Enzymes- Properties, Classification and Significance

Enzymes are biological catalysts that increase the rate of chemical reactions without being changed themselves in the process. They are essential for life because they allow biochemical reactions to occur under mild conditions (such as low temperature, atmospheric pressure and neutral pH) that are compatible with living cells.

Enzymes are mostly proteins, although some RNA molecules can also act as enzymes. Enzymes have specific three-dimensional shapes that determine their functions and interactions with other molecules. The region of an enzyme that binds and transforms the substrate (the molecule that undergoes the reaction) is called the active site. The active site is usually a small part of the enzyme that is formed by amino acid residues that may be far apart in the linear sequence of the protein.

Enzymes are highly specific for their substrates and products, meaning that they only catalyze one type of reaction or a few closely related reactions. This specificity is determined by the shape and properties of the active site, which can only accommodate certain substrates and facilitate certain transformations. For example, sucrase is an enzyme that catalyzes the hydrolysis of sucrose into glucose and fructose, but it cannot hydrolyze other disaccharides such as lactose or maltose.

Enzyme activity can be regulated by various factors, such as temperature, pH, substrate concentration, inhibitors, activators and feedback mechanisms. Enzymes usually have an optimal temperature and pH at which they function most efficiently. For example, pepsin is an enzyme that digests proteins in the stomach and has an optimal pH of 2, while trypsin is an enzyme that digests proteins in the small intestine and has an optimal pH of 8.5. Enzyme activity can also be modulated by molecules that bind to the enzyme and either enhance or reduce its catalytic ability. These molecules can be either reversible or irreversible inhibitors or activators. Furthermore, some enzymes are regulated by feedback mechanisms, where the product of a reaction inhibits or activates the enzyme that catalyzes it or another enzyme in the same pathway.

Enzymes are classified into six major classes based on the type of reaction they catalyze: oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. Each enzyme has a unique name and a four-digit classification number assigned by the Enzyme Commission (EC). For example, trypsin has the EC number 3.4.21.4, which indicates that it belongs to the class of hydrolases (3), sub-class of proteases (4), sub-sub-class of serine proteases (21) and serial number (4).

Enzymes have many roles and applications in biological systems and beyond. They are involved in metabolism, gene expression, signal transduction, cell division, defense mechanisms and many other processes. They also have industrial and medical uses, such as in food production, biotechnology, diagnostics, drug delivery and therapy.

Many enzymes require the presence of small, non-protein molecules or ions to carry out their catalytic function. These molecules or ions are called cofactors. Cofactors can be classified into two main types: coenzymes and prosthetic groups.

Coenzymes

• Coenzymes are organic molecules that bind loosely to enzymes and help them catalyze reactions.
• Coenzymes often act as carriers or donors of chemical groups or electrons between different enzymes.
• Coenzymes can be reused by different enzymes in a metabolic pathway.
• Many coenzymes are derived from vitamins, such as NAD+ from niacin, FAD from riboflavin, and coenzyme A from pantothenic acid.
• Examples of coenzymes are NAD+, FAD, coenzyme A, thiamine pyrophosphate, biotin, and pyridoxal phosphate.

Prosthetic groups

• Prosthetic groups are organic molecules or metal ions that bind tightly or covalently to enzymes and form part of their structure.
• Prosthetic groups often play an important role in the catalytic mechanism of enzymes by facilitating substrate binding, stabilizing transition states, or participating in redox reactions.
• Prosthetic groups are usually not interchangeable between different enzymes and remain attached to the same enzyme throughout its lifetime.
• Examples of prosthetic groups are heme, flavin, metal ions (such as Fe2+, Zn2+, Cu2+, etc.), and biotin.

• Some enzymes require the presence of small, non-protein units or cofactors to carry out their particular reaction. Cofactors may be either one or more inorganic ions, such as Zn2+ or Fe2+, or a complex organic molecule called a coenzyme.
• A coenzyme that is covalently attached to the enzyme is called a prosthetic group (for example, heme in hemoglobin).
• A complete catalytically-active enzyme together with its cofactor or prosthetic group is called a holoenzyme . A holoenzyme is complete and catalytically active.
• The protein part of the enzyme on its own without its cofactor or prosthetic group is termed an apoenzyme . An apoenzyme is an inactive enzyme, and activation of the enzyme occurs upon binding of an organic or inorganic cofactor.
• Most cofactors are not covalently bound but instead are tightly bound. However, some organic prosthetic groups such as an iron ion or a vitamin can be covalently bound.
• Examples of holoenzymes include DNA polymerase and RNA polymerase which contain multiple protein subunits. The complete complexes contain all the subunits necessary for activity.

