Enzyme

Etymology and history

  As early as the late 1700s and early 1800s, the digestion of meat by stomach secretions^[5] 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.^[6]

In the 19th century, when studying the 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."^[7]

In 1878 German physiologist Wilhelm Kühne (1837–1900) first used 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 that lacked any living yeast cells to ferment sugar. 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.^[8] He named the enzyme that brought about the fermentation of sucrose "DNA polymerase forms DNA polymers).

Having shown that enzymes could function outside a living cell, the next step was to determine their biochemical nature. Many early workers noted that enzymatic activity was associated with proteins, but several scientists (such as Nobel laureate Stanley, who worked on the digestive enzymes pepsin (1930), trypsin and chymotrypsin. These three scientists were awarded the 1946 Nobel Prize in Chemistry.^[10]

This discovery that enzymes could be crystallized eventually allowed their structures to be solved by structural biology and the effort to understand how enzymes work at an atomic level of detail.

Structures and mechanisms

See also: Enzyme catalysis

 

Enzymes are generally globular proteins and range from just 62 amino acid residues in size, for the amino acids) is directly involved in catalysis.^[15] 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 three-dimensional structure of the protein. Depending on the enzyme, denaturation may be reversible or irreversible.

Specificity

Enzymes are usually very specific as to which reactions they catalyze and the chemoselectivity.^[16]

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 ribosomes.^[21]

Some enzymes that produce secondary metabolites are described as promiscuous, as they can act on a relatively broad range of different substrates. It has been suggested that this broad substrate specificity is important for the evolution of new biosynthetic pathways.^[22]

"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.^[23] 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. The "lock and key" model has proven inaccurate and the induced fit model is the most currently accepted enzyme-substrate-coenzyme figure.

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 continually reshaped by interactions with the substrate as the substrate interacts with the enzyme.^[24] As a result, the substrate does not simply bind to a rigid active site, the amino acid side chains which make up the active site are moulded 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.^[25] The active site continues to change until the substrate is completely bound, at which point the final shape and charge is determined.^[26]

Mechanisms

Enzymes can act in several ways, all of which lower ΔG^‡:^[27]

• Lowering the activation energy by creating an environment in which the transition state is stabilized (e.g. straining the shape of a substrate - by binding the transition-state conformation of the substrate/product molecules, the enzyme distorts the bound substrate(s) into their transition state form, thereby reducing the amount of energy required to complete the transition).
• Lowering the energy of the transition state, but without distorting the substrate, by creating an environment with the opposite charge distribution to that of the transition state.
• Providing an alternative pathway. For example,temporarily reacting with the substrate to form an intermediate ES complex, which would be impossible in the absence of the enzyme.
• Reducing the reaction entropy change by bringing substrates together in the correct orientation to react. Considering ΔH^‡ alone overlooks this effect.

Interestingly, this entropic effect involves destabilization of the ground state,^[28] and its contribution to catalysis is relatively small.^[29]

Transition State Stabilization

The understanding of the origin of the reduction of ΔG^‡ requires one to find out how the enzymes can stabilize its transition state more than the transition state of the uncatalyzed reaction. Apparently, the most effective way for reaching large stabilization is the use of electrostatic effects, in particular, by having a relatively fixed polar environment that is oriented toward the charge distribution of the transition state.^[30] Such an environment does not exist in the uncatalyzed reaction in water.

Dynamics and function

Recent investigations have provided new insights into the connection between internal dynamics of enzymes and their mechanism of catalysis.^[31][32][33] 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.^{[34][35][36][37]} 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 and developing new drugs.

It should be clarified, however, that the dynamical time-dependent processes are not likely to help to accelerate enzymatic reactions, since such motions randomize and the rate constant is determined by the probability (P) of reaching the transition state, (P = exp {ΔG^‡/RT}).^[38] Furthermore, the reduction of ΔG^‡ requires having relatively smaller motions (in relation to the corresponding motions in solution reactions) for the transition between the reactant and the product states. Thus, it is not clear that motional or dynamical effects contribute to the catalysis of the chemical step.

Allosteric modulation

protein subunits that interact with the allosteric enzyme and thus influence catalytic activity.

