enzyme - Structures and mechanisms, Cofactors and coenzymes, Thermodynamics, Kinetics, Inhibition, Biological function, Control of activity

A specialized protein molecule produced by a living cell, which acts as a biological catalyst for biochemical reactions. Each enzyme is specific to a particular reaction or group of similar reactions. The molecule undergoing a reaction (the substrate) binds on to an active site on the enzyme to form a short-lived compound molecule, thereby greatly increasing the rate of the reaction. Enzyme activity is strongly influenced by substrate concentration, acidity (pH), temperature, and the presence of other substances (co-factors). The names of enzymes typically end in -ase, and are derived from the substrates on which they act; for example, lipase is an enzyme that breaks down lipid (fat).

In these reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts these into different molecules, the products. Almost all processes in the cell need enzymes in order to occur at significant rates. Since enzymes are extremely 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 providing an alternative path of lower activation energy for a reaction and thus dramatically accelerate the rate of the reaction. 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. Most natural enzymes accelerate their reaction many millions of times faster compared to the uncatalyzed reaction. As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. Enzymes are known to catalyze about 4,000 biochemical reactions.

Enzyme activity can be affected by other molecules. Inhibitors are molecules that decrease enzyme activity, and activators are molecules that increase activity. Drugs and poisons are often enzyme inhibitors. Enzyme activity is also affected by temperature, pH, and the concentration 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 in steak tenderizers breakdown long meat proteins, making them easier to chew). enzymes are usually named according to the reaction they carry out. Typically the suffix -ase is added to the name of the substrate (e.g., lactase is the enzyme that cleaves lactose) or the type of reaction (e.g., DNA polymerase forms DNA polymers).

Having shown that enzymes could function outside a living cell, the next step was to determine their biochemical nature. Many early workers noted that enzymatic activity was associated with proteins, but several scientists (such as Nobel laureate Richard Willstätter) argued that proteins were merely carriers for the true enzymes and that proteins per se were incapable of catalysis. Sumner showed that the enzyme urease was a pure protein and crystallized it; The conclusion that pure proteins can be enzymes was definitively proved by Northrop and Stanley, who worked on the digestive enzymes pepsin (1930), trypsin and chymotrypsin.

This discovery that enzymes could be crystalised eventually allowed their structures to be solved by x-ray crystallography. This high-resolution structure of lysozyme marked the beginning of the field of structural biology and the effort to understand how enzymes work at an atomic level of detail.

Structures and mechanisms

See also: Enzyme catalysis

The activities of enzymes are determined by their three-dimensional structure.

Most enzymes are much larger than the substrates they act on, and only a very small portion of the enzyme (around 3–4 amino acids) is directly involved in catalysis. Enzymes can also contain sites that bind cofactors, which are needed for catalysis. Some enzymes also have binding sites for small molecules, which are often direct or indirect products or substrates of the reaction catalyzed. This binding can serve to increase or decrease the enzyme's activity, providing a means for feedback regulation.

Like all proteins, enzymes are made as long, linear chains of amino acids that fold to produce a three-dimensional product. Most enzymes can be denatured—that is, unfolded and inactivated—by heating, which destroys the three-dimensional structure of the protein.

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.

Some of the enzymes showing the highest specificity and accuracy are involved in the copying and expression of the genome. Here, an enzyme such as DNA polymerase catalyses a reaction in a first step and then checks that the product is correct in a second step.

Some enzymes that produce secondary metabolites are described as promiscuous, as they can act on a relatively broad range of different substrates.

"Lock and key" model

Enzymes are very specific, and it was suggested by Emil Fischer in 1894 that this was because both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. However, while this model explains enzyme specificity, it fails to explain the stabilization of the transition state that enzymes achieve.

Induced fit model

In 1958 Daniel Koshland suggested a modification to the lock and key model: since enzymes are rather flexible structures, the active site can be reshaped by interactions with the substrate as the substrate interacts with the enzyme. As a result, the amino acid side chains which make up the active site are molded into the precise positions that enable the enzyme to perform its catalytic function.

Dynamics and function

Recent investigations have provided new insights into the connection between internal dynamics of enzymes and their mechanism of catalysis. An enzyme's internal dynamics are described as the movement of internal parts (e.g. amino acids, a group of amino acids, a loop region, an alpha helix, neighboring beta-sheets or even entire domain) of these biomolecules, which can occur at various time-scales ranging from femtoseconds to seconds. Networks of protein residues throughout an enzyme's structure can contribute to catalysis through dynamic motions. Protein motions are vital to many enzymes, but whether small and fast vibrations or larger and slower conformational movements are more important depends on the type of reaction involved. These new insights also have implications in understanding allosteric effects, producing designer enzymes and developing new drugs.

