<h5 id="41-enzymes-classification-and-mode-of-action">4.1 Enzymes: Classification and Mode of Action</h5> <ul> <li> <p>The text discusses the role of certain metal ions and coenzymes in enzyme catalysis.</p> </li> <li> <p>A prosthetic group is a coenzyme or metal ion that is tightly bound to an enzyme protein through a covalent bond.</p> </li> <li> <p>Examples of metal ions that can act as prosthetic groups include $\mathrm{Fe}^{2+}$, $\mathrm{Fe}^{3+}$, $\mathrm{Cu}^{2+}$, $\mathrm{Mg}^{2+}$, $\mathrm{Mn}^{2+}$, $\mathrm{K}^{+}$, $\mathrm{Mo}^{2+}$, and $\mathrm{Zn}^{2+}$.</p> </li> <li> <p>Enzymes that use these prosthetic groups include catalase, peroxidase, cytochrome oxidase, DNA polymerase, arginase, pyruvate kinase, nitrogenase, nitrate reductase, carbonic anhydrase, alcohol dehydrogenase, and urease.</p> </li> <li> <p>These enzymes and prosthetic groups play important roles in various biological processes, such as redox reactions, DNA replication, and nitrogen fixation.</p> </li> </ul> <p>======</p> <h5 id="411-classification-of-enzymes">4.1.1 Classification of enzymes</h5> <ul> <li>Oxidoreductases are enzymes that catalyze oxidation-reduction reactions, which involve the transfer of electrons.</li> <li>Transferases are enzymes that catalyze the transfer of groups from one molecule to another.</li> <li>Hydrolases are enzymes that catalyze hydrolytic reactions, which involve the transfer of functional groups to water.</li> <li>Lyases are enzymes that catalyze the addition or removal of groups to form double bonds.</li> <li>Isomerases are enzymes that catalyze the transfer of groups within molecules to yield isomeric forms.</li> <li>Ligases are enzymes that catalyze the condensation of two molecules coupled through ATP hydrolysis.</li> </ul> <p>======</p> <h5 id="isozymes">Isozymes</h5> <ul> <li>Isoenzymes, also known as isozymes, are enzymes that exist in multiple forms within the same species, tissue, or cell.</li> <li>They catalyze the same reaction but have different amino acid compositions, resulting in different physicochemical properties.</li> <li>An example of a glycolytic enzyme, hexokinase, exists in four isozyme forms in various tissues.</li> <li>Lactate dehydrogenase (LDH), involved in anaerobic glucose metabolism, has two isozyme forms in humans - one is found in the heart and the other in skeletal muscles.</li> <li>The existence of isozymes allows for functional specialization and adaptation to different physiological needs in different tissues.</li> </ul> <p>======</p> <h5 id="enzyme-active-site">Enzyme active site</h5> <ul> <li>The enzyme catalytic reaction occurs at a specific site called the active site.</li> <li>The active site is a three-dimensional structure, made up of portions of a polypeptide chain, and is a small part of the total enzyme size.</li> <li>It forms a pocket or cleft in the enzyme molecule where the substrate can fit, and involves various non-covalent bonds such as electrostatic interactions, hydrogen bonds, Van Der Waals forces, and hydrophobic interactions.</li> <li>The active site often has a nonpolar environment, which facilitates the binding of the substrate and catalysis, although some polar residues may be present.</li> <li>This type of environment is not found in any other region of the enzyme molecule.</li> </ul> <p>======</p> <h5 id="fischers-lock-and-key-model">Fischer’s Lock and Key Model</h5> <ul> <li>In 1894, Emil Fischer introduced the Lock and Key Model for substrate and enzyme interaction.</li> <li>The model suggests that complementary structural features are present between enzyme and substrate.</li> <li>The active site is pre-shaped to fit the substrate, allowing the substrate to fit into its complementary site on the enzyme.</li> <li>This interaction results in the formation of an enzyme-substrate complex.</li> <li>The Lock and Key Model is represented by the image of a key fitting into a lock.</li> </ul> <p>======</p> <h5 id="koshlands-induced-fit-model">Koshland’s Induced Fit Model</h5> <ul> <li>Daniel Koshland proposed the Induced Fit Hypothesis in 1958.