Enzyme

Ribbon diagram of the catalytically perfect enzyme TIM.

Factor D enzyme crystal prevents the immune system from inappropriately running out of control. An enzyme (from Greek nsimo (), formed by n = at or in and simo = leaven or yeast) is a protein that catalyzes, or speeds up, a chemical reaction. Enzymes are essential to sustain life, because most chemical reactions in biological cells would occur too slowly or would lead to different products without enzymes. A malfunction (mutation, overproduction, underproduction or deletion) of a single critical enzyme can lead to severe diseases. For example, phenylketonuria is caused by an enzyme malfunction in the enzyme phenylalanine hydroxylase, which catalyses the first step in the degradation of phenylalanine. If this enzyme does not function, the resulting build-up of phenylalanine leads to mental retardation.

Like all catalysts, enzymes work by lowering the activation energy of a reaction, thus allowing the reaction to proceed much faster. Enzymes may speed up reactions by a factor of many thousands. An enzyme, like any catalyst, remains unaltered by the completed reaction and can therefore continue to function. Because enzymes, like all catalysts, do not affect the relative energy between the products and reagents, they do not affect equilibrium of a reaction. However, the advantage of enzymes compared to most other catalysts is their sterio-, regio- and chemoselectivity and specificity. Enzyme activity can be affected by other molecules. Inhibitors are molecules that decrease or abolish enzyme activity; activators are molecules that increase the activity. Suicide inhibitors are inhibitors that incorporate themselves into the enzyme, permanently deactivating it. Inhibitors can be either natural or man-made. Many drugs are enzyme inhibitors. Aspirin, for example, inhibits an enzyme that produces the inflammation messenger prostaglandin, thus suppressing pain and inflammation. Enzymes are also used in everyday products such as washing detergents, where they speed up chemical reactions involved in cleaning the clothes (for example, breaking down starch stains). For industrial purposes the properties of Enzymes are emulated to form new kinds of catalytic molecules named Synzymes and Abzyme. More than 5,000 enzymes are known. To name different enzymes, one typically uses the ending -ase with the name of the chemical being transformed (substrate), e.g., lactase is the enzyme that catalyzes the cleavage of lactose.

Etymology and history

Eduard Buchner The word enzyme comes from Greek: "in leaven". As early as the late-1700s and early-1800s, the digestion of meat by stomach secretions and the conversion of starch to sugars by plant extracts and saliva were observed.

Studying the fermentation of sugar to alcohol by yeast, Louis Pasteur came to the conclusion that this fermentation was catalyzed by "ferments" in the yeast, which were thought to function only in the presence of living organisms. In 1897, Hans and Eduard Buchner inadvertently used yeast extracts to ferment sugar, despite the absence of living yeast cells. They were interested in making extracts of yeast cells for medical purposes, and, as one possible way of preserving them, they added large amounts of sucrose to the extract. To their surprise, they found that the sugar was fermented, even though there were no living yeast cells in the mixture. The term "enzyme" was used to describe the substance(s) in yeast extract that brought about the fermentation of sucrose. An example of an enzyme would be amylase

3D-Structure
In enzymes, as with other proteins, function is determined by structure. An enzyme can be:

A monomeric protein, i.e., containing only one polypeptide chain, made up of about hundred amino acids or more; or an oligomeric protein consisting of several polypeptide chains, different or identical, that act together as a unit.

As with any protein, each monomer is actually produced as a long, linear chain of amino acids, which folds in a particular fashion to produce a three-dimensional product. Individual monomers may then combine via non-covalent interactions to form a multimeric protein.

