Enzymes



An enzyme is a protein that catalyzes, or speeds up, a chemical reaction. The word comes from the Greek ένζυμο, énsymo, which comes from én ("at" or "in") and simo ("leaven" or "yeast"). Certain RNAs also have catalytic activity, but to differentiate them from protein enzymes, they are referred to as RNA enzymes or ribozymes.

Enzymes are essential to sustain life because most chemical reactions in biological cells would occur too slowly, or would lead to different products, without enzymes. A malfunction (mutation, overproduction, underproduction or deletion) of a single critical enzyme can lead to a severe disease. For example, the most common type of phenylketonuria is caused by a single amino acid mutation in the enzyme phenylalanine hydroxylase, which catalyses the first step in the degradation of phenylalanine. The resulting build-up of phenylalanine and related products can lead to mental retardation, if the disease is untreated.

Like all catalysts, enzymes work by lowering the activation energy of a reaction, thus allowing the reaction to proceed much faster. Enzymes may speed up reactions by a factor of many millions. An enzyme, like any catalyst, remains unaltered by the completed reaction and can therefore continue to function. Because enzymes, like all catalysts, do not affect the relative energy between the products and reagents, they do not affect equilibrium of a reaction. However, the advantage of enzymes compared to most other catalysts is their sterio-, regio- and chemoselectivity and specificity.

Enzyme activity can be affected by other molecules. Inhibitors are naturally occuring or synthetic molecules that decrease or abolish enzyme activity; activators are molecules that increase activity. Some irreversible inhibitors bind enzymes very tightly, effectively inactivating them. Many drugs and poisons act by inhibiting enzymes. Aspirin inhibits the COX-1 and COX-2 enzymes that produce the inflammation messenger prostaglandin, thus suppressing pain and inflammation. The poison cyanide inhibits cytochrome c oxidase, which effectively blocks cellular respiration.

While all enzymes have a biological role, some enzymes are used commercially for other purposes. Many household cleaners use enzymes to speed up chemical reactions ( i.e., breaking down protein or starch stains in clothes).

More than 5,000 enzymes are known. Typically the suffix -ase is added to the name of the substrate (e.g., lactase is the enzyme that catalyzes the cleavage of lactose) or the type of reaction (e.g., DNA polymerase catalyzes the formation of DNA polymers). However, this is not always the case, especially when enzymes modify multiple substrates. For this reason Enzyme Commission or EC numbers are used to classify enzymes based on the reactions they catalyze. Even this is not a perfect solution, as enzymes from different species or even very similar enzymes in the same species may have identical EC numbers.

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

Studying the fermentation of sugar to alcohol by yeast, Louis Pasteur came to the conclusion that this fermentation was catalyzed by "ferments" in the yeast, which were thought to function only in the presence of living organisms.

In 1897, Hans and Eduard Buchner inadvertently used yeast extracts to ferment sugar, despite the absence of living yeast cells. They were interested in making extracts of yeast cells for medical purposes, and, as one possible way of preserving them, they added large amounts of sucrose to the extract. To their surprise, they found that the sugar was fermented, even though there were no living yeast cells in the mixture. The term "enzyme" was used to describe the substance(s) in yeast extract that brought about the fermentation of sucrose.

3D-Structure
In enzymes, as with other proteins, function is determined by structure. An enzyme can be:
 * A monomeric protein, i.e., containing only one polypeptide chain, typically one hundred or more amino acids; or
 * an oligomeric protein consisting of several polypeptide chains, different or identical, that act together as a unit.

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

Most enzymes are larger than the substrates they act on and that only a very small portion of the enzyme, around 10 amino acids, come into direct contact with the substrate(s). This region, where binding of the substrate(s) and then the reaction occurs, is known as the active site of the enzyme. Some enzymes contain sites that bind cofactors, which are needed for catalysis. Certain enzymes 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 (depending on the molecule and enzyme), providing a means for feedback regulation.

Although not all enzymes are vulnerable to heat most are. Rising the temperature of the substance that contains the enzyme can usually induce it to lose its tertiary structure. Once the substance is again cooled off the enzyme will often fold back, but not necessarily in its previous folding, making it inactive. Two important exceptions to this are enzymes that do not unfold with heat (termophiles), and enzymes that fold back to the original structure.

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

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

Induced fit model
In 1958 Daniel Koshland suggested a modification to the "lock and key" model. Enzymes are rather flexible structures. The active site of an enzyme could be modified as the substrate interacts with the enzyme. The amino acids sidechains which make up the active site are molded into a precise shape which enables the enzyme to perform its catalytic function. In some cases the substrate molecule changes shape slightly as it enters the active site.