Allosteric modulation

Allosteric enzymes change their structure in response to binding of effectors. Modulation can be direct, where the effector binds 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.

Cofactors and coenzymes

Cofactors

Some enzymes do not need any additional components to show full activity. Organic cofactors (coenzymes) are usually prosthetic groups, which are tightly bound to the enzymes that they assist.

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 in its active site.

Enzymes that require a cofactor but do not have one bound are called apoenzymes. Most cofactors are not covalently attached to an enzyme, but are very tightly bound. However, organic prosthetic groups can be covalently bound (e.g., thiamine pyrophosphate in the enzyme pyruvate dehydrogenase).

Coenzymes

Coenzymes are small molecules that transport chemical groups from one enzyme to another.

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 cofactor NADH.

Thermodynamics

As with all catalysts, all reactions catalyzed by enzymes must be "spontaneous" (containing a net negative Gibbs free energy). In the presence of an enzyme, a reaction runs in the same direction as it would without the enzyme, just more quickly. Furthermore, enzymes can couple two or more reactions, so that a thermodynamically favorable reaction can be used to "drive" a thermodynamically unfavorable one.

Enzymes catalyze the forward and backward reactions equally. Under these conditions it is possible that the enzyme will only catalyze the reaction in one direction.

Kinetics

Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products. The rate data used in kinetic analyses are obtained from enzyme assays.

The major contribution of Michaelis and Menten was to think of enzyme reactions in two stages. In the first, the substrate binds reversibly to the enzyme, forming the enzyme-substrate complex. The enzyme then catalyzes the chemical step in the reaction and releases the product.

Enzymes can catalyze up to several million reactions per second. For example, the reaction catalysed by orotidine 5'-phosphate decarboxylase will consume half of its substrate in 78 million years if no enzyme is present. Saturation happens because, as substrate concentration increases, more and more of the free enzyme is converted into the substrate-bound ES form. At the maximum velocity (Vmax) of the enzyme, all enzyme active sites are saturated with 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. This is given 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, and this can show how tight the binding of the substrate is to the enzyme.

The efficiency of an enzyme can be expressed in terms of kcat/Km. 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. 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.

Some enzymes operate with kinetics which are faster than diffusion rates, which would seem to be impossible. 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

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

Non-competitive inhibition

Non-competitive inhibitors can bind either to the active site, or to other parts of the enzyme far away from the substrate-binding site. Moreover, non-competitive inhibitors bind to the enzyme-substrate (ES) complex and to the free enzyme. Their binding to this site changes the shape of the enzyme and stops the active site binding substrate(s). Consequently, since there is no direct competition between the substrate and inhibitor for the enzyme, the extent of inhibition depends only on the inhibitor concentration and will not be affected by the substrate concentration.

Irreversible inhibitors

Some enzyme inhibitors react with the enzyme and form a covalent adduct with the protein.

An example of an inhibitor being 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. 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. If an enzyme produces too much of one substance in the organism, that substance may act as an inhibitor for the enzyme that produces it, causing production of the substance to slow down or stop when there is sufficient amount.

Biological function

Enzymes serve a wide variety of functions inside living organisms.

Viruses can contain enzymes for infecting cells, such as the HIV integrase and reverse transcriptase, or for viral release from cells, like the influenza 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 is inabsorbable in the intestine but enzymes hydrolyse the starch chains into smaller molecules such as maltose and eventually glucose, which can then be absorbed. In ruminants which have a herbivorous diets, bacteria in the gut produce another enzyme, cellulase to break down the cellulose cell walls of plant fibre.

Several enzymes can work together in a specific order, creating metabolic pathways. In a metabolic pathway, one enzyme takes the product of another enzyme as a substrate. After the catalytic reaction, the product is then passed on to another enzyme. Sometimes more than one enzyme can catalyse the same reaction in parallel, this can allow more complex regulation: with for example a low contant activity being provided by one enzyme but an inducible high activity from a second enzyme.

Enzymes determine what steps occur in these pathways. Without enzymes, metabolism would neither progress through the same steps, nor be fast enough to serve the needs of the cell. Consequently, the network of metabolic pathways within each cell depends on the set of functional enzymes that are present.

