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Osmosis

Osmosis is the net movement of solvent molecules through a partially permeable membrane into a region of higher solute concentration, in order to equalize the solute concentrations on the two sides.[1][2][3] It may also be used to describe a physical process in which any solvent moves, without input of energy,[4] across a semipermeable membrane (permeable to the solvent, but not the solute) separating two solutions of different concentrations.[5] Although osmosis does not require input of energy, it does use kinetic energy [6] and can be made to do work.[7] One frame of a computer simulation of osmosis Net movement of solvent is from the less concentrated (hypotonic) to the more concentrated (hypertonic) solution, which tends to reduce the difference in concentrations. This effect can be countered by increasing the pressure of the hypertonic solution, with respect to the hypotonic. The osmotic pressure is defined to be the pressure required to maintain an equilibrium, with no net movement of solvent. Osmotic pressure is a colligative property, meaning that the osmotic pressure depends on the molar concentration of the solute but not on its identity. Osmosis is essential in biological systems, as biological membranes are semipermeable. In general, these membranes are impermeable to large and polar molecules, such as ions, proteins, and polysaccharides, while being permeable to non-polar and/or hydrophobic molecules like lipids as well as to small molecules like oxygen, carbon dioxide, nitrogen, nitric oxide, etc. Permeability depends on solubility, charge, or chemistry, as well as solute size. Water molecules travel through the plasma membrane, tonoplast membrane (vacuole) or protoplast by diffusing

across the phospholipid bilayer via aquaporins (small transmembrane proteins similar to those in facilitated diffusion and in creating ion channels). Osmosis provides the primary means by which water is transported into and out of cells. The turgor pressure of a cell is largely maintained by osmosis, across the cell membrane, between the cell interior and its relatively hypotonic environment.[8] Jean-Antoine Nollet first documented observation of osmosis in 1748.[9] The word "osmosis" descends from the words "endosmose" and "exosmose", which were coined by French physician Ren Joachim Henri Dutrochet (17761847) from the Greek words (endon : within), (exo : outside), and (osmos : push, impulsion).*10+*11+*12+*13+*14+

Basic explanations
Osmosis may occur when there is a partially permeable membrane, such as a cell membrane. When a cell is submerged in water, the water molecules pass through the cell membrane from an area of low solute concentration (outside the cell) to one of high solute concentration (inside the cell); this is called osmosis. The cell membrane is selectively permeable, so only necessary materials are let into the cell and wastes are [8] left out.

separated by a semipermeable membrane has less entropy than a similar system having two solutions of equal concentration. The system with the differing concentrations is said to be more ordered, and thus has less entropy. The second law of thermodynamics requires the presence of an osmotic flow that will take the system from an ordered state of low entropy to a disordered state of higher entropy. Thermodynamic equilibrium is achieved when the entropy gradient between the two solutions becomes zero. Particle size has no bearing on osmotic pressure; this is the fundamental [15] postulate of colligative properties.

Examples of osmosis
Water passing through a semi-permeable membrane to create an isotonic environment

When the membrane has a volume of pure water on both sides, water molecules pass in and out in each direction at exactly the same rate; there is no net flow of water through the membrane. Osmosis can be explained using the concept of thermodynamic free energy: the less concentrated solution contains more free energy, so its solvent molecules will tend to diffuse to a place of lower free energy in order to equalize free energy. Since the semipermeable membrane only allows solvent molecules to pass through it, the result is a net flow of water to the side with the more concentrated solution. Assuming the membrane does not break, this net flow will slow and finally stop as the pressure on the more concentrated side lessens and the movement in each direction becomes equal: this state is called dynamic equilibrium. Osmosis can also be explained using the notion of entropy, from statistical mechanics. A system that has two solutions of different concentrations

