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• Simple Enzyme Experiments - Sharon B. Miller, Tri-County Technical College


• Cloning of a Cellulase Enzyme - BioTeach


• Factors that Influence the Activity of an Enzyme - Wiley


• The Action of Amylase on Starch - biotopics.co.uk


• Enzyme Activity: An Inquiry Based Approach - Nancy Contolini, Brookfield High School


• Properties of Enzymes - CSME


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• Enzyme Grabbers - Randyll Warehime, Woodrow Wilson Biology Institute


• Regulation of Enzyme Activity - Michelle Thornton

Ribbon diagram of the catalytically perfect enzyme TIM.

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 catalyzes 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 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 occurring 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 ( e.g., 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

Eduard Buchner

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

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

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

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. Many enzymes can be unfolded or inactivated by heating, which destroys the three-dimensional structure of the protein.

Cartoon showing the active site of an enzyme.

Most enzymes are larger than the substrates they act on and 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.

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

Fischer's lock and key hypothesis of enzyme action.

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

A diagram showing a more realistic situation for induced fit hypothesis. Incorrect substrates, either too big or too small in size, do not fit with the active site.

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 structure 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.

Catalysis Models

Understanding the fundamental processes which allow enzymes to function so efficiently has long intringued scientists. In 1894, Emil Fischer offered the first popular model to explain enzyme function generally. This model was born of his fascination with certain glycolytic enzymes and their abilities to distinguish between different sugar stereoisomers. Consider a reaction in which a ‘round’ substrate or ground state is converted to a ‘square’ product via a ‘triangular’ transition state:

In his textbook 'lock-and-key' analogy, the enzyme is the 'lock' capable of accepting only one or a very few 'keys'.

While suggesting that the enzyme active site is complementary to the reaction ground state and thereby offering an explanation for the remarkable specificity of enzymes, this model is grossly oversimplified in that it says nothing about what happens after the enzyme binds the substrate. Questions concerning the means by which the bound substrate is transformed to the transition state are ignored.

This view of enzyme catalysis predominated until 1930 when J.B.S. Haldane turned his thoughts to the subject. In his reasoning, it made more sense for the active site to exist not in a ground state complementarity but in a complementarity to the reaction transition state as its stabilization results in rate enhancement. In this case, the active site is therefore triangular.

He suggested no active role for the bulk of the enzyme that does not constitute the active site and it is generally believed that the large enzyme mass is necessary for the optimal orientation of the active site functionalities for binding the transition state.

In the late 1950s, D. Koshland introduced the ‘induced fit’ modification of the Haldane model. In the Haldane/Koshland model, the enzyme catalytic groups are not quite optimized for stabilization of the transition state until the ground state binds.

In the Haldane/Koshland view, the substrate induces the transition state complementarity via local rearrangements of the active site functionalities, is subjected to this transition state stabilizing force, and converted to product. This modification is deficient, however, in that it ignores the great potential for reaction facilitation via global enzyme conformational changes.

In 1993, M. Britt introduced the ‘shifting specificity’ model for enzyme catalysis. In this view, the enzyme is a conformationally high energy structure with an active site complementarity to the reaction ground state. Binding the ground state to the active site necessarily induces a global conformational change which transforms the active site from a ground state complementarity to a transition state complementarity in the process transforming the ground state to the transition state. The enzyme achieves a conformational energy minimum at the reaction transition state thereby aiding in the facilitation of the reaction.

Placing this model in an historical perspective, the first step is simply Fischer's lock and key analogy. We now know that this does not necessarily imply a dead end for the reaction given the potential that enzymes possess for adopting different conformations. Binding of the ground state then results in an active site geometry which prefers the binding of the transition state, as Haldane requires. There is an 'induced fit' of sorts but is one from a full-fledged ground state complementarity and one which actively involves the entire enzyme molecule. Furthermore, the conformational change is one which leads to a significantly lower enzyme-localized energy at the transition state.

Thermodynamics

Diagram of a catalytic reaction, showing the energy niveau at each stage of the reaction. The substrates usually need a large amount of energy to reach the transition state, which then reacts to form the end product. The enzyme stabilizes the transition state, reducing the energy of the transition state and thus the energy required to get over this barrier.

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.

(in tissues - high CO₂ concentration)

(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 further 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 (V_max) of the enzyme. In this state, all enzyme active sites are saturated with substrate. However, V_max 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 (K_m), which is the substrate concentration required for an enzyme to reach one half its maximum velocity. Each enzyme has a characteristic K_m for a given substrate.