Control of activity

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

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. For example, bacteria may become resistant to antibiotics such as penicillin because enzymes called beta-lactamases are induced that hydrolyse 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. 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. 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. 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.

Factors affecting rates of reactions

Salt Concentration: Most enzymes can not tolerate extremely high salt concentrations. Effects of Temperature: All enzymes work within a range of temperature specific to the organism. This is due to the denaturating (alteration) of protein structure resulting from the breakdown of the weak ionic and hydrogen bonding that stabilize the three dimensional structure of the enzyme. Denaturating of enzymes, regardless of the cause, leads to the death of the cell. Effects of pH: Most enzymes are sensitive to pH and have specific ranges of activity. The pH can stop enzyme activity by denaturating (altering) the three dimensional shape of the enzyme by breaking weak bonds such as ionic, and hydrogen. Enzyme Saturation: Increasing the substrate concentration increases the rate of reaction (enzyme activity). However, enzyme saturation limits reaction rates. An enzyme is saturated when the active sites of all the molecules are occupied most of the time.

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. 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.

Another example is when germline mutations in genes coding for DNA repair enzymes cause hereditary cancer syndromes such as xeroderma pigmentosum.

Naming conventions

An enzyme's name is often derived from its substrate or the chemical reaction it catalyzes, with the word ending in -ase. This may result in different enzymes, called isoenzymes, with the same function having the same basic name. Furthermore, the normal physiological reaction an enzyme catalyzes may not be the same as under artifical conditions.

The International Union of Biochemistry and Molecular Biology have developed a nomenclature for enzymes, the EC numbers; The first number broadly classifies the enzyme based on its mechanism:

The top-level 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

Enzymes are used in the chemical industry and other industrial applications when extremely specific catalysts are required. However, enzymes in general are limited in the number of reactions they have evolved to catalyse and also by their lack of stability in organic solvents and at high temperatures. Consequently, protein engineering is an active area of research and involves attempts to create new enzymes with novel properties, either through rational design or in vitro evolution.

Application	Enzymes used	Uses
Baking industry alpha-amylase catalyzes the release of sugar monomers from starch	Fungal alpha-amylase enzymes are normally inactivated at about 50 degrees Celsius, but are destroyed during the baking process.
	Proteases	Biscuit manufacturers use them to lower the protein level of flour.
Baby foods	Trypsin	To predigest baby foods.	Enzymes from barley are released during the mashing stage of beer production.
Industrially produced barley enzymes	Widely used in the brewing process to substitute for the natural enzymes found in barley.
Fruit juices	Cellulases, pectinases	Clarify fruit juices
Dairy industry Roquefort cheese	Rennin, derived from the stomachs of young ruminant animals (like calves and lambs).
	Microbially produced enzyme	Now finding increasing use in the dairy industry. margin: 0;" cellspacing="0">
	Glucose		Fructose

Amylases, amyloglucosideases and glucoamylases	Converts starch into glucose and various syrups.
Meat tenderizers	Papain	To soften meat for cooking.
Biological detergent Laundry soap	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.
	Contact lens cleaners	Proteases	To remove proteins on contact lens to prevent infections.
	Rubber industry	Catalase	To generate oxygen from peroxide to convert latex into foam rubber.
	Photographic industry	Protease (ficin)	Dissolve gelatin off scrap film, allowing recovery of its silver content.	Restriction enzymes, DNA ligase and polymerases	Used to manipulate DNA in genetic engineering, important in pharmacology, agriculture and medicine. Structure and Mechanism in Protein Science : A Guide to Enzyme Catalysis and Protein Folding. Theory of Enzyme Catalysis.- Molekuliarnaya Biologia, (1972), 431-439 (In Russian, English summary) Warshel, A., Computer Modeling of Chemical Reactions in enzymes and Solutions John Wiley & ISBN 0-471-18440-3 Thermodynamics Reactions and Enzymes Chapter 10 of On-Line Biology Book at Estrella Mountain Community College.	Kinetics and inhibition Athel Cornish-Bowden, Fundamentals of Enzyme Kinetics. Function and control of enzymes in the cell Price, N. and Stevens, L., Fundamentals of Enzymology: Cell and Molecular Biology of Catalytic Proteins Oxford University Press, (1999), ISBN 0-19-850229-X Nutritional and Metabolic Diseases Enzyme-naming conventions Enzyme Nomenclature, Recommendations for enzyme names from the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology. Press, New York, (1959) Industrial applications History of industrial enzymes, Article about the history of industrial enzymes, from the late 1900's to the present times.