Effect of different solutions on blood cells

Plant cell under different environments

Osmotic pressure is the main cause of support in many plants. The osmotic entry of water raises the turgor pressure exerted against the cell wall, until it equals the osmotic pressure, creating a steady state. When a plant cell is placed in a hypertonic solution, the water in the cells moves to an area higher in solute concentration and the cell shrinks, and in doing so, becomes flaccid. This means the cell has become plasmolyzed the cell membrane has completely left the cell wall due to lack of water pressure on it; the opposite of turgid. Also, osmosis is responsible for the ability of plant roots to draw water from the soil. Since there are many fine roots, they have a large surface area, and water enters the roots by osmosis. Osmosis can also be seen when potato slices are added to a high concentration of salt solution. The water from inside the potato moves to the salt solution, causing the potato to shrink and to lose its 'turgor pressure'. The more concentrated the salt solution, the bigger the difference in size and weight of the potato slice. In unusual environments, osmosis can be very harmful to organisms. For example, freshwater and saltwater aquarium fish placed in water of a different salinity than that to which they are adapted to will die quickly, and in the case of saltwater fish, dramatically. Another example of a harmful osmotic effect is the use of table salt to kill leeches and slugs. Suppose an animal or a plant cell is placed in a solution of sugar or salt in water. 1. If the medium is hypotonic a dilute solution, with a higher water concentration than the cell the cell will gain water through osmosis.

2. If the medium is isotonic a solution with exactly the same water concentration as the cell there will be no net movement of water across the cell membrane. 3. If the medium is hypertonic a concentrated solution, with a lower water concentration than the cell the cell will lose water by osmosis. Essentially, this means that if a cell is put in a solution which has a solute concentration higher than its own, then it will shrivel up, and if it is put in a solution with a lower solute concentration than its own, the cell will expand and burst. Electronucleal exchange is the passive diffusion of cations and anions across a semi-permeable membrane according to electrical charge. Chemical gardens demonstrate the effect of osmosis in inorganic chemistry.

Factors
Osmotic pressure
Main article: Osmotic pressure As mentioned before, osmosis may be opposed by increasing the pressure in the region of high solute concentration with respect to that in the low solute concentration region. The force per unit area, or pressure, required to prevent the passage of water through a selectively permeable membrane and into a solution of greater concentration is equivalent to the osmotic pressure of the solution, or turgor.Osmotic pressure is a colligative property, meaning that the property depends on the concentration of the solute, but not on its identity.

Osmotic gradient
The osmotic gradient is the difference in concentration between two solutions on either side of a semipermeable membrane, and is used to tell the difference in percentages of the concentration of a specific particle dissolved in a solution. Usually the osmotic gradient is used while comparing solutions that have a semipermeable membrane between them allowing water to diffuse between the two solutions, toward the hypertonic solution (the solution with the higher concentration). Eventually, the force of the column of water on the hypertonic side of the semipermeable membrane will equal the force of diffusion on the hypotonic (the side with a lesser concentration) side, creating equilibrium. When equilibrium is reached, water continues to flow, but it flows both ways in equal amounts as well as force, therefore stabilizing the solution.

pressure than the feed solution is used to induce a net flow of water through a semi-permeable membrane, such that the feed solution becomes concentrated as the draw solution becomes dilute. The diluted draw solution may then be used directly (as with an ingestible solute like glucose), or sent to a secondary separation process for the removal of the draw solute. This secondary separation can be more efficient than a reverse osmosis process would be alone, depending on the draw solute used and the feedwater treated. Forward osmosis is an area of ongoing research, focusing on applications in desalination, water purification, water treatment, food processing, etc.

Variation
Reverse osmosis
Main article: Reverse osmosis Reverse osmosis is a separation process that uses pressure to force a solvent through a semi-permeable membrane that retains the solute on one side and allows the pure solvent to pass to the other side. More formally, it is the process of forcing a solvent from a region of high solute concentration through a membrane to a region of low solute concentration by applying a pressure in excess of the osmotic pressure.

