the restriction enzymes selectively cut up foreign DNA in a process called restriction digestion; meanwhile, host DNA is protected by a modification enzyme (a methyltransferase) that modifies the prokaryotic DNA and blocks cleavage. Together, these two processes form the restriction modification system.Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially. These enzymes are routinely used for DNA modification in laboratories, and they are a vital tool in molecular cloning.

Substrate (chemistry)

In chemistry, a substrate is typically the chemical species being observed in a chemical reaction, which reacts with a reagent to generate a product. In synthetic and organic chemistry, the substrate is the chemical of interest that is being modified. In biochemistry, an enzyme substrate is the material upon which an enzyme acts. When referring to Le Chatelier's principle, the substrate is the reagent whose concentration is changed. The term substrate is highly context-dependent. It essentially refers to the part of the molecule that is precursor to a product.

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