Cartoon showing the active site of an enzyme. Most enzymes are far larger molecules than the substrates they act on and that only a very small portion of the enzyme, around 10 amino acids, come into direct contact with the substrate(s). This region, where binding of the substrate(s) and than the reaction occurs, is known as the active site of the enzyme. Sometimes enzymes contain additionally other binding sites. Some enzymes have a binding site for a cofactor, which is needed for catalysis. Some enzymes have a binding site that serve regulatory functions, which increase or decrease the enzyme's activity. These

typically bind small molecules, often direct or indirect products or substrates of the reaction catalyzed. This provides a means for feedback regulation. The amino acid sidechains of an enzyme are either involved in forming the active site or a binding site, or are needed to form the 3D-structure of the protein. Some amino acid sidechains are not needed for function or structure of the enzyme.

Specificity
Enzymes are usually specific as to the reactions they catalyze and the substrates that are involved in these reactions. Shape and charge complementarity of enzyme and substrate are responsible for this specificity.

"Lock and key" hypothesis

Fischer's lock and key hypothesis of enzyme action. Enzymes are very specific and it was suggested by Emil Fischer in 1890 that this was because the enzyme had a particular shape into which the substrate(s) fit exactly. This is often referred to as "the lock and key" hypothesis. An enzyme combines with its substrate(s) to form a short lived enzyme-substrate complex.

Induced fit hypothesis

Diagrams to show Koshland's induced fit hypothesis of enzyme action.

In 1958 Daniel Koshland suggested a modification to the "lock and key" hypothesis. Enzymes are rather flexible structures. The active site of an enzyme could be modified as the substrate interacts with the enzyme. The amino acids sidechains 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 changes shape slightly as it enters the active site. A suitable analogy would be that of a hand changing the shape of a glove as the glove is put on.

Modifications
Many enzymes contain not only a protein part but need additionally various modifications. These modifications are made posttranslational, i.e. after the polypeptide chain was synthesized. Additional groups can be synthesized onto the polypeptide chain. E.g. phosphorylation or glycolisation of the enzyme. Another kind of posttranslational modification is the cleavage and splicing of the polypeptide chain. E.g. 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 prevents the enzyme from harmful digestion of the pancreas or other tissue. This type of inactive precursor to an enzyme is known as a zymogen.

Enzyme cofactors
Some enzymes do not need any additional components to exhibit full activities. However, many enzymes are chemically inactive, and they require additional components to become active. An enzyme cofactor is the non-protein component of an enzyme essential for its catalytic activity. There are three types of cofactors, namely activators, coenzymes, prosthetic groups.

Activators
Certain enzymes require inorganic ions as cofactors. These inorganic ions are called activators. They are mainly metallic monovalent or divalent cations which are either loosely or firmly bound to the enzymes. For example in blood clotting, calcium ions, known as factor IV, are required to activate thrombokinase to convert prothrombin into thrombin.

Prosthetic groups

Structure of heme. Non-protein organic cofactors which are firmly bound to the enzyme molecules are called prosthetic groups. They combine to form an integral part in performing catalytic functions. FAD, a prosthetic group containing heavy metals, is a prosthetic group having similar function as NAD and NADP in carrying hydrogen. Heme is a prosthetic group responsible for carring electrons in the cytochrome system.

Coenzymes
The cofactors of some other enzymes are non-protein organic molecules known as coenzymes, which are not bonded to enzyme molecules like prosthetic groups. Being vitamin-derivatives, they usually serve as carriers to transfer atoms or functional groups from one enzyme to a substrate. Common examples are NAD (derived from nicotinic acid, a member of vitamin B complex) and NADP, which act as hydrogen carriers and Coenzyme A that transfers the acetyl groups. Those inactive protein parts of enzymes are called apoenzymes. An apoenzyme works effectively only in the presence of non-protein cofactors. An apoenzyme together with its cofactor constitutes a holoenzyme, i.e., an active enzyme. Most of the cofactors are either regenerated or chemically unchanged at the end of the reactions.

Allosteric modulation
Allosteric enzymes have either effector binding sites, or multiple protein subunits that interact with each other and thus influence catalytic activity.