</li> <li>The hypothesis suggests that the structure of a substrate is complementary to that of the active site in the enzyme-substrate complex, not in the free enzyme.</li> <li>The interaction between the substrate and the enzyme induces conformational changes in the enzyme, aligning the amino acid residues or other groups for substrate binding and catalysis.</li> <li>The relationship between a substrate and an active site is likened to a hand and a woollen glove.</li> <li>During interaction, the structure of one component, i.e., substrate or hand, remains rigid, while the shape of the second component, i.e., active site or glove, becomes flexible to become complementary to that of the first.</li> </ul> <p>======</p> <h5 id="enzyme-specificity">Enzyme specificity</h5> <ul> <li>Enzymes are highly specific in their action.</li> <li>There are four types of enzyme specificity: group specificity, absolute specificity, stereospecificity, and geometrical specificity.</li> <li>Group specificity is when enzymes act on several different closely related substrates.</li> <li>Absolute specificity is when enzymes act only on one particular substrate.</li> <li>Stereospecificity, also known as stereochemical or optical specificity, occurs when substrate exists in two stereochemical forms and only one of the isomers will undergo reaction by a particular enzyme.</li> <li>Geometrical specificity is when enzymes are specific towards cis and trans forms, such as fumarase catalyzing the interconversion of fumarate and malate.</li> </ul> <p>======</p> <h5 id="412-factors-affecting-enzyme-activity">4.1.2 Factors affecting enzyme activity</h5> <ul> <li>The rate of enzyme catalyzed reactions can be affected by factors such as temperature, pH, substrate concentration, and modulators.</li> <li>Temperature: The rate of reaction increases with temperature until it reaches a maximum and then falls, creating a bell-shaped curve. The temperature at which the maximum rate occurs is the enzyme’s optimum temperature, which is typically between 40-45°C for most enzymes.</li> <li>pH: Enzyme activity is affected by pH, with a bell-shaped curve obtained when plotting enzyme activity against pH. Each enzyme has a unique optimum pH at which the rate of reaction is greatest.</li> <li>Substrate concentration: The rate of reaction increases as substrate concentration increases, up to a certain point where further increases have no effect on the rate of reaction. This is because enzyme molecules become saturated and work at their maximum possible rate.</li> <li>The enzyme’s optimum temperature and pH vary depending on the specific enzyme, with many enzymes in higher organisms showing optimum reaction rates around neutral pH (6-8). However, there are exceptions such as pepsin (pH 1-2), acid phosphatases (pH 4-5), and alkaline phosphatases (pH 10-11). Enzymes can become inactive at extreme temperatures and pH levels.</li> </ul> <p>======</p> <h5 id="413-unit-of-enzyme-activity">4.1.3 Unit of enzyme activity</h5> <ul> <li>The enzyme unit (U) is a unit of enzyme activity defined as the amount of enzyme that catalyzes the conversion of 1 micromole of substrate per minute under standard conditions.</li> <li>The International Union of Biochemistry (I.U.B.) adopted the enzyme unit as the unit of enzyme activity in 1964.</li> <li>However, the use of the enzyme unit is discouraged in favor of the katal, as the minute is not an SI unit.</li> <li>One katal (kat) is the amount of enzyme that catalyzes 1 mole of substrate per second.</li> <li>Therefore, 1 kat is equal to 60,000,000 U.</li> </ul> <p>======</p> <h5 id="414-specific-activity">4.1.4 Specific activity</h5> <ul> <li>Specific activity is a unit of enzyme measurement.</li> <li>It is defined as the moles of product formed by an enzyme in a given amount of time under given conditions per milligram of proteins.</li> <li>Specific activity represents a measurement of enzyme purity in the mixture.