• Isoenzymes are different forms of an enzyme that catalyze the same reaction, but have different physical or kinetic properties, such as isoelectric point, pH optimum, substrate affinity or effect of inhibitors.
• Isoenzymes are usually derived from different genes and often occur in different tissues of the body. They may have different regulatory mechanisms or different roles in metabolism.
• Isoenzymes can be used as biomarkers to diagnose diseases or monitor organ function. For example, the enzyme lactate dehydrogenase (LDH) has five isoenzymes that can be separated by electrophoresis. The relative levels of these isoenzymes can indicate tissue damage or disease in the heart, liver, muscle or red blood cells.
• Another example of an enzyme with isoenzymes is hexokinase, which catalyzes the first step of glycolysis. There are four isoenzymes of hexokinase in mammals, each with a different tissue distribution and kinetic behavior. Hexokinase I and II are found in most tissues and have a high affinity for glucose. Hexokinase III is found mainly in the liver and has a lower affinity for glucose. Hexokinase IV (also called glucokinase) is found only in the liver and pancreas and has a very low affinity for glucose. Hexokinase IV is regulated by insulin and glucagon and plays a key role in glucose homeostasis.
• Isoenzymes can also be used to study the evolutionary relationships among organisms. For example, the enzyme alcohol dehydrogenase (ADH) has multiple isoenzymes in different species that reflect their adaptation to different environments. ADH isoenzymes can be compared by their amino acid sequences, molecular weights, substrate specificities and inhibition patterns. ADH isoenzymes can also be used to trace the origin and migration of human populations.

The active site of an enzyme is the region that binds the substrate and converts it into product. It is usually a relatively small part of the whole enzyme molecule and is a three-dimensional entity formed by amino acid residues that can lie far apart in the linear polypeptide chain. The active site is often a cleft or crevice on the surface of the enzyme that forms a predominantly nonpolar environment which enhances the binding of the substrate.

The substrate(s) is bound in the active site by multiple weak forces (electrostatic interactions, hydrogen bonds, van der Waals bonds, hydrophobic interactions; and in some cases by reversible covalent bonds. The binding of the substrate to the active site also involves a precise alignment of the substrate with respect to the catalytic residues of the enzyme. These residues are usually located at the bottom or center of the active site and are responsible for facilitating the chemical transformation of the substrate into product.

The active site of an enzyme is highly specific for its substrate(s) and can discriminate between very similar molecules based on their size, shape, charge, polarity and functional groups. The specificity of an enzyme is determined by the properties and spatial arrangement of the amino acid residues forming the active site. Subtle changes in these residues can alter the substrate specificity or catalytic efficiency of an enzyme.

The active site of an enzyme can also undergo conformational changes upon binding of the substrate or other molecules. These changes can affect the shape, size and accessibility of the active site as well as the orientation and proximity of the catalytic residues. Conformational changes can be induced by the substrate itself (induced fit model) or by other molecules that bind to a different site on the enzyme (allosteric regulation).

The active site of an enzyme is therefore a dynamic and flexible structure that adapts to its substrate(s) and catalyzes their conversion into product(s) with high specificity and efficiency. The active site is also subject to regulation by various factors that can modulate its activity and function.

One of the remarkable features of enzymes is their substrate specificity, which means their ability to select and bind only specific molecules from a group of similar chemical compounds. The substrate specificity of enzymes is determined by the active site, the region of the enzyme that binds the substrate and catalyzes its conversion into product.

The active site is composed of a unique combination of amino acid residues that form a three-dimensional structure that fits the substrate like a jigsaw puzzle piece. The amino acid residues in the active site can have different properties, such as size, charge, polarity, and hydrophobicity, that create a specific chemical environment that attracts and stabilizes the substrate.

The substrate binds to the active site by multiple weak interactions, such as hydrogen bonds, electrostatic forces, van der Waals forces, and hydrophobic interactions. These interactions are highly specific and depend on the precise spatial arrangement and orientation of the substrate and the active site.