Forward osmosis
Main article: Forward osmosis Osmosis may be used directly to achieve separation of water from a "feed" solution containing unwanted solutes. A "draw" solution of higher osmotic

Enzyme
"Biocatalyst" redirects here. For the use of natural catalysts in organic chemistry, see Biocatalysis.
Human glyoxalase I. Two zinc ions that are needed for the enzyme to catalyze its reaction are shown as purple spheres, and an enzyme inhibitor called Shexylglutathione is shown as a space-filling model, filling the two active sites.

increase activity. Many drugs and poisons are enzyme inhibitors. Activity is also affected by temperature, pressure, chemical environment (e.g., pH), and theconcentration of substrate. Some enzymes are used commercially, for example, in the synthesis of antibiotics. In addition, some household products use enzymes to speed up biochemical reactions (e.g., enzymes in biological washing powders break down protein or fat stains on clothes; enzymes inmeat tenderizers break down proteins into smaller molecules, making the meat easier to chew).

Enzymes are biological molecules that catalyze (i.e., increase the [1][2] rates of) chemical reactions. In enzymatic reactions, themolecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in abiological cell need enzymes in order to occur at rates sufficient for life. Since enzymes are selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell. Like all catalysts, enzymes work by lowering the activation energy (Ea ) for a reaction, thus dramatically increasing the rate of the reaction. As a result, products are formed faster and reactions reach their equilibrium state more rapidly. Most enzyme reaction rates are millions of times faster than those of comparable un-catalyzed reactions. As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter theequilibrium of these reactions. However, enzymes do differ from most other catalysts in that they are highly specific for their substrates. [3] Enzymes are known to catalyze about 4,000 biochemical reactions. A few RNA molecules called ribozymes also catalyze reactions, with an [4][5] important example being some parts of the ribosome. Synthetic [6] molecules called artificial enzymes also display enzyme-like catalysis. Enzyme activity can be affected by other molecules. Inhibitors are molecules that decrease enzyme activity; activators are molecules that

Structures and mechanisms


Ribbon diagram showing human carbonic anhydrase II. The grey sphere is the zinc cofactor in the active site. Diagram drawn from PDB 1MOO.

Enzymes are in general globular proteins and range from just 62 amino acid residues in size, for the monomer of 4-oxalocrotonate [17] tautomerase, to over 2,500 residues in the animal fatty acid [18] synthase. A small number of RNA-based biological catalysts exist, with the most common being theribosome; these are referred to as either RNAenzymes or ribozymes. The activities of enzymes are determined by [19] their three-dimensional structure. However, although structure does determine function, predicting a novel enzyme's activity just from its [20] structure is a very difficult problem that has not yet been solved. Most enzymes are much larger than the substrates they act on, and only a small portion of the enzyme (around 24 amino acids) is directly involved [21] in catalysis. 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 long, linear chains of amino acids that fold to produce a three-dimensional product. Each unique amino acid sequence produces a specific structure, which has unique properties. Individual protein chains may sometimes group together to form a protein complex. Most enzymes can be denaturedthat is, unfolded and inactivatedby heating or chemical denaturants, which disrupt the threedimensional structure of the protein. Depending on the enzyme, denaturation may be reversible or irreversible. Structures of enzymes with substrates or substrate analogs during a reaction may be obtained using Time resolved crystallography methods.

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.

"Lock and key" model


Enzymes are very specific, and it was suggested by the Nobel laureate organic chemist Emil Fischer in 1894 that this was because both the enzyme and the substrate possess specific complementary geometric [29] shapes that fit exactly into one another. 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.

Specificity
Enzymes are usually very specific as to which reactions they catalyze and the substrates that are involved in these reactions. Complementary shape, charge and hydrophilic/hydrophobic characteristics of enzymes and substrates are responsible for this specificity. Enzymes can also show impressive levels [22] of stereospecificity, regioselectivity andchemoselectivity. 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 asDNA polymerase catalyzes a reaction in a first step and then checks that the [23] product is correct in a second step. This two-step process results in average error rates of less than 1 error in 100 million reactions in high[24] fidelity mammalian polymerases. Similar proofreading mechanisms are [25] also found in RNA polymerase, aminoacyl tRNA [26] [27] synthetases and ribosomes.