Modifications
Many enzymes contain not only a protein part but need additionally various modifications. These modifications are made posttranslational, i.e., after the polypeptide chain is synthesized. Additional groups can be synthesized onto the polypeptide chain, e.g., phosphorylation or glycosylation of the enzyme.

Another kind of posttranslational modification is the cleavage and splicing 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 prevents the enzyme from harmful digestion of the pancreas or other tissue. This type of inactive precursor to an enzyme is known as a zymogen.

Enzyme cofactors
Some enzymes do not need any additional components to exhibit full activity. However, others require non-protein molecules to be bound for activity. Cofactors can be either inorganic (e.g., metal ions and Iron-sulfur clusters) or organic compounds, which are also known as coenzymes.

Enzymes that require a cofactor, but do not have one bound are called apoenzymes. An apoenzyme together with its cofactor(s) constitutes a holoenzyme (i.e, the active form). Most cofactors are not covalently bound to an enzyme, but are closely associated. However, some cofactors known as prosthetic groups are covalently bound (e.g., thiamine pyrophosphate in certain enzymes).

Most cofactors are either regenerated or chemically unchanged at the end of the reactions. Many cofactors are vitamin-derivatives and serve as carriers to transfer electrons, atoms, or functional groups from an enzyme to a substrate. Common examples are NAD and NADP, which are involved in electron transfer and coenzyme A, which is involved in the transfer of acetyl groups.

Allosteric modulation
Allosteric enzymes change their stucture in response to binding of effectors. Modulation can be direct, where effectors bind directly to binding sites in the enzyme, or indirect, where the effector binds to other proteins or protein subunits that interact with the allosteric enzyme and thus influence catalytic activity.

Thermodynamics


As with all catalysts, all reactions catalyzed by enzymes must be "spontaneous" (containing a net negative Gibbs free energy). With the enzyme, they run in the same direction as they would without the enzyme, just more quickly. However, the uncatalyzed, "spontaneous" reaction might lead to different products than the catalyzed reaction. Furthermore, enzymes can couple two or more reactions, so that a thermodynamically favorable reaction can be used to "drive" a thermodynamically unfavorable one. For example, the cleavage of the high-energy compound ATP is often used to drive other, energetically unfavorable chemical reactions.

Enzymes catalyze the forward and backward reactions equally. They do not alter the equilibrium itself, but only the speed at which it is reached. Carbonic anhydrase catalyzes its reaction in either direction depending on the conditions.
 * $$\mathrm{CO_2 + H_2O

{}^\mathrm{\quad Carbonic\ anhydrase} \!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \overrightarrow{\qquad\qquad\qquad\qquad} H_2CO_3}$$ (in tissues - high CO2 concentration)
 * $$\mathrm{H_2CO_3

{}^\mathrm{\quad Carbonic\ anhydrase} \!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \overrightarrow{\qquad\qquad\qquad\qquad} CO_2 + H_2O}$$ (in lungs - low CO2 concentration)

Kinetics
In 1913, Leonor Michaelis and Maud Menten proposed a quantitative theory of enzyme kinetics, which is referred to as Michaelis-Menten kinetics. Their work was futher developed by G. E. Briggs and J. B. S. Haldane, who derived numerous kinetic equations that are still widely used today.

Enzymes can perform up to several million catalytic reactions per second; to determine the maximum speed of an enzymatic reaction, the substrate concentration is increased until a constant rate of product formation is achieved. This is the maximum velocity (Vmax) of the enzyme. In this state, all enzyme active sites are saturated with substrate. However, Vmax is only one kinetic parameter that biochemists are interested in. The amount of substrate needed to achieve a given rate of reaction is also of interest. This can be expressed by the Michaelis-Menten constant (Km), which is the substrate concentration required for an enzyme to reach one half its maximum velocity. Each enzyme has a characteristic Km for a given substrate.

The efficiency of an enzyme can be expressed in terms of kcat/Km. The quantity kcat, also called the turnover number, incorporates the rate constants for all steps in the reaction, and is the quotient of Vmax and the total enzyme concentration. kcat/Km is a useful quantity for comparing different enzymes against each other, or the same enzyme with different substrates, because it takes both affinity and catalytic ability into consideration. The theoretical maximum for kcat/Km, called diffusion limit, is about 108 to 109 (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 that reach this kcat/Km value are called catalytically perfect or kinetically perfect. Example of such enzymes are triose-phosphate isomerase, carbonic anhydrase, acetylcholinesterase, catalase, fumarase, ß-lactamase, and superoxide dismutase.