The binding of the substrate to the active site also induces a conformational change in the enzyme, resulting in a better fit between the two molecules. This phenomenon is called induced fit and it enhances the catalytic efficiency and specificity of the enzyme .

The substrate specificity of enzymes can vary in degree, depending on how selective they are for their substrates. Some enzymes are very specific and can bind only one type of substrate, while others are more flexible and can bind several substrates with similar structures or functional groups. The degree of specificity can also depend on the environmental conditions, such as temperature, pH, and ionic strength, that can affect the shape and charge of the enzyme and the substrate .

The substrate specificity of enzymes is crucial for their biological functions, as it ensures that only the desired reactions take place in the cells and prevents unwanted side reactions. The substrate specificity of enzymes also allows them to be regulated by various mechanisms, such as allosteric modulation, covalent modification, feedback inhibition, and compartmentalization .

The substrate specificity of enzymes also has many applications in biotechnology, medicine, and industry. For example, enzymes can be used as biosensors to detect specific molecules in biological samples, such as glucose or cholesterol. Enzymes can also be used as biocatalysts to synthesize useful compounds, such as antibiotics or biofuels. Enzymes can also be used as therapeutic agents to treat diseases caused by enzyme deficiencies or dysfunctions .

Enzymes are biological catalysts that speed up the rate of chemical reactions by lowering the activation energy required for the reaction to proceed. Activation energy is the minimum amount of energy needed to convert the reactants into products. Enzymes do not alter the equilibrium constant or the free energy change of the reaction, but they allow the reaction to reach equilibrium faster.

The mechanism of action of enzymes involves two main steps: binding and catalysis. In the binding step, the enzyme recognizes and binds its specific substrate(s) in its active site, forming an enzyme-substrate complex. In the catalysis step, the enzyme facilitates the conversion of the substrate(s) into product(s) by stabilizing the transition state and lowering the activation energy. The product(s) then dissociate from the enzyme, leaving it free to bind another substrate molecule and repeat the cycle.

The binding step is governed by the principle of molecular recognition, which states that molecules interact with each other based on their shape and chemical properties. The active site of an enzyme has a complementary shape and charge distribution to its substrate(s), allowing them to fit together like a lock and key or an induced fit. The binding is also influenced by factors such as temperature, pH, ionic strength, and presence of inhibitors or activators.

The catalysis step is governed by the principle of transition state stabilization, which states that enzymes lower the activation energy by providing an alternative reaction pathway that involves a more stable transition state. The transition state is a high-energy intermediate that forms between the reactants and products. Enzymes stabilize the transition state by providing favorable interactions such as electrostatic attraction, hydrogen bonding, covalent bonding, or metal ion coordination. The catalysis step may involve one or more mechanisms such as acid-base catalysis, covalent catalysis, metal ion catalysis, or proximity and orientation effects.

The overall mechanism of action of enzymes can be represented by a simple equation:

E + S ⇌ ES ⇌ EP ⇌ E + P

where E is the enzyme, S is the substrate, ES is the enzyme-substrate complex, EP is the enzyme-product complex, and P is the product.

The rate of an enzyme-catalyzed reaction depends on several factors such as enzyme concentration, substrate concentration, temperature, pH, and presence of inhibitors or activators. The relationship between these factors can be described by mathematical models such as Michaelis-Menten kinetics or allosteric regulation. These models help us understand how enzymes work and how they can be controlled in biological systems.

The substrate-enzyme binding is the process by which an enzyme recognizes and attaches to its specific substrate(s) and catalyzes a chemical reaction. The substrate(s) is the molecule(s) that undergoes a change in the presence of the enzyme. The enzyme is the protein that acts as a biological catalyst and speeds up the reaction rate.

The substrate binds to the enzyme by interacting with amino acids in the binding site. The binding site on enzymes is often referred to as the active site because it contains amino acids that both bind the substrate and aid in its conversion to product. The active site is usually a relatively small part of the whole enzyme molecule and is a three-dimensional entity formed by amino acid residues that can lie far apart in the linear polypeptide chain. The active site is often a cleft or crevice on the surface of the enzyme that forms a predominantly nonpolar environment which enhances the binding of the substrate.