The efficiency of an enzyme can be expressed in terms of k_cat/K_m. The quantity k_cat, also called the turnover number, incorporates the rate constants for all steps in the reaction, and is the quotient of V_max and the total enzyme concentration. k_cat/K_m 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 k_cat/K_m, 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 k_cat/K_m 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

A competitive inhibitor binds reversibly to the enzyme, preventing the binding of substrate. On the other hand, binding of substrate prevents binding of the inhibitor, thus substrate and inhibitor compete for the enzyme.

Diagram showing the mechanism of non-competitive 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 K_m value to increase, but does not effect V_max.

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-Competitive inhibition causes a decrease in V_max, but does not change the K_m 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 V_max, but an unaffected K_m 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 V_max and the K_m 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 V_max and an increase in the K_m 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/

Industrial Applications

Application	Enzymes used	Uses	Notes and examples
Biological detergent	Primarily proteases, produced in an extracellular form from bacteria	Used for presoak conditions and direct liquid applications helping with removal of protein stains from clothes.
	Amylase enzymes	Detergents for machine dish washing to remove resistant starch residues.
Baking industry	Fungal alpha-amylase enzymes: normally inactivates about 50 degrees Celsius, destroyed during baking process	Catalyze breakdown of starch in the flour to sugar. Yeast action on sugar produces carbon dioxide. Used in production of white bread, buns, and rolls	alpha-amylase catalyzes the release of sugar monomers from starch
	Protease enzymes	Biscuit manufacturers use them to lower the protein level of flour.
Baby foods	Trypsin	To predigest baby foods
Brewing industry	Enzymes from barley are released during the mashing stage of beer production.	They degrade starch and proteins to produce simple sugar, amino acids and peptides that are used by yeast to enhance fermentation.	Germinating barley used for malt.
	Industrially produced barley enzymes.	Widely used in the brewing process to substitute for the natural enzymes found in barley.
	Amylase, glucanases, proteases	Split polysaccharides and proteins in the malt
	Betaglucosidase	Improve the filtration characteristics.
	Amyloglucosidase	Low-calorie beer
	Proteases	Remove cloudiness during storage of beers.
Fruit juices	Cellulases, pectinases	Clarify fruit juices
Dairy industry	Rennin, derived from the stomachs of young ruminant animals (calves, lambs)	Manufacture of cheese, used to split protein	Note: As animals age rennin production decreases and is replaced by another protease, pepsin, which is not suitable for cheese production. In recent years the increase in cheese consumption, as well as increased beef production, has resulted in a shortage of rennin and escalating prices.
	Microbially produced enzyme	Now finding increasing use in the dairy industry	Roquefort cheese
	Lipases	Is implemented during the production of Roquefort cheese to enhance the ripening of the blue-mould cheese.
	Lactases	Break down lactose to glucose and galactose
Starch industry	Amylases, amyloglucosideases and glucoamylases	Converts starch into glucose and various syrups	Glucose Fructose
	Glucose isomerase	Converts glucose into fructose (high fructose syrups derived from starchy materials have enhanced sweetening properties and lower calorific values)
Rubber industry	Catalase	To generate oxygen from peroxide to convert latex to foam rubber
Paper industry	Amylases	Degrade starch to lower viscosity product needed for sizing and coating paper
Photographic industry	Protease (ficin)	Dissolve gelatin off the scrap film allowing recovery of silver present

See also

• List of enzymes
• Enzyme kinetics

References

• Koshland D. The Enzymes, v. I, ch. 7, Acad. Press, New York, 1959
• Perutz M. Proc. Roy. Soc., B (1967) 167, 448,
• Cha, Y., Murray, C. J. & Klinman, J. P. Science (1989) 243, 1325-1330 .
• Leonor Michaelis and Maud Menten, Die Kinetik der Invertinwirkung, Biochem. Z. (1913) 49, 333-369.
• G. E. Briggs and J. B. S. Haldane, A note on the kinetics of enzyme action, Biochem. J., (1925) 19, 339-339.
• M.V. Volkenshtein, R.R. Dogonadze, A.K. Madumarov, Z.D. Urushadze, Yu.I. Kharkats. Theory of Enzyme Catalysis.- Molekuliarnaya Biologia, (1972), 431-439 (In Russian, English summary)

External links

Wikimedia Commons has media related to:

Category:Enzymes

• ExPASy enzyme database, links to Swiss-Prot sequence data, entries in other databases and to related literature searches
• PDBsum links to the known 3-D structure data of enzymes in the Protein Data Bank
• BRENDA, comprehensive compilation of information and literature references about all known enzymes; requires payment by commercial users
• Weizmann Institute's Genecards Database, extensive database of protein properties and their associated genes.
• Cytochrome P450 enzymes site lists over 4000 versions of enzymes from this cytochrome in plants and animals