Cofactors and coenzymes

Main articles: Cofactor (biochemistry) and Coenzyme

Cofactors

Some enzymes do not need any additional components to show full activity. However, others require non-protein molecules called cofactors to be bound for activity.^[39] Cofactors can be either adenosine triphosphate. These molecules act to transfer chemical groups between enzymes.^[40]

An example of an enzyme that contains a cofactor is 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 (this is 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 organic molecules that transport chemical groups from one enzyme to another.^[42] Some of these chemicals such as 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 coenzyme NADH.^[43]

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

Main articles: Chemical equilibrium

 

As all catalysts, enzymes do not alter the position of the chemical equilibrium of the reaction. Usually, in the presence of an enzyme, the reaction runs in the same direction as it would without the enzyme, just more quickly. However, in the absence of the enzyme, other possible uncatalyzed, "spontaneous" reactions might lead to different products, because in those conditions this different product is formed faster.

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 hydrolysis of ATP is often used to drive other 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.

(in tissues; high CO₂ concentration)

(in lungs; low CO₂ concentration)

Nevertheless, if the equilibrium is greatly displaced in one direction, that is, in a very exergonic reaction, the reaction is effectively irreversible. Under these conditions the enzyme will, in fact, only catalyze the reaction in the thermodynamically allowed direction.

Kinetics

Main article: Enzyme 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 1902 Victor Henri ^[44] proposed a quantitative theory of enzyme kinetics, but his experimental data were not useful because the significance of the hydrogen ion concentration was not yet appreciated. After Peter Lauritz Sørensen had defined the logarithmic pH-scale and introduced the concept of buffering in 1909^[45] the German chemist Leonor Michaelis and his Canadian postdoc J. B. S. Haldane, who derived kinetic equations that are still widely used today.^[47]

The major contribution of Henri 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 complex. The enzyme then catalyzes the chemical step in the reaction and releases the product.

  Enzymes can catalyze up to several million reactions per second. For example, the reaction catalyzed 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.^[48] Enzyme rates depend on solution conditions and substrate concentration. Conditions that denature the protein abolish enzyme activity, such as high temperatures, extremes of pH or high salt concentrations, while raising substrate concentration tends to increase activity. 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 form. At the maximum velocity (V_max) 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. 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 superoxide dismutase.

Michaelis-Menten kinetics relies on the Michaelis-Menten kinetics may be applied.^{[50][51][52][53]}

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.^[54][55] Quantum tunneling for protons has been observed in tryptamine.^[56] 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

 

 

Main article: Enzyme inhibitor

Enzyme reaction rates can be decreased by various types of enzyme inhibitors.

Competitive inhibition

In competitive inhibition, the inhibitor and substrate compete for the enzyme (i.e., they can not bind at the same time). Often competitive inhibitors strongly resemble the real substrate of the enzyme. For example, tetrahydrofolate. The similarity between the structures of folic acid and this drug are shown in the figure to the right bottom. Note that binding of the inhibitor need not be to the substrate binding site (as frequently stated), if binding of the inhibitor changes the conformation of the enzyme to prevent substrate binding and vice versa. In competitive inhibition the maximal velocity of the reaction is not changed, but higher substrate concentrations are required to reach a given velocity, increasing the apparent K_m.

Uncompetitive inhibition

In uncompetitive inhibition the inhibitor can not bind to the free enzyme, but only to the ES-complex. The EIS-complex thus formed is enzymatically inactive. This type of inhibition is rare, but may occur in multimeric enzymes.

Non-competitive inhibition

Non-competitive inhibitors can bind to the enzyme at the same time as the substrate, i.e. they never bind to the active site. Both the EI and EIS complexes are enzymatically inactive. Because the inhibitor can not be driven from the enzyme by higher substrate concentration (in contrast to competitive inhibition), the apparent V_max changes. But because the substrate can still bind to the enzyme, the K_m stays the same.

Mixed inhibition

This type of inhibition resembles the non-competitive, except that the EIS-complex has residual enzymatic activity.

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. Enzymes which are subject to this form of regulation are often multimeric and have allosteric binding sites for regulatory substances. Their substrate/velocity plots are not hyperbolar, but sigmoidal (S-shaped).