The substrate specificity of enzymes is determined by the properties and spatial arrangement of the amino acid residues forming the active site of an enzyme. A specific chemical substrate matches this site like a jigsaw puzzle piece and makes the enzyme specific to its substrate. Substrate specificity is often determined by changes in relatively few amino acids in the active site. This is clearly seen in the three digestive enzymes trypsin, chymotrypsin and elastase, which have different substrate preferences but share a common catalytic mechanism.

The binding of substrate induces a conformational change in the active site of the enzyme. This phenomenon is known as induced fit and it states that the binding of substrate causes the enzyme to adjust its shape slightly to achieve optimum fit. In addition, the enzyme may distort the substrate, forcing it into a conformation similar to that of the transition state. The transition state is an unstable intermediate stage between reactants and products that has higher energy than both. By stabilizing the transition state, the enzyme lowers the activation energy required for the reaction to proceed.

The substrate(s) is bound in the active site by multiple weak forces (electrostatic interactions, hydrogen bonds, van der Waals bonds, hydrophobic interactions; and in some cases by reversible covalent bonds) . These forces result into the formation of an enzyme-substrate complex, which is a temporary association between the enzyme and its substrate(s). Once bound, active residues within the active site of the enzyme act on the substrate molecule to transform it first into the transition state complex and then into product(s), which are released . The enzyme is now free to bind another molecule of substrate and begin its catalytic cycle again .

The substrate-enzyme binding is influenced by several factors, such as temperature, pH, concentration of substrate and enzyme, presence of inhibitors or activators, and allosteric regulation. These factors can affect the shape, stability, affinity, and activity of enzymes and their substrates, and thus modulate the rate and outcome of enzymatic reactions. Understanding how enzymes bind their substrates is essential for elucidating their mechanisms of action, their regulation, and their applications in biotechnology and medicine.

The lock and key model and the induced fit model are two ways of explaining how an enzyme binds its substrate and catalyzes a reaction. Both models assume that the enzyme has a specific shape that is complementary to the substrate, but they differ in how flexible the enzyme is.

• In the lock and key model, the enzyme and the substrate are thought to fit together like a key into its lock. The enzyme has a rigid and fixed shape that matches the substrate exactly. Only one substrate can bind to one enzyme, and the binding is very specific. The enzyme does not change its shape or conformation when it binds the substrate.

• In the induced fit model, the enzyme and the substrate are thought to interact dynamically. The enzyme has a flexible shape that can adjust to fit the substrate. The binding of the substrate induces a conformational change in the enzyme, making it more complementary to the substrate. The enzyme can bind to different substrates that have similar shapes, and the binding is more versatile. The enzyme changes its shape or conformation when it binds the substrate.

The reality is that different enzymes show features of both models, with some complementarity and some conformational change. The induced fit model is more widely accepted as it can explain more experimental observations, such as:

• The binding of substrates often involves distortion or strain, which lowers the activation energy of the reaction.
• The binding of substrates often involves multiple weak interactions, which are influenced by the shape and charge of the molecules.
• The binding of substrates often involves cooperative effects, where the binding of one substrate affects the binding of another.
• The binding of substrates often involves allosteric regulation, where the binding of a molecule at a site other than the active site affects the activity of the enzyme.

The lock and key model and the induced fit model are useful simplifications to understand how enzymes work, but they do not capture all the complexity and diversity of enzyme-substrate interactions. Enzymes are dynamic and adaptable molecules that can adjust their shape and function to different situations.

Enzyme nomenclature is a system of naming enzymes that is based on the type of reaction they catalyze and the substrate they act on. The system was introduced by the International Union of Biochemistry and Molecular Biology (IUBMB) in 1961 and has been updated periodically .

The enzyme nomenclature consists of four numbers separated by dots, called the Enzyme Commission (EC) number. The first number indicates the major class of the enzyme, which corresponds to the general type of reaction it catalyzes. There are six major classes of enzymes:

• EC 1: Oxidoreductases, which catalyze oxidation-reduction reactions
• EC 2: Transferases, which catalyze transfer of functional groups
• EC 3: Hydrolases, which catalyze hydrolysis reactions
• EC 4: Lyases, which catalyze cleavage or formation of double bonds
• EC 5: Isomerases, which catalyze isomerization reactions
• EC 6: Ligases, which catalyze ligation or joining of molecules

The second number indicates the subclass of the enzyme, which corresponds to the specific type of reaction or group transfer it catalyzes within the major class. For example, within the oxidoreductases class, there are subclasses for enzymes that act on CH-OH groups, aldehydes, ketones, CH-NH2 groups, etc.