Kinetics
In 1913, Leonor Michaelis and Maud Menten proposed a quantitative theory of enzyme kinetics which is still widely used today (usually referred to as MichaelisMenten kinetics). Enzymes can perform up to several million catalytic reactions per second; to determine the maximum speed of an enzymatic reaction, the substrate concentration is increased until a constant rate of product formation is achieved. This is the maximum velocity (Vmax) of the enzyme. In this state, all enzyme active sites are saturated with substrate. However, Vmax is only one kinetic parameter that biochemists are interested in. The amount of substrate needed to achieve a given rate of reaction is also of interest. This can be expressed by the Michaelis-Menten constant (KM), which is the substrate concentration required for an enzyme to reach one half its maximum velocity. Each enzyme has a characteristic KM for a given substrate. Since Vmax cannot be measured directly, both KM and Vmax are usually determined by extrapolating from a limited data set, using what is known as a double reciprocal, or Lineweaver-Burk plot. The efficiency of an enzyme can be expressed in terms of kcat/Km. The quantity kcat, also called the turnover number, incorporates the rate constants for all steps in the reaction, and is the product of Vmax and the total enzyme concentration. kcat/Km is a useful quantity for comparing different enzymes against each other, or the same enzyme with different substrates, because it takes both affinity and catalytic ability into consideration. The theoretical maximum for kcat/Km, called diffusion limit, is about 108 to 109 (l mol-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 that reach this kcat/Km value are called catalytically perfect or kinetically perfect. Example of such enzymes are triosephosphate isomerase, carbonic anhydrase, acetylcholinesterase, catalase, fumarase, lactamase, and superoxide dismutase.

Thermodynamics

Diagram of a catalytic reaction, showing the energy niveau at each stage of the reaction. The substrates (A and B) usually need a large amount of energy to reach the

transition state (TS), which then reacts to form the end product (C and D). The enzyme stabilizes the transition state, reducing the energy niveau of the transition state and thus the energy required to get over this barrier. Because the lower energy niveau is easier to reach and therefore occurs more frequently, the reaction is more likely to take place, thus increasing the reaction speed. As with all catalysts, all reactions catalyzed by enzymes must be "spontaneous" (containing a net negative Gibbs free energy). With the enzyme, they run in the same direction as they 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. Many reactions catalyzed by an enzyme are reversible.

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 which catalyzes a reaction in either direction depending on the conditions at the time. (in tissues - high CO2 concentration) (in concentration) lungs low CO2

Inhibition
Enzymes reaction rates can be changed by competitive inhibition, non-competitive inhibition, uncompetitive inhibition and mixed inhibition.

Competitive inhibition

Competitive inhibition. A competitive inhibitor binds reversibly to the enzyme, preventing the binding of substrate. On the other hand, binding of substrate prevents binding of the inhibitor, thus substrate and inhibitor compete for the enzyme. The inhibitor may bind to the substrate binding site as shown in the figure above, thus preventing substrate binding. An example for competitive inhibition is the enzyme succinate dehydrogenase by malonate. Succinate dehydrogenase catalyses the oxidation of succinate to fumarate.

Action of the enzyme succinate dehydrogenase on succinate (right) and competitive inhibition of the enzyme by malonate (bottom).

Uncompetitive inhibition
Uncompetitive inhibition occurs when the inhibitor binds only to the enzymesubstrate complex, not to the free enzyme, the enzyme-inhibitor-substrate (EIS) complex is catalytically inactive. This mode of inhibition is rare.

Non-competitive inhibition

Diagram showing the mechanism of non-competitive inhibition. Non-competitive inhibitors never bind to the active center, but to other parts of the enzyme that can be far away from the substrate binding site, consequently, there is no competition between the substrate and inhibitor for the enzyme. The extent of inhibition depends entirely on the inhibitor concentration and will not be affected by the substrate concentration. However, these inhibitors bind only loosely with the enzyme and can be removed to resume the enzymatic activities. For example, cyanide combines with the copper prosthetic groups of the enzyme cytochrome c oxidase, thus inhibiting respiration. By changing the conformation (the three-dimensional structure) of the enzyme, the inhibitors either disable the ability of the enzyme to bind or turn over its substrate. The EI and EIS-complex have no catalytic activity.