Diagrams to show the induced fit hypothesis of enzyme action

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 [30] interacts with the enzyme. 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 [31] site. The active site continues to change until the substrate is completely [32] bound, at which point the final shape and charge is determined. Induced fit may enhance the fidelity of molecular recognition in the presence of [33] competition and noise via the conformational proofreadingmechanism.

and the enzyme becomes denatured. Some enzymes like thermolabile enzymes work best at low temperatures. It is interesting that this entropic effect involves destabilization of the [35] [36] ground state, and its contribution to catalysis is relatively small.

Transition state stabilization


The understanding of the origin of the reduction of G requires one to find out how the enzymes can stabilize its transition state more than the transition state of the uncatalyzed reaction. It seems that the most effective way for reaching large stabilization is the use of electrostatic effects, in particular, when having a relatively fixed polar environment that [37] is oriented toward the charge distribution of the transition state. Such an environment does not exist in the uncatalyzed reaction in water.

Mechanisms
Enzymes can act in several ways, all of which lower G (Gibbs [34] energy): Lowering the activation energy by creating an environment in which the transition state is stabilized (e.g. straining the shape of a substrateby 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 H alone overlooks this effect. Increases in temperatures speed up reactions. Thus, temperature increases help the enzyme function and develop the end product even faster. However, if heated too much, the enzymes shape deteriorates

Dynamics and function


See also: Protein dynamics The internal dynamics of enzymes has been suggested to be linked with [38][39][40] their mechanism of catalysis. Internal dynamics are the movement of parts of the enzyme's structure, such as individual amino acid residues, a group of amino acids, or even an entire protein domain. These movements occur at various time-scales ranging from femtoseconds to seconds. Networks of protein residues throughout an enzyme's structure [41][42][43][44] can contribute to catalysis through dynamic motions. This is simply seen in the kinetic scheme of the combined process, enzymatic activity and dynamics; this scheme can have several independent Michaelis-Menten-like reaction pathways that are connected [45][46][47] through fluctuation rates. 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. However, although

these movements are important in binding and releasing substrates and products, it is not clear if protein movements help to accelerate the [48] chemical steps in enzymatic reactions. These new insights also have implications in understanding allosteric effects and developing new medicines.

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 [51] be bound for activity. Cofactors can be either inorganic (e.g., metal ions and iron-sulfur clusters) or organic compounds (e.g., flavin and heme). Organic cofactors can be either prosthetic groups, which are tightly bound to an enzyme, or coenzymes, which are released from the enzyme's active site during the reaction. Coenzymes include NADH, NADPH and adenosine triphosphate. [52] These molecules transfer chemical groups between enzymes. An example of an enzyme that contains a cofactor is carbonic anhydrase, and is shown in the ribbon diagram above with a zinc cofactor bound as [53] part of its active site. These tightly bound molecules are usually found in the active site and are involved in catalysis. For example, flavin and heme cofactors are often involved in redox reactions. Enzymes that require a cofactor but do not have one bound are called apoenzymes or apoproteins. 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., biotin in the enzyme pyruvate carboxylase). 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.

Allosteric modulation

Allosteric transition of an enzyme between R and T states, stabilized by an agonist, an inhibitor and a substrate (the MWC model)

Main article: Allosteric regulation Allosteric sites are sites on the enzyme that bind to molecules in the cellular environment. The sites form weak, noncovalent bonds with these molecules, causing a change in the conformation of the enzyme. This change in conformation translates to the active site, which then affects the [49] reaction rate of the enzyme. Allosteric interactions can both inhibit and activate enzymes and are a common way that enzymes are controlled in [50] the body.