The third number indicates the subsubclass of the enzyme, which corresponds to the specific substrate or cofactor involved in the reaction. For example, within the subclass of transferases that act on aldehyde or ketone residues, there are subsubclasses for enzymes that transfer one-carbon groups, aldehydes, ketones, etc.

The fourth number is a serial number that uniquely identifies the enzyme within its subsubclass. For example, within the subsubclass of transferases that transfer one-carbon groups as methyl groups, there are enzymes with serial numbers from 1 to 29.

Each enzyme also has a recommended name that reflects its function and specificity. The recommended name usually consists of the name of the substrate or group transferred followed by the name of the acceptor or donor molecule and the suffix "-ase". For example, hexokinase is an enzyme that transfers a phosphate group from ATP to hexose sugars. Some enzymes have alternative names that do not follow this pattern, such as trypsin or chymotrypsin.

To rationalize enzyme names and avoid confusion, a system of enzyme nomenclature has been internationally agreed by the Nomenclature Commission of the International Union of Biochemistry. This system places all enzymes into one of six major classes based on the type of reaction they catalyze. Each enzyme is then uniquely identified with a four-digit classification number, preceded by the prefix EC—for enzyme classification—that indicates its group, subgroup, and so forth. For example, alcohol dehydrogenase has the EC number 3.4.21.4, where the first number (3) denotes that it is an oxidoreductase, the second number (4) that it is a dehydrogenase, the third number (21) that it is a serine protease with a critical serine residue at the active site, and the fourth number (4) indicates that it was the fourth enzyme to be assigned to this class.

The six major classes of enzymes and their subclasses are as follows :

• Oxidoreductases: These enzymes catalyze oxidation-reduction reactions where electrons are transferred from one molecule (the reductant) to another molecule (the oxidant). The most common name used for these enzymes is dehydrogenase, which indicates that they remove hydrogen atoms from their substrates. Other names include oxidase, which indicates that oxygen is the electron acceptor, and reductase, which indicates that the enzyme reduces its substrate. Examples of oxidoreductases are alcohol dehydrogenase, which oxidizes ethanol to acetaldehyde, and cytochrome c oxidase, which transfers electrons from cytochrome c to oxygen in the electron transport chain.

• Transferases: These enzymes catalyze the transfer of a functional group from one molecule (the donor) to another molecule (the acceptor). The functional group may be an atom or a group of atoms, such as a methyl, phosphate, or amino group. The name of these enzymes usually indicates the donor and acceptor molecules involved in the transfer. Examples of transferases are hexokinase, which transfers a phosphate group from ATP to glucose, and transaminase, which transfers an amino group from one amino acid to another.

• Hydrolases: These enzymes catalyze the hydrolysis of various bonds by adding water to their substrates. The bonds that can be hydrolyzed include ester, peptide, glycosidic, and phosphodiester bonds. The name of these enzymes usually indicates the substrate or the bond that is hydrolyzed. Examples of hydrolases are lipase, which hydrolyzes lipids into fatty acids and glycerol, and protease, which hydrolyzes proteins into amino acids.

• Lyases: These enzymes catalyze the cleavage of various bonds by means other than hydrolysis or oxidation. They can also catalyze the reverse reaction, where they join two molecules by forming a new bond. The bonds that can be cleaved or formed include carbon-carbon, carbon-oxygen, carbon-nitrogen, and carbon-sulfur bonds. The name of these enzymes usually indicates the substrate or the bond that is cleaved or formed. Examples of lyases are aldolase, which cleaves fructose-1,6-bisphosphate into glyceraldehyde-3-phosphate and dihydroxyacetone phosphate in glycolysis, and synthase, which forms a bond between two substrates without using ATP.

