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Enzymes are Biological Catalysts
Introduction Catalysts are substances that increase the rates of chemical reactions. Life requires that many chemical reactions occur within organisms. The human body employs over a thousand chemical reactions. Many of these reactions would occur too slowly to be useful in the absence of a catalyst. Nature provides humans and other biological organisms with proteins that are capable of catalyzing reactions. Enzymes are protein, organic or biological catalysts that increase the velocity of a chemical reaction and are not consumed during the reaction they catalyze. This process is called catalysis and enzymes thus catalyze biochemical reactions. Scientists who specialize in studying enzymes are called enzymologists. In enzymatic reactions, the molecules present at the beginning of the reaction (reactants) are called substrates. Enzymes convert substrates into different molecules, called products. All processes in nature require enzymes in order to occur at significant rates. Enzyme kinetics is a fundamental way of describing, predicting and calculating how enzymes bind substrates, turn these into products and also how fast and efficiently this is happening. Properties of Enzymes
1. Enzymes are Proteins: Most of the enzyme are simple or conjugated proteins. Some enzymes are simple proteins. On hydrolysis they yield amino acids only. Eg. Amylase, Urease etc. Many enzymes possess chemical group that are non-amino acid in nature, ie, the protein part of the enzyme is conjugated with a non protein part. These are called conjugated proteins or holoenzymes. Several types of groups are associated with enzymes (Figure ). These groups are related to the structure and function of an enzyme. Fig. 7.1: Schematic representation of an enzyme. Apoenzyme: The protein part of an enzyme molecule is known as apoenzyme and is inactive. Cofactor: The nonprotein part of an enzyme is called a cofactor. These cofactors are basically the additional chemical groups, which appear in those enzymes that are conjugated protein molecules. Some cofactors may be metal ions such as Mg2+, Zn2+^ or complex organic molecules, such as nicotinamide adenine dinucleotide. Whatever be the nature of a cofactor, both are required for enzyme activity (participate in substrate binding or in catalysis). The term holoenzyme refers to the active enzyme with its non-protein component, whereas the enzyme without its non-protein moiety is termed an apoenzyme and is inactive.
PROPERTIES AND CHARACTERISTICS OF ENZYMES BY C. B. LUKONG (PhD) @2019/ Holoenzyme: The combination of an apoenzyme and the cofactor is known as the holoenzyme and is active: Apoenzyme + Cofactor Holoenzyme Coenzyme: When the cofactor of an enzyme is a nonprotein organic molecule, the cofactor is known as a coenzyme. They are often derivatives of vitamins (for example, NAD, FAD, coenzyme A. Prosthetic group: A cofactor, which is tightly bound to the apoenzyme, is generally called a prosthetic group. They are either small organic molecules or inorganic metal ions. Examples include pyridoxal phosphate, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), thiamin pyrophosphate, and biotin. Metal ions constitute the most common type of prosthetic group. The roughly one-third of all enzymes that contain tightly bound Fe, Co, Cu, Mg, Mn, and Zn are termed metalloenzymes. Activator: In case the cofactor of an enzyme molecule is a metal ion such as, Mg2+, Ca2+, K+, Na+^ etc and associate reversibly, the cofactor is known as an activator. Enzymes that require a reversibly metal ion cofactor are termed metal-activated enzymes to distinguish them from the metalloenzymes for which bound metal ions serve as prosthetic groups. Active site: Enzyme molecules contain a special pocket or cleft called the active site where all the events of the catalytic process occur. The active site contains amino acid side chains that create a three- dimensional surface complementary to the substrate. The active site binds the substrate, forming an enzyme-substrate (ES) complex. ES is converted to enzyme-product (EP), which subsequently dissociates to enzyme and product The active site consists of a few amino acid side chains, some known as binding groups and the other as catalytic groups. The binding side chains can bind to different parts of the substrate by hydrogen bonds and electrostatic or hydrophobic interactions. Thus, binding amino acids can either be polar or nonpolar. However, the amino acid side chains of the catalytic groups are of polar type only, because they bring about changes in the electronic structure of the substrate, which is a pre-stage to chemical transformations.