Coenzymes
Space-filling model of the coenzyme NADH

Thermodynamics

Coenzymes are small organic molecules that can be loosely or tightly bound to an enzyme. Tightly bound coenzymes can be called allosteric groups. Coenzymes transport chemical groups from one enzyme to [54] another. Some of these chemicals such as riboflavin, thiamine and folic acid are vitamins (compounds that cannot be synthesized by the body and must be acquired from the diet). The chemical groups carried include + the hydride ion (H ) carried by NAD or NADP , the phosphate group carried by adenosine triphosphate, the acetyl group carried by coenzyme A, formyl, methenyl or methyl groups carried by folic acid and the methyl group carried by S-adenosylmethionine. Since coenzymes are chemically changed as a consequence of enzyme action, it is useful to consider coenzymes to be a special class of substrates, or second substrates, which are common to many different enzymes. For example, about 700 enzymes are known to use the [55] coenzyme NADH. 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 Sadenosylmethionine by methionine adenosyltransferase. This continuous regeneration means that even small amounts of coenzymes are used very intensively. For example, the human body turns over its own weight in ATP [56] each day.
The energies of the stages of a chemical reaction. Substrates need a lot of potential energy to reach a transition state, which then decays into products. The enzyme stabilizes the transition state, reducing the energy needed to form products.

Main articles: Activation energy, Thermodynamic equilibrium, and 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 [57] 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 in effect irreversible. Under these conditions, the enzyme will, in fact, catalyze the reaction only in the thermodynamically allowed direction.

Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products. The rate data used in kinetic analyses are commonly obtained from enzyme assays, where since the 90s, the dynamics of many enzymes are studied on the level of individual molecules. In 1902 Victor Henri proposed a quantitative theory of enzyme [58] kinetics, but his experimental data were not useful because the significance of the hydrogen ion concentration was not yet appreciated. After Peter Lauritz Srensen had defined the logarithmic pH-scale and introduced the concept of buffering in [59] 1909 the German chemist Leonor Michaelis and his Canadian postdoc Maud Leonora Menten repeated Henri's experiments and confirmed his equation, which is referred to as Henri-Michaelis[60] Menten kinetics (termed also Michaelis-Menten kinetics). Their work was further developed by G. E. Briggs and J. B. S. Haldane, who derived kinetic equations that are still widely considered [61] today a starting point in solving enzymatic activity. 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. Note that the simple Michaelis Menten mechanism for the enzymatic activity is considered today a basic idea, where many examples show that the enzymatic activity involves structural dynamics. This is incorporated in the enzymatic mechanism while introducing several Michaelis Menten pathways [45][46][47] that are connected with fluctuating rates. Nevertheless, there is a mathematical relation connecting the behavior obtained from the basic Michaelis Menten mechanism (that was indeed

Kinetics
Main article: Enzyme kinetics

Mechanism for a single substrate enzyme catalyzed reaction. The enzyme (E) binds a substrate (S) and produces a product (P).

proved correct in many experiments) with the generalized Michaelis Menten mechanisms involving dynamics and [62] activity; this means that the measured activity of enzymes on the level of many enzymes may be explained with the simple Michaelis-Menten equation, yet, the actual activity of enzymes is richer and involves structural dynamics.

is low. 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 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. However, Vmax 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 (Km), which is the substrate concentration required for an enzyme to reach one-half its maximum reaction rate. Each enzyme has a characteristic Km for a given substrate, and this can show how tight the binding of the substrate is to the enzyme. Another useful constant is kcat, 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 kcat/Km. This is also called the specificity constant and incorporates the rate constants for all steps in the reaction. Because the specificity constant reflects both affinity and catalytic ability, it is useful for comparing different enzymes against each other, or the same enzyme with different substrates. The theoretical maximum for the specificity constant is called the diffusion limit and is about 8 9 1 1 10 to 10 (M s ). 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 triosephosphate isomerase, carbonic

Saturation curve for an enzyme reaction showing the relation between the substrate concentration (S) and rate (v)