Partially competitive inhibition

The mechanism of partially competitive is similar to that of non-competitive inhibition, except that the EIS-complex has catalytic activity, which may be lower or even higher (partially competitive activation) than that of the ES-complex.

Irreversible inhibitors
Some inhibitor bind irreversibly with the enzyme molecules, inhibiting the catalytic activities permanently. The enzymatic reactions will stop sooner or later and are not affected by an increase in substrate concentration. These are irreversible inhibitors. Examples are heavy metal ions including silver, mercury and lead ions. Another example of irreversible inhibition is provided by the nerve gas diisopropylfluorophosphate (DFP) designed for use in warfare. It combines with the amino acid serine (contains the SH group) at the active site of the enzyme acetylcholinesterase. The enzyme deactivates the neurotransmitter acetylcholine. Neurotransmitters are needed to continue the passage of nerve impulses from one neurone to another across the synapse. Once the impulse has been transmitted, acetylcholinesterase functions to deactivate the acetycholine almost immediately by breaking it down. If the enzyme is inhibited, acetylcholine accumulates and nerve impulses cannot be stopped, causing prolonged muscle contration. Paralysis occurs and death may result since the respiratory muscles are affected. Some insecticides currently in use, including those known as organophosphates (e.g. parathion), have a similar effect on insects, and can also cause harm to nervous and muscular system of humans who are overexposed to them.

Metabolic pathways and allosteric enzymes

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. The end product(s) of such a 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. 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.

Common feedback inhibition mechanisms, (1) The basic feedback inhibition mechanism, where the product (P) inhibits the committed step (AB). (2) Sequential feedback inhibition. The end products P1 and P2 inhibit the first committed step of their individual pathway (CD or CF). If both products are present in abundance, all pathways from C are blocked. This leads to a buildup of C, which in turn inhibits the first common committed step AB. (3) Enzyme multiplicity. Each end product inhibits both the first individual committed step and one of the enzymes performing the first common committed step. (4) Concerted feedback inhibition. Each end product inhibits the first individual committed step. Together, they inhibit the first common committed step. (5) Cumulative feedback inhibition. Each end product inhibits the first individual committed step. Also, each end product partially inhibits the first common committed step. Enzymes that are regulated by end-production inhibition are usually allosteric enzymes. An allosteric enzyme molecule has an active site and also an allosteric site. The allosteric site can bind with allosteric effectors that affect the activity of the enzyme molecule. Allosteric effectors include allosteric activators and allosteric inhibitors. The binding with an allosteric activator activates an enzyme molecule because the active site is in the right conformation to bind with substrate molecules. The binding with an allosteric inhibitor inactivates the enzyme molecule because the conformation of the active site is altered. The activation and inhibition of an allosteric enzyme are reversible.

Allosteric inhibition. In the example ATCase, the enzyme of the first reaction in the pathway, is an allosteric enzyme, and CTP, the end product, is an allosteric inhibitor of ATCase.

Enzyme naming conventions

By common convention, an enzyme's name consists of 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 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's function may not be that observed under laboratory conditions. This can result in the same enzyme being identified with two different names: one stemming from the formal laboratory identification as described above, the other representing its behavior in the cell. For instance the enzyme formally known as xylitol:NAD+ 2oxidoreductase (D-xylulose-forming) is more commonly referred to in the cellular physiological sense as D-xylulose reductase, reflecting the fact that the function of the enzyme in the cell is actually the reverse of what is often seen under in vitro conditions. The International Union of Biochemistry and Molecular Biology has developed a nomenclature for enzymes, the EC numbers; each enzyme is described by a sequence of four numbers, preceded by "EC". The first number broadly classifies the enzyme based on its mechanism: Group Reaction catalyzed Typical reaction Enzyme example(s) with trivial name