2. They are Catalysts: In the presence of enzymes, certain chemical reactions proceed faster than in their absence. An enzyme changes only the rate of the reaction, not the direction or equilibirium. Like all catalysts, enzymes work by lowering only the activation energy for a reaction. This is illustrated in Fig. 1. Figure 1 : The Lowering of the activation energy (ΔG‡) and the standard free-energy (ΔGo') in the enzyme catalyzed and uncatalyzed reaction. Catalysts, like enzymes, act by lowering the energy difference between the substrates (reactants) and the transition state. This lowers the activation barrier for the reaction, allowing it to proceed more rapidly. Since lowering of the kinetic barrier also accelerates the reverse reaction, the equilibrium of the reaction remains unchanged. As with all catalysts, enzymes are not consumed by the
PROPERTIES AND CHARACTERISTICS OF ENZYMES BY C. B. LUKONG (PhD) @2019/
- Stereochemical specificity: stereospecific enzymes act only on a particular steric or optical isomer and not on their isomeric counterparts. Eg. D.amino acid oxidase oxidises D. amino acids. L. Amino acid oxidase oxidises L. amino acid and not D. amino acid. Enzyme-Substrate Interaction Models Two models have been proposed to explain how an enzyme binds its substrate: Lock and key and the induced-fit models (Figure ). Figure 1: Enzyme-substrate binding models. (a) Lock and Key Model (b) Induced Fit Model. The Lock and Key Model In 1894, Email Fischer had proposed the “lock and key” hypothesis to explain the specificity of enzyme-substrate interaction. Enzymes are very specific, and Email Fischer suggested that this was because the shape of the enzyme active site is complementary in conformation to the substrate, so that enzyme and substrate recognize and fit exactly into one another (Figure a). The two shapes are considered as rigid and fixed, and perfectly complement each other when brought together in the right alignment. This is comparable to a lock which opens up with only one particular key. The enzyme is analogous to a lock while the substrate is the key that fits that lock. Although this model explains enzyme specificity, it fails to explain the stabilization of the transition state that accompany substrate binding and catalysis. The ‘lock and key’ model has proven inaccurate and the induced fit model is the most currently accepted model for enzyme–substrate–coenzyme interaction. Induced Fit Model Since enzymes are rather flexible structures, Daniel E. Koshland, Jr in 1958, suggested a modification to the lock and key model; the induced fit model (Figure b) which states that the enzyme continually changes shape on binding substrate, so that the conformation of substrate and enzyme is only complementary after the substrate is completely bound. The enzyme in turn may induce reciprocal changes in its substrate, forcing it into a conformation similar to that of the transition state and joins the energy of binding to facilitate the transformation of substrate into product. The induced fit model is analogous to placing a hand (substrate) into a glove (enzyme), which then causes or more appropriately ‘induces’ a fit. However, the specificity is still retained as a left-handed glove
PROPERTIES AND CHARACTERISTICS OF ENZYMES BY C. B. LUKONG (PhD) @2019/ will not fit in the right hand and vice versa. The induced fit model has been fully confirmed by biophysical studies of enzyme motion during substrate binding. Factors Influencing Enzyme Activities Several factors affect the rate at which enzymatic reactions proceed, such as temperature, pH, enzyme concentration, substrate concentration and the presence of any inhibitors or activators. Temperature: An enzyme is active within a narrow range of temperature. The temperature at which an enzyme shows its highest activity is called optimum temperature. It generally corresponds to the body temperature of warm-blooded animals, e.g., 37 °C in human beings. Enzyme activity decreases above and below this temperature. An increase in temperature affects the rate of an enzyme-controlled reaction in two ways; a. As the temperature increases the kinetic energy of the substrate and enzyme molecules also increases and so they move fast. The faster these molecules move, the more they collide with one another and therefore the greater the rate of reaction. b. Secondly as temperature increases more atoms which make up the enzyme molecules vibrate. These vibrations break the hydrogen bonds and other forces which hold the molecules in their precise shape hence changing enzyme active sites. The three-dimensional shape of the enzyme molecules is therefore changed by these vibrations as the bonds, hydrogen bonds and hydrophobic interactions, which were holding it get broken to such an extent that the active site no longer allows the substrate to fit. Under these conditions the enzyme is said to be denatured by the increasing temperature and therefore loses its catalytic x-tics. Therefore, increasing the temperature beyond the optimum temperature rapidly denatures enzymes and very low temperatures inactivate enzymes. At the optimum temperature enzymes attain their maximum activity thereby providing the maximum rate of the reaction. Inactivated enzymes are not denatured and therefore they can regain their catalytic properties when higher temperatures are provided. Optimum pH: Every enzyme has an optimum pH when it is most effective. A rise or fall in pH reduces enzyme activity by changing the degree of ionization of its side chains. Most of the intracellular enzymes function near neutral pH with the exception of several digestive enzymes which work either in acidic range of pH or alkaline, e.g., 2.0 pH for pepsin, 8.5 for trypsin. The hydrogen bonds which make up the three-dimensional molecular shape of the enzyme may be broken by the concentration of hydrogen ions present. PH is the measure of the hydrogen ion concentration. By breaking the hydrogen bonds which give enzyme molecules their shape, any change in the pH can effectively denature enzymes. Each enzyme works best at a particular pH and deviations from this optimum pH may result into denaturing of these enzymes. Enzyme Concentration: The rate of a biochemical reaction rises with the increase in enzyme concentration up to a point called limiting or saturation point. Beyond this, increase in enzyme concentration has little effect. Provided that the substrate concentration is maintained at a high level and other conditions such as pH and temperature are maintained constant, the rate of a reaction increases with increase in enzyme concentration until when the rate remains constant. Usually, the enzyme concentration is much lower than the substrate concentration. Therefore, as the enzyme concentration increases, the rate of substrate is either being exhausted in the reaction or greatly reduced thereby limiting the reaction. Substrate concentration The rate of enzyme-controlled reaction increases with increase in the substrate concentration for a given quantity of an enzyme until such a concentration when all the active sites of an enzyme are saturated. At such concentration the rate of reaction becomes constant or levels. After leveling of the rate of the reaction, the rate can only be increased by increasing enzyme concentration which would provide new active sites for the substrate. The increase in substrate concentration increases the interaction between the enzyme molecules and the substrate molecules which increases the rate of collision between the enzyme and the substrate so as to form the products. Product Concentration: If the products are allowed to remain in the area of the reaction, the rate of forward reaction will fall. Reverse reaction can also start.