Enzymes can catalyze up to several million reactions per second. For example, the uncatalyzed decarboxylation of orotidine 5'monophosphate has a half life of 78 million years. However, when the enzyme orotidine 5'-phosphate decarboxylase is added, the [63] same process takes just 25 milliseconds. 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 when [S]

anhydrase, acetylcholinesterase, catalase, fumarase, lactamase, and superoxide dismutase. Michaelis-Menten kinetics relies on the law of mass action, which is derived from the assumptions of free diffusion and thermodynamically driven random collision. However, many biochemical or cellular processes deviate significantly from these conditions, because of macromolecular crowding, phaseseparation of the enzyme/substrate/product, or one or two[64] dimensional molecular movement. In these situations, [65][66][67][68] a fractal Michaelis-Menten kinetics may be applied. 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 quantummechanical tunneling explanation, whereby a proton or an electron can tunnel through activation barriers, although for proton tunneling this model remains somewhat [69][70] controversial. Quantum tunneling for protons has been [71] observed in tryptamine. 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

Competitive inhibitors bind reversibly to the enzyme, preventing the binding of substrate. On the other hand, binding of substrate prevents binding of the inhibitor. Substrate and inhibitor compete for the enzyme.

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 [73] 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, methotrexate is a competitive inhibitor of the enzyme dihydrofolate reductase, which catalyzes the reduction of dihydrofolate to tetrahydrofolate. The similarity between the structures of folic acid and this drug are shown in the figure to the right bottom. 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. For example,strychnine acts as an allosteric inhibitor of the glycine receptor in the mammalian spinal cord and brain stem. Glycine is a major post-synaptic inhibitory neurotransmitter with a specific receptor site. Strychnine binds to an alternate site that reduces the affinity of the glycine receptor for glycine, resulting in [74] convulsions due to lessened inhibition by the glycine. In competitive inhibition the maximal rate of the reaction is not changed, but higher substrate concentrations are required to reach a given maximum rate, increasing the apparent Km. Uncompetitive inhibition In uncompetitive inhibition, the inhibitor cannot bind to the free enzyme, 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

Types of inhibition. This classification was introduced by W.W. Cleland.[72]

Non-competitive inhibitors can bind to the enzyme at the binding site at the same time as the substrate,but not 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 Vmax changes. But because the substrate can still bind to the enzyme, the Km stays the same. Mixed inhibition This type of inhibition resembles the non-competitive, except that the EIS-complex has residual enzymatic activity.This type of inhibitor does not follow Michaelis-Menten equation. 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 that 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).

Uses of inhibitors Since inhibitors modulate the function of enzymes they are often used as drugs. A common example of an inhibitor that is used as a drug is aspirin, which inhibits the COX-1 and COX-2 enzymes that produce the inflammation messenger prostaglandin, thus suppressing pain and inflammation. However, 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 [76] blocks cellular respiration.

Biological function
Enzymes serve a wide variety of functions inside living organisms. They are indispensable for signal transduction and cell regulation, [77] often via kinases and phosphatases. They also generate movement, with myosin hydrolyzing ATP to generate muscle contractionand also moving cargo around the cell as part of [78] the cytoskeleton. Other ATPases in the cell membrane are ion pumps involved inactive transport. Enzymes are also involved in more exotic functions, such as luciferase generating light [79] in fireflies. Viruses can also contain enzymes for infecting cells, such as the HIV integrase and reverse transcriptase, or for viral release from cells, like theinfluenza virus neuraminidase. 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 asmaltose and eventually glucose, which can

Irreversible inhibitors react with the enzyme and form a covalent adduct with the protein. The inactivation is irreversible. These compounds include eflornithine a drug used to treat the parasitic disease sleeping [75] sickness. Penicillin and 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.