EC 1 To catalyze oxidation/reduction Oxidoreductase reactions; transfer of H and O s atoms or electrons from one substance to another EC Transferases

AH + B A Dehydrogenase, + BH oxidase (reduced) A + O AO (oxidized)

2 Transfer of a functional group AB + C A Transaminase, from one substance to another. + BC kinase The group may be methyl-, acyl-, amino- or phospate group 3 Formation of two products from AB + H2O Lipase, amylase, a substrate by hydrolysis AOH + BH peptidase 4 Non-hydrolytic addition or RCOCOOH removal of groups from RCOH + substrates. C-C, C-N, C-O or C- CO2 S bonds may be cleaved 5 Intramolecule rearrangement, AB BA i.e. isomerization changes within a single molecule Isomerase, mutase

EC Hydrolases EC Lyases

EC Isomerases EC Ligases

6 Join together two molecules by X + Y+ ATP Synthetase synthesis of new C-O, C-S, C-N XY + ADP or C-C bonds with simultaneous + Pi breakdown of ATP

Applications
Application Biological detergent Enzymes used Uses Notes and examples Used for presoak conditions and Primarily direct liquid proteases, produced applications in an extracellular helping with form from bacteria removal of protein stains from clothes. Detergents for machine dishwashing to remove resistant starch residues Biological washing powders contain protease. Note: The amylases and proteases used in detergents are allergenic for the process workers, although, encapsulation techniques have reduced this problem.

Amylase enzymes

Baking industry

Fungal alphaamylase enzymes: normally inactivates about 50 degrees Celsius, destroyed during baking process

Catalyze breakdown of starch in the flour to sugar. Yeast action on sugar produces carbon dioxide. Used in production of alpha-amylase catalyzes white bread, monomers (n) from starch buns, and rolls Biscuit manufacturers use them to lower the protein level of flour. To predigest baby foods

the

release

sugar

Protease enzymes

Baby foods Brewing industry

Trypsin

Enzymes from barley are released during the mashing stage of beer production.

They degrade starch and proteins to produce simple sugar, amino acids and peptides that are used by yeast to enhance Germinating barley used for malt. fermentation. enzymes

Industrially

produced

now widely used in the brewing process to substitute for the natural enzymes found in barley: Amylase, glucanases, proteases Betaglucosidase Amyloglucosidase Split polysaccharide s and proteins in the malt Improve the filtration characteristics. Low-calorie beer Remove cloudiness during storage of beers. Clarify juices fruit

Proteases Cellulases, pectinases

Fruit juices

Note: As animals age rennin production decreases Rennin, derived and is replaced by another protease, pepsin, which from the stomachs Manufacture of is not suitable for cheese production. In recent years of young ruminant cheese, used to the increase in cheese consumption, as well as animals (calves, split protein increased beef production, has resulted in a shortage lambs, kids) of rennin and escalating prices. Microbially produced enzyme Dairy industry Lipases Now finding increasing use in the dairy industry Is implemented during the production of Roquefort cheese to enhance the ripening of the blue-mould cheese. Break down lactose to glucose and galactose Converts starch into glucose and various syrups

Roquefort cheese

Lactases Starch industry Amylases, amyloglucosidease s and glucoamylases

Glucose isomerase Converts glucose in fructose (high

fructose syrups Glucose derived from starchy materials have enhanced sweetening properties and lower calorific values) Immobilised enzymes

Fructose

Production of Note: Although this process is widely used in the high fructose USA and Japan, legislation in the EEC restricts its syrups use to protect sugar beet farmers. To generate oxygen from peroxide to convert latex to foam rubber

Rubber industry

Catalase

Paper industry

Amylases

Degrade starch to lower viscosity product needed for sizing and coating paper Paper factories use amylase Dissolve gelatin off the scrap film allowing recovery of silver present

Photographic Protease (ficin) industry