PROPERTIES AND CHARACTERISTICS OF ENZYMES BY C. B. LUKONG (PhD) @2019/ The substrate and the reaction they catalyzed e.g. Lactate dehydrogenase, Pyruvate decarboxylase etc. Other enzymes have trivial or historical names with no direct relationship to the source, function, substrate or reaction catalyzed by the enzyme e.g. trypsin, thrombin, pepsin etc. Recent studies on the mechanism of enzyme catalysed reactions have led to a more rational classification of enzymes. In 1961, the International Union of Biochemistry (IUB) established a commission on enzyme nomenclature to adopt a systematic classification and nomenclature of all the existing and yet to be discovered enzymes. This system is based on the substrate and reaction specificity. Although, this Enzyme Commission (EC) system is complex, it is precise, descriptive and informative. The EC system classifies enzymes into six major classes (should be written in specific order only) 1. Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases. Again, each class is divided into subclasses according to the type of reaction catalysed. Each enzyme is assigned a recommended name usually a short for everyday use, a systematic name which identify the reaction it catalyses and a classification number which is used where accurate and unambiguous identification of an enzyme is required. 1) Oxidoreductases: Oxidoreductases are enzymes which involved in biological oxidations and reductions. Oxidation means addition of oxygen or removal of hydrogen. Reduction means addition of hydrogen or removal of oxygen. The important sub classes are: a) Dehydrogenases : Dehydrogenases are enzymes that catalyze the removal of hydrogen from one substrate and pass it on to a second substrate. AH2 + B BH2+ A Eg. Alcohol dehydrogenase enzymes. b) Oxidase: Oxidase are enzymes which catalyze the removal of hydrogen from a substrate and pass it directly to oxygen. AH2 +O2 AH2O Eg. Cytochrome oxidase enzyme c) Oxygenases: These are enzymes which catalyze the incorporation of oxygen directly into substrate. 2) Transferases These enzymes transfer a group from one substrate to another substrate. The reaction cam be represented as follows. AX + B A + BX Transferases transfers amino group from one amino acid to another amino acid. 3) Hydrolases Hydrolases are enzymes which catalyze hydrolysis. ie. The direct addition of water molecule across a bond which is cleaved. Hydrolases are divied into three sub classes. They are proteases. esterases and carbohydrases. a) Proteases: These are enzymes that attack the peptide bonds on proteins and peptides. b) Esterases: These enzymes catalyze hydrolysis of ester linkage. Eg. Liver esterase, lipase, nuclease etc. 4) Lyases Lyases are the enzymes which catalyze either the removal of a group of atoms from their substrate leaving double bonds or add groups to double bonds without hydrolysis, oxidation or reduction. Eg. Aldose, enolase. 5) Isomerse These enzymes catalyze intramolecular rearrangements. That is they catalyze the interconversion of a compound to one of its isomers. 6) Ligases or Synthetases These enzymes catalyze synthesis reactions by joining two molecules coupled with the break down of a pyrophosphate bond of ATP ADP. Significance of Enzymes
- In the absence of an enzyme, biochemical reactions hardly proceed at all, whereas in its presence the rate can be increased up to 10^7 - fold. Thus, they are crucial for normal metabolism of living systems.
- Besides in the body, extracted and purified enzymes have many applications.
PROPERTIES AND CHARACTERISTICS OF ENZYMES BY C. B. LUKONG (PhD) @2019/
- Medical applications of enzymes include:
- To treat enzyme related disorders.
- To assist in metabolism
- To assist in drug delivery.
- To diagnose & detect diseases.
- In manufacture of medicines.
- Industrial applications of enzymes include:
- Amylase, lactases, cellulases are enzymes used to break complex sugars into simple sugars.
- Pectinase like enzymes which act on hard pectin is used in fruit juice manufacture.
- Lipase enzymes act on lipids to break them in fatty acids and glycerol. Lipases are used to remove stains of grease, oils, butter.
- Enzymes are used in detergents and washing soaps.
- Protease enzymes are used to remove stains of protein nature like blood, sweat etc.