• Isomerases: These enzymes catalyze the interconversion of different isomers of a molecule. Isomers are molecules that have the same molecular formula but different arrangements of atoms. The types of isomerism that can be catalyzed by these enzymes include structural isomerism, geometric isomerism, and optical isomerism. The name of these enzymes usually indicates the substrate or the type of isomerism involved. Examples of isomerases are phosphoglucose isomerase, which converts glucose-6-phosphate into fructose-6-phosphate in glycolysis, and cis-trans isomerase, which converts cis-fatty acids into trans-fatty acids.

• Ligases: These enzymes catalyze the joining of two molecules by forming a new bond with the input of energy from ATP or another nucleoside triphosphate. The bonds that can be formed include carbon-carbon, carbon-oxygen, carbon-nitrogen, and carbon-sulfur bonds. The name of these enzymes usually indicates the substrates or the bond that is formed. Examples of ligases are DNA ligase, which joins two DNA strands by forming a phosphodiester bond between them, and carboxylase, which adds a carboxyl group to a substrate using bicarbonate and ATP.

These six classes of enzymes cover most but not all types of enzymatic reactions. A seventh class called translocases has been proposed to include enzymes that catalyze the movement of ions or molecules across membranes or within cells. However, this class has not been officially accepted by the Nomenclature Commission yet.

Enzymes are essential for life as they catalyze almost all the biochemical reactions that occur in living organisms. They also have many applications in various fields such as medicine, industry, biotechnology and environmental management. Some of the significance of enzymes are:

• Enzymes regulate the metabolic pathways that sustain life by controlling the rate and specificity of chemical reactions. For example, enzymes are involved in the synthesis and degradation of carbohydrates, lipids, proteins and nucleic acids, as well as in energy production and utilization .
• Enzymes modulate the cellular responses to various stimuli such as hormones, neurotransmitters, growth factors and stress signals. For example, enzymes are involved in signal transduction, gene expression, cell cycle regulation and apoptosis .
• Enzymes maintain the homeostasis of the internal environment by regulating the pH, temperature, osmolarity and redox state of the cells and tissues. For example, enzymes are involved in acid-base balance, thermoregulation, osmoregulation and antioxidant defense .
• Enzymes participate in the defense mechanisms against pathogens and foreign substances by catalyzing the production and degradation of immune molecules such as antibodies, cytokines and complement components. For example, enzymes are involved in inflammation, phagocytosis, antigen presentation and adaptive immunity .

Enzymes also have many applications outside the biological systems due to their remarkable catalytic properties such as high efficiency, specificity, selectivity and mild reaction conditions. Some of the applications of enzymes are:

• Enzymes are used in medicine to treat enzyme-related disorders, to assist in metabolism and drug delivery, to diagnose and detect diseases, and to manufacture medicines. For example, enzymes are used to replace or supplement deficient or defective enzymes in patients with metabolic diseases such as phenylketonuria, lactose intolerance and Gaucher`s disease . Enzymes are also used to degrade or modify drugs or toxins for targeted delivery or enhanced efficacy . Enzymes are also used to measure the levels of biomarkers or pathogens in blood, urine or other samples for diagnosis or monitoring of diseases such as diabetes, hepatitis and HIV . Enzymes are also used to synthesize or modify drugs or natural products for improved pharmacological properties or reduced side effects .
• Enzymes are used in industry to produce or modify various products such as food, beverages, textiles, paper, leather, detergents and biofuels. For example, enzymes are used to hydrolyze starch into glucose or fructose for sweetening agents . Enzymes are also used to modify proteins such as casein for cheese making or gluten for bread making . Enzymes are also used to degrade cellulose into glucose for ethanol production or paper pulp . Enzymes are also used to remove stains or odors from fabrics or surfaces by breaking down fats, proteins or other organic compounds .
• Enzymes are used in biotechnology to manipulate DNA or RNA for genetic engineering or molecular biology. For example, enzymes are used to cut, paste, copy or amplify DNA fragments for cloning, sequencing or gene expression . Enzymes are also used to synthesize or modify RNA molecules for transcription, translation or interference .
• Enzymes are used in environmental management to degrade or detoxify pollutants or waste materials. For example, enzymes are used to break down pesticides or herbicides into harmless products . Enzymes are also used to degrade plastics or other synthetic polymers into biodegradable monomers .

In summary, enzymes play a vital role in biological systems and have a wide range of applications in various fields due to their remarkable catalytic properties.

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