OsmosisOsmosis 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 René Joachim Henri Dutrochet (1776–1847) from the Greek words ένδον (endon : within), έξο (exo : outside), and ωσμος (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

left out.[8]

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

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

postulate of colligative properties.[15]

Examples of osmosis

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.

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

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.

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 S-

hexylglutathione is shown as a space-filling model, filling the two active sites.

Enzymes are biological molecules that catalyze (i.e., increase the

rates of) chemical reactions.[1][2] 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. Enzymes

are known to catalyze about 4,000 biochemical reactions.[3] A

few RNA molecules called ribozymes also catalyze reactions, with an

important example being some parts of the ribosome.[4][5] Synthetic

molecules called artificial enzymes also display enzyme-like catalysis.[6]

Enzyme activity can be affected by other molecules. Inhibitors are

molecules that decrease enzyme activity; activators are molecules that

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).

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 tautomerase,[17] to over 2,500 residues in the animal fatty acid synthase.[18] A small

number of RNA-based biological catalysts exist, with the most common

being theribosome; these are referred to as either RNA-enzymes

or ribozymes. The activities of enzymes are determined by their three-

dimensional structure.[19]However, although structure does determine

function, predicting a novel enzyme's activity just from its structure is a

very difficult problem that has not yet been solved.[20]

Most enzymes are much larger than the substrates they act on, and only a

small portion of the enzyme (around 2–4 amino acids) is directly involved

in catalysis.[21] 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 denatured—that is, unfolded and

inactivated—by heating or chemical denaturants, which disrupt the three-

dimensional 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.

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 of stereospecificity, regioselectivity andchemoselectivity.[22]

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

product is correct in a second step.[23] This two-step process results in

average error rates of less than 1 error in 100 million reactions in high-

fidelity mammalian polymerases.[24] Similar proofreading mechanisms are

also found in RNA polymerase,[25] aminoacyl tRNA

synthetases [26]  and ribosomes.[27]

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

shapes that fit exactly into one another.[29] 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.

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

interacts with the enzyme.[30] As a result, the substrate does not simply

bind to a rigid active site; the amino acid side-chains that make up the

active site are molded into the precise positions that enable the enzyme to

perform its catalytic function. In some cases, such as glycosidases, the

substrate molecule also changes shape slightly as it enters the active site.[31] The active site continues to change until the substrate is completely

bound, at which point the final shape and charge is determined.[32] Induced

fit may enhance the fidelity of molecular recognition in the presence of

competition and noise via the conformational proofreadingmechanism.[33]

Mechanisms

Enzymes can act in several ways, all of which lower ΔG‡ (Gibbs energy):[34]

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.

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 enzyme’s shape deteriorates

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

ground state,[35] and its contribution to catalysis is relatively small.[36]

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. 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 is oriented

toward the charge distribution of the transition state.[37] Such an

environment does not exist in the uncatalyzed reaction in water.

Dynamics and function

See also: Protein dynamics

The internal dynamics of enzymes has been suggested to be linked with

their mechanism of catalysis.[38][39][40] 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

can contribute to catalysis through dynamic motions.[41][42][43][44] 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

through fluctuation rates.[45][46][47]

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

chemical steps in enzymatic reactions.[48] These new insights also have

implications in understanding allosteric effects and developing new

medicines.

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

reaction rate of the enzyme.[49] Allosteric interactions can both inhibit and

activate enzymes and are a common way that enzymes are controlled in

the body.[50]

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.[51] 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.

These molecules transfer chemical groups between enzymes.[52]

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

part of its active site.[53] 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.

Coenzymes

Space-filling model of the coenzyme NADH

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

another.[54] 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

coenzyme NADH.[55]

Coenzymes are usually continuously regenerated and their concentrations

maintained at a steady level inside the cell: for example, NADPH is

regenerated through the pentose phosphate pathway and S-

adenosylmethionine by methionine adenosyltransferase. This continuous

regeneration means that even small amounts of coenzymes are used very

intensively. For example, the human body turns over its own weight in ATP

each day.[56]

Thermodynamics

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

often used to drive other chemical reactions.[57]

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.

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).

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

kinetics,[58] 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[59] 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-

Menten kinetics (termed also Michaelis-Menten kinetics).[60] Their

work was further developed by G. E. Briggs and J. B. S. Haldane,

who derived kinetic equations that are still widely considered

today a starting point in solving enzymatic activity.[61]

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

that are connected with fluctuating rates.[45][46][47] Nevertheless,

there is a mathematical relation connecting the behavior obtained

from the basic Michaelis Menten mechanism (that was indeed

proved correct in many experiments) with the generalized

Michaelis Menten mechanisms involving dynamics and

activity; [62] 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.

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

same process takes just 25 milliseconds.[63] 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]

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

108 to 109 (M−1 s−1). At this point every collision of the enzyme with

its substrate will result in catalysis, and the rate of product

formation is not limited by the reaction rate but by the diffusion

rate. Enzymes with this property are called catalytically

perfect or kinetically perfect. Example of such enzymes are triose-

phosphate isomerase, carbonic

anhydrase, acetylcholinesterase, catalase, fumarase, β-

lactamase, and superoxide dismutase.

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, phase-

separation of the enzyme/substrate/product, or one or two-

dimensional molecular movement.[64] In these situations,

a fractal Michaelis-Menten kinetics may be applied.[65][66][67][68]

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.[69]

[70] Quantum tunneling for protons has been observed

in tryptamine.[71] 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.

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

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).[73] 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

convulsions due to lessened inhibition by the glycine.[74] 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

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).

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

sickness.[75] 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.

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

blocks cellular respiration.[76]

Biological function

Enzymes serve a wide variety of functions inside living organisms.

They are indispensable for signal transduction and cell regulation,

often via kinases and phosphatases.[77] They also generate

movement, with myosin hydrolyzing ATP to generate muscle

contractionand also moving cargo around the cell as part of

the cytoskeleton.[78] 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

in fireflies.[79] 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

then be absorbed. Different enzymes digest different food

substances. In ruminants, which have herbivorous diets,

microorganisms in the gut produce another enzyme, cellulase, to

break down the cellulose cell walls of plant fiber.[80]

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 beta-

lactamases are induced that hydrolyze the crucial beta-

lactam 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

the mitochondrion, through β-oxidation.[81]

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

to changes in blood sugar.[82] Another example of post-

translational modification is the cleavage of the

polypeptide chain. Chymotrypsin, a digestive protease, is

produced in inactive form as chymotrypsinogen in

the pancreas and transported in this form to

the stomach where it is activated. This stops the enzyme

from digesting the pancreas or other tissues before it

enters the gut. This type of inactive precursor to an

enzyme is known as a zymogen.

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

the lysosome.[83]

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

untreated.[84]

Another example of enzyme deficiency is pseudocholinesterase,

in which there is slow metabolic degradation of exogenous

choline.[citation needed]

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

therapy for celiac disease) in vivo.[85]

Acid–base titrationAn 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.

Acid–base titrations can also be used to find percent purity of chemicals.

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

substance.[1]

[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)

[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.

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.

Acid–base 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 of weak acid

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

four categories [2]:

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

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):

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.

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

3. at equivalence CaVa = CbVb

The previous 3 relationships are used to generate the equivalence

point pH formula below:

Ca = concentration of acid and Cb = concentration of base

Kw = dissociation constant for water and Ka = for the acid

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:

Single formula. More accurately, a single formula[3] that

describes the titration of a weak acid with a strong base

from start to finish is given below:

φ = fraction of completion of the titration (φ

< 1 is before the equivalence point, φ = 1 is

the equivalence point, and φ > 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

αA- = 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.