 

Aspirin also act in this manner. With these drugs, the compound is bound in the active site and the enzyme then converts the inhibitor into an activated form that reacts irreversibly with one or more amino acid residues.

Uses of inactivators

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 antibiotics and other anti-infective drugs are just specific poisons that kill a pathogen but not its host.

An example of an inactivator being used as a drug is cellular respiration.^[60]

Biological function

Enzymes serve a wide variety of functions inside living organisms. They are indispensable for neuraminidase.

An important function of enzymes is in the digestive systems of animals. Enzymes such as cellulase to break down the cellulose cell walls of plant fiber.^[64]

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 constant 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 glucose-6-phosphate is found to be the only significant product. Consequently, the network of metabolic pathways within each cell depends on the set of functional enzymes that are present.

Control of activity

There are five main ways that enzyme activity is controlled in the cell.

1. Enzyme production (drug interactions.
2. 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 the Golgi apparatus and used by a different set of enzymes as a source of energy in the β-oxidation.^[65]
3. Enzymes can be regulated by inhibitors and activators. 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. Negative feedback mechanism can effectively adjust the rate of synthesis of intermediate metabolites according to the demands of the cells. This helps allocate materials and energy economically, and prevents the manufacture of excess end products. Like other homeostatic devices, the control of enzymatic action helps to maintain a stable internal environment in living organisms.
4. Enzymes can be regulated through 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.
5. Some enzymes may become activated when localized to a different environment (eg. from a reducing (cytoplasm) to an oxidising (periplasm) environment, high pH to low pH etc). For example, hemagglutinin of the influenza virus undergoes a conformational change once it encounters the acidic environment of the host cell vesicle causing its activation.^[67]

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 phenylalanine, results in build-up of phenylalanine and related products. This can lead to mental retardation if the disease is untreated.^[68]

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 often derived from its substrate or the chemical reaction it catalyzes, with the word ending in -ase. Examples are glucose into the sweetener fructose, is a xylose isomerase in vivo.

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". The first number broadly classifies the enzyme based on its mechanism:

The top-level classification is

• EC 1 oxidation/reduction reactions
• EC 2 functional group (e.g. a methyl or phosphate group)
• EC 3 hydrolysis of various bonds
• EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation
• EC 5 isomerization changes within a single molecule
• EC 6 covalent bonds

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

Industrial applications

Enzymes are used in the 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.^[69][70]

Application	Enzymes used	Uses
Baking industry	Fungal alpha-amylase enzymes are normally inactivated at about 50 degrees Celsius, but are destroyed during the baking process.	Catalyze breakdown of starch in the flour to sugar. Yeast action on sugar produces carbon dioxide. Used in production of white bread, buns, and rolls.
	Proteases	Biscuit manufacturers use them to lower the protein level of flour.
Baby foods	Trypsin	To predigest baby foods.
Brewing industry	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 for fermentation.
	Industrially produced barley enzymes	Widely used in the brewing process to substitute for the natural enzymes found in barley.
	Amylase, glucanases, proteases	Split polysaccharides and proteins in the malt.
	Betaglucanases and arabinoxylanases	Improve the wort and beer filtration characteristics.
	Amyloglucosidase and pullulanases	Low-calorie beer and adjustment of fermentability.
	Proteases	Remove cloudiness produced during storage of beers.
	Acetolactatedecarboxylase (ALDC)	Avoid the formation of diacetyl
Fruit juices	Cellulases, pectinases	Clarify fruit juices
Dairy industry	Rennin, derived from the stomachs of young ruminant animals (like calves and lambs).	Manufacture of cheese, used to hydrolyze protein.
	Microbially produced enzyme	Now finding increasing use in the dairy industry.
	Lipases	Is implemented during the production of Roquefort cheese to enhance the ripening of the blue-mould cheese.
	Lactases	Break down glucose and galactose.
Meat tenderizers	Papain	To soften meat for cooking.
Starch industry Glucose Fructose	Amylases, amyloglucosideases and glucoamylases	Converts glucose and various syrups.
	Glucose isomerase	Converts glucose into fructose in production of high fructose syrups from starchy materials. These syrups have enhanced sweetening properties and lower calorific values than sucrose for the same level of sweetness.
Paper industry	Cellulases and ligninases	Degrade starch to lower viscosity, aiding sizing and coating paper. Xylanases reduce bleach required for decolorising; cellulases smooth fibers, enhance water drainage, and promote ink removal; lipases reduce pitch and lignin-degrading enzymes remove lignin to soften paper.
Biofuel industry	Cellulases	Used to break down cellulose into sugars that can be fermented (see cellulosic ethanol).
	Ligninases	Use of lignin waste
Biological detergent	Primarily proteases, produced in an extracellular form from bacteria	Used for presoak conditions and direct liquid applications helping with removal of protein stains from clothes.
	Amylases	Detergents for machine dish washing to remove resistant starch residues.
	Lipases	Used to assist in the removal of fatty and oily stains.
	Cellulases	Used in biological fabric conditioners.
Contact lens cleaners	Proteases	To remove proteins on contact lens to prevent infections.
Rubber industry	Catalase	To generate peroxide to convert latex into foam rubber.
Photographic industry	Protease (ficin)	Dissolve silver content.
Molecular biology	Restriction enzymes, DNA ligase and polymerases	Used to manipulate DNA in genetic engineering, important in pharmacology, agriculture and medicine. Essential for restriction digestion and the polymerase chain reaction. Molecular biology is also important in forensic science.