then be absorbed. Different enzymes digest different food substances. In ruminants, which have herbivorous diets, microorganisms in the gut produce another enzyme, cellulase, to [80] break down the cellulose cell walls of plant fiber. 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 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. 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 asglycolysis could not exist independently of enzymes. Glucose, for example, can react directly with ATP to becomephosphorylated at one or more of its carbons. In the absence of enzymes, this occurs so slowly as to be insignificant. However, if hexokinase is added, these slow reactions continue to take place except that phosphorylation at carbon 6 occurs so rapidly that, if the mixture is tested a short time later, glucose-6-phosphate is found to be the only significant product. As a consequence, 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 (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 and inhibition (see enzyme induction). For example, bacteria may become resistant to antibiotics such as penicillin because enzymes called betalactamases are induced that hydrolyze the crucial betalactam ring within the penicillin molecule. Another example are enzymes in the liver called cytochrome P450 oxidases, which are important in drug metabolism. Induction or inhibition of these enzymes can cause 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 [81] the mitochondrion, through -oxidation. 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. The control of enzymatic action helps to maintain a stable internal environment in living organisms. 4. Enzymes can be regulated through post-translational modification. This can include phosphorylation, myristoylation and glycosylation. 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 [82] to changes in blood sugar. Another example of posttranslational 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. 5. Some enzymes may become activated when localized to a different environment (e.g., from a reducing (cytoplasm) to an oxidizing (periplasm) environment, high pH to low pH, etc.). For example,hemagglutinin in the influenza virus is activated by a conformational

change caused by the acidic conditions, these occur when it is taken up inside its host cell and enters [83] the lysosome.

Involvement in disease
Since the tight control of enzyme activity is essential for homeostasis, any malfunction (mutation, overproduction, underproduction or deletion) of a single critical enzyme can lead to a genetic disease. The importance of enzymes is shown by the fact that a lethal illness can be caused by the malfunction of just one type of enzyme out of the thousands of types present in our bodies. One example is the most common type of phenylketonuria. A mutation of a single amino acid in the enzyme phenylalanine hydroxylase, which catalyzes the first step in the degradation of phenylalanine, results in build-up of phenylalanine and related products. This can lead to mental retardation if the disease is [84] untreated. Another example of enzyme deficiency is pseudocholinesterase, in which there is slow metabolic degradation of exogenous [citation needed] choline. 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. Oral administration of enzymes can be used to treat several diseases (e.g. pancreatic insufficiency and lactose intolerance). Since enzymes are proteins themselves they are potentially subject to inactivation and digestion in the gastrointestinal

environment. Therefore a non-invasive imaging assay was developed to monitor gastrointestinal activity of exogenous enzymes (prolyl endopeptidase as potential adjuvant [85] therapy for celiac disease) in vivo.

Acidbase titration
An acid-base titration is the determination of the concentration of an acid or base by exactly neutralizing the acid/base with an acid or base of known concentration. This allows for quantitative analysis of the concentration of an unknown acid or base solution. It makes use of the neutralization reaction that occurs between acids and bases and the knowledge of how acids and bases will react if their formulas are known. Acidbase titrations can also be used to find percent purity of chemicals.

[edit]Method Before starting the titration a suitable pH indicator must be chosen. The equivalence point of the reaction, the point at which equivalent amounts of the reactants have reacted, will have a pH dependent on the relative strengths of the acid and base used. The pH of the equivalence point can be estimated using the following rules: A strong acid will react with a strong base to form a neutral (pH=7) solution. A strong acid will react with a weak base to form an acidic (pH<7) solution. A weak acid will react with a strong base to form a basic (pH>7) solution.

Alkalimetry and acidimetry


Alkalimetry, sometimes spelled alkimetry, is the specialized analytic use of acid-base titration to determine the concentration of a basic (synonymous to alkaline) substance. Acidimetry, sometimes spelled acidometry, is the same concept of specialized analytic acid-base titration, but for an acidic [1] substance. [edit]Equipment The key equipment used in a titration are: Burette White tile used to see a colour change in the solution Pipette pH indicator (the one used varies depending on the reactants) Erlenmeyer flask/ Conical flask Titrant or titrator (a standard solution of known concentration, a common one is aqueous sodium carbonate) Analyte or titrand (solution of unknown concentration)

When a weak acid reacts with a weak base, the equivalence point solution will be basic if the base is stronger and acidic if the acid is stronger. If both are of equal strength, then the equivalence pH will be neutral. However, weak acids are not often titrated against weak bases because the colour change shown with the indicator is often quick, and therefore very difficult for the observer to see the change of colour. The point at which the indicator changes colour is called the end point. A suitable indicator should be chosen, preferably one that will experience a change in colour (an end point) close to the equivalence point of the reaction. First, the burette should be rinsed with the standard solution, the pipette with the unknown solution, and the conical flask with distilled water. Secondly, a known volume of the unknown concentration solution should be taken with the pipette and placed into the conical flask, along with a small amount of the indicator chosen.