The quantum-mechanical (physical) model of enzyme catalysis explains how certain enzymes work faster than previously thought possible. This is achieved by a process known as tunneling, which allows electron and proton transfers to "tunnel" through activation barriers rather go over them.

Inhibition


Enzymes reaction rates can be decreased by competitive, non-competitive, partially competitive, uncompetitive inhibition, and mixed inhibition.

Competitive inhibition
In competitive inhibition, the inhibitor binds to the substrate binding site as shown (right part b), thus preventing substrate binding. Malonate is a competitive inhibitor of the enzyme succinate dehydrogenase, which catalyzes the oxidation of succinate to fumarate.

Competitive inhibition causes the Km value to increase, but does not effect Vmax.

Non-competitive inhibition
Non-competitive inhibitors never bind to the active center, but to other parts of the enzyme that can be far away from the substrate binding site, consequently, there is no competition between the substrate and inhibitor for the enzyme. The extent of inhibition depends entirely on the inhibitor concentration and will not be affected by the substrate concentration. For example, cyanide combines with the copper prosthetic groups of the enzyme cytochrome c oxidase, thus inhibiting cellular respiration. This type of inhibition is typically irreversible, meaning that the enzyme will no longer function.

By changing the conformation (the three-dimensional structure) of the enzyme, the inhibitors either disable the ability of the enzyme to bind or turn over its substrate. The enzyme-inhibitor (EI) and enzyme-inhibitor-substrate (EIS) complex have no catalytic activity.

Non-Competive inhibition causes a decrease in Vmax, but does not change the Km value.

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

This inhibition typically displays a lower Vmax, but an unaffected Km value.

Uncompetitive inhibition
Uncompetitive inhibition occurs when the inhibitor binds only to the enzyme-substrate complex, not to the free enzyme, the EIS complex is catalytically inactive. This mode of inhibition is rare and causes a decrease in both Vmax and the Km value.

Mixed inhibition
Mixed inhibitors can bind to both the enzyme and the ES complex. It has the properties of both competitive and uncompetitive inhibition.

Both a decrease in Vmax and an increase in the the Km value are seen in mixed inhibition.

Metabolic pathways and allosteric enzymes
Several enzymes can work together in a specific order, creating metabolic pathways. In a metabolic pathway, one enzyme takes the product of another enzyme as a substrate. After the catalytic reaction, the product is then passed on to another enzyme. The end product(s) of such a pathway are often inhibitors for one of the first enzymes of the pathway (usually the first irreversible step, called committed step), thus regulating the amount of end product made by the pathways. Such a regulatory mechanism is called a negative feedback mechanism, because the amount of the end product produced is regulated by its own concentration. Negative feedback mechanism can effectively adjust the rate of synthesis of intermediate metabolites according to the demands of the cells. This helps with effective allocations of materials and energy economy, and it prevents the excess manufacture of end products. Like other homeostatic devices, the control of enzymatic action helps to maintain a stable internal environment in living organisms.

Enzyme naming conventions
By common convention, an enzyme's name consists of a description of what it does, with the word ending in -ase. Examples are alcohol dehydrogenase and DNA polymerase. Kinases are enzymes that transfer phosphate groups. This results in different enzymes with the same function having the same basic name; they are therefore distinguished by other characteristics, such as their optimal pH (alkaline phosphatase) or their location (membrane ATPase). Furthermore, the reversibility of chemical reactions means that the normal physiological direction of an enzyme's function may not be that observed under laboratory conditions. This can result in the same enzyme being identified with two different names: one stemming from the formal laboratory identification as described above, the other representing its behavior in the cell. For instance the enzyme formally known as xylitol:NAD+ 2-oxidoreductase (D-xylulose-forming) is more commonly referred to in the cellular physiological sense as D-xylulose reductase, reflecting the fact that the function of the enzyme in the cell is actually the reverse of what is often seen under in vitro conditions.

The International Union of Biochemistry and Molecular Biology has developed a nomenclature for enzymes, the EC numbers; each enzyme is described by a sequence of four numbers, preceded by "EC". The first number broadly classifies the enzyme based on its mechanism:

The toplevel classification is
 * EC 1 Oxidoreductases: catalyze oxidation/reduction reactions
 * EC 2 Transferases: transfer a functional group (e.g. a methyl or phosphate group)
 * EC 3 Hydrolases: catalyze the hydrolysis of various bonds
 * EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation
 * EC 5 Isomerases: catalyze isomerization changes within a single molecule
 * EC 6 Ligases: join two molecules with covalent bonds

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