See also

• List of enzymes
• Enzyme assay
• Enzyme catalysis
• RNA Biocatalysis
• SUMO enzymes
• K_i Database
• protein engineering

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Further reading

Etymology and history New Beer in an Old Bottle: Eduard Buchner and the Growth of Biochemical Knowledge, edited by Athel Cornish-Bowden and published by Universitat de València (1997): ISBN 84-370-3328-4, A history of early enzymology. Williams, Henry Smith, 1863–1943. A History of Science: in Five Volumes. Volume IV: Modern Development of the Chemical and Biological Sciences, A textbook from the 19th century. Kleyn, J. and Hough J. The Microbiology of Brewing. Annual Review of Microbiology (1971) Vol. 25: 583–608 Enzyme structure and mechanism Fersht, A. Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding. W. H. Freeman, 1998 ISBN 0-7167-3268-8 Walsh, C., Enzymatic Reaction Mechanisms. W. H. Freeman and Company. 1979. ISBN 0-7167-0070-0 Page, M. I., and Williams, A. (Eds.), 1987. Enzyme Mechanisms. Royal Society of Chemistry. ISBN 0-85186-947-5 Bugg, T. Introduction to Enzyme and Coenzyme Chemistry, 2004, Blackwell Publishing Limited; 2nd edition. ISBN 1-40511-452-5 Warshel, A., Computer Modeling of Chemical Reactions in enzymes and Solutions John Wiley & Sons Inc. 1991. ISBN 0-471-18440-3 Thermodynamics Reactions and Enzymes Chapter 10 of On-Line Biology Book at Estrella Mountain Community College.

Kinetics and inhibition Athel Cornish-Bowden, Fundamentals of Enzyme Kinetics. (3rd edition), Portland Press (2004), ISBN 1-85578-158-1. Irwin H. Segel, Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. Wiley-Interscience; New Ed edition (1993), ISBN 0-471-30309-7. John W. Baynes, Medical Biochemistry, Elsevier-Mosby; 2th Edition (2005), ISBN 0-7234-3341-0, p. 57. Function and control of enzymes in the cell Price, N. and Stevens, L., Fundamentals of Enzymology: Cell and Molecular Biology of Catalytic Proteins Oxford University Press, (1999), ISBN 0-19-850229-X Nutritional and Metabolic Diseases Chapter of the on-line textbook "Introduction to Genes and Disease" from the NCBI. Enzyme-naming conventions Enzyme Nomenclature, Recommendations for enzyme names from the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology. Koshland D. The Enzymes, v. I, ch. 7, Acad. Press, New York, (1959) Industrial applications History of industrial enzymes, Article about the history of industrial enzymes, from the late 1900s to the present times.

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Categories: Metabolism