The known solution should then be allowed out of the burette, into the conical flask. At this stage we want a rough estimate of the amount of this solution it took to neutralize the unknown solution. The solution should be let out of the burette until the indicator changes colour and the value on the burette should be recorded. This is the first (or rough) titre and should be discluded from any calculations. At least three more titrations should be performed, this time more accurately, taking into account roughly where the end point will occur. The initial and final readings on the burette (prior to starting the titration and at the end point, respectively) should be recorded. Subtracting the initial volume from the final volume will yield the amount of titrant used to reach the endpoint. The end point is reached when the indicator just changes color permanently. This is best achieved by washing a hanging drop from the tip of the burette into the flask right at the end of the titration to achieve a drop that is smaller in volume than what can usually be achieved by just dripping solution off the burette. Acidbase titration is performed with a phenolphthalein indicator, when it is a strong acid strong base titration, a bromthymol blue indicator in weak acid weak base reactions, and a methyl orange indicator for strong acid weak base reactions. If the base is off the scale, i.e. a pH of >13.5, and the acid has a pH >5.5, then an Alizarine yellow indicator may be used. On the other hand, if the acid is off the scale, i.e. a pH of <0.5, and the base has a pH <8.5, then a Thymol Blue indicator may be used. [edit]Titration

The pH of a weak acid solution being titrated with a strong base solution can be found at different points along the way. These points fall into one of [2] four categories : 1. initial pH 2. pH before the equivalence point 3. pH at the equivalence point 4. pH after the equivalence point 1. The initial pH is approximated for a weak acid solution in water using the equation

of weak acid

where Ka is the dissociation constant and F is the concentration of the acid.

2. The pH before the equivalence point depends on the amount of weak acid remaining and the amount of conjugate base formed. The pH can be calculated by the following formula (which is a variation of the Henderson-Hasselbalch equation): Ca = concentration of acid and Cb = concentration of base Kw = dissociation constant for water and Ka = for the acid

where: pKa is the negative log of the acid dissociation constant of the weak acid. nOH- added is the number of moles of added strong base in the solution. nHA initial is the number of moles the weak acid initially present.

Note that when an acid neutralizes a base, the pH may or may not be neutral (pH = 7). The pH depends on the strengths of the acid and base. 4. After the equivalence point, the solution will contain two bases: the conjugate base of the acid and the strong base of the titrant. However, the base of the titrant is stronger than the conjugate base of the acid. Therefore, the pH in this region is controlled by the strong base. As such the pH can be found using the following:

When the numerator of the log term equals the denominator ( ), then the ratio goes to 1 and the log term goes to zero. Thus the pH will equal the pKa which occurs half-way to the equivalence point. 3. At the equivalence point, the weak acid is consumed and converted to its weak conjugate base. The pH will be greater than 7 and can be calculated from an equation derived from the following relationships: 1. pH + pOH = 14 2. KaKb = 10
14

Single formula. More accurately, a single formula that describes the titration of a weak acid with a strong base from start to finish is given below:

[3]

3. at equivalence CaVa = CbVb The previous 3 relationships are used to generate the equivalence point pH formula below:

= fraction of completion of the titration ( < 1 is before the equivalence point, = 1 is the equivalence point, and > 1 is after the equivalence point) Ca, Cb = the concentrations of the acid and base respectively Va, Vb = the volumes of the acid and base respectively A- = the fraction of the weak acid that is ionized Ka = the dissociation constant for the acid [H ], [OH ] = concentrations of the H and OH ions respectively
+ +

This formula is somewhat cumbersome, but does describe the titration curve as a single equation.

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