</li> <li>Higher specific activity indicates higher enzyme purity.</li> <li>It is inversely proportional to the amount of non-enzymatic protein present in the preparation.</li> </ul> <p>======</p> <h5 id="415-mechanism-of-enzyme-action">4.1.5 Mechanism of enzyme action</h5> <ul> <li>Enzymes affect the rate of reaction by altering the energy of activation, not the overall change in free energy of the reaction.</li> <li>The Michaelis-Menten equation, v0 = Vmax[S]/(Km + [S]), describes the kinetics of a one-substrate reaction, where v0 is the initial velocity, Vmax is the maximum velocity, [S] is the substrate concentration, and Km is the Michaelis constant.</li> <li>At low substrate concentration ([S] « Km), the reaction rate is directly proportional to the substrate concentration. At high substrate concentration ([S] » Km), the reaction rate is maximum and independent of substrate concentration.</li> <li>Km represents the substrate concentration at which half of the maximum reaction rate is obtained, and Vmax represents the turnover number of an enzyme, which is the number of substrate molecules converted into product by an enzyme molecule in a unit time when the enzyme is fully saturated with substrate.</li> <li>The Michaelis-Menten theory of enzyme kinetics is the simplest one that accounts for kinetic properties of many enzymes, explaining the formation of an enzyme-substrate complex (ES) as an intermediate during catalysis.</li> </ul> <p>======</p> <h5 id="416-enzyme-inhibition">4.1.6 Enzyme inhibition</h5> <ul> <li>Enzyme inhibitors are substances that decrease the rate of enzyme-catalyzed reactions.</li> <li>Enzyme inhibition can be classified into two types: reversible and irreversible inhibition.</li> <li>In irreversible inhibition, the inhibitor binds permanently to the enzyme, preventing further reactions. An example is the antibiotic penicillin, which binds to the enzyme transpeptidase and inhibits bacterial cell wall synthesis.</li> <li>In reversible inhibition, the inhibitor can rapidly dissociate from the enzyme-inhibitor complex. This type has three subtypes: competitive, non-competitive, and uncompetitive inhibition.</li> <li>Competitive inhibition occurs when the inhibitor binds to the active site of the enzyme, preventing the substrate from binding. Non-competitive inhibition occurs when the inhibitor binds to a different site on the enzyme, while uncompetitive inhibition occurs when the inhibitor binds only to the enzyme-substrate complex.</li> </ul> <p>======</p> <h5 id="i-competitive-inhibition">(i) Competitive inhibition</h5> <ul> <li>In competitive inhibition, the inhibitor (I) and substrate (S) have similar structures and compete for the same active site on an enzyme.</li> <li>The enzyme can form either an enzyme-substrate (ES) complex or an enzyme-inhibitor (EI) complex, but not both.</li> <li>Competitive inhibitors decrease the rate of reaction by reducing the number of active enzyme molecules bound to a substrate.</li> <li>At high substrate concentration, the probability of inhibitor molecule binding to the enzyme decreases, and $V_{max}$ remains unchanged.</li> <li>The $K_{m}$ (Michaelis-Menten constant), which is the substrate concentration at which $v_{0} = 1/2 V_{max}$, increases in the presence of a competitive inhibitor and is denoted as $K_{m}’$.</li> </ul> <p>======</p> <h5 id="ii-non-competitive-inhibition">(ii) Non-Competitive inhibition</h5> <ul> <li>Non-competitive inhibition occurs when an inhibitor (I) binds to an enzyme (E) at a site different from the substrate (S) binding site, resulting in the formation of EI and ESI complexes.</li> <li>The binding of the non-competitive inhibitor does not prevent the substrate from binding to the enzyme, but the ESI complex does not form a product.</li> <li>Non-competitive inhibition reduces the Vmax (maximum reaction velocity) as the inhibitor lowers the concentration of active enzyme, but it does not affect the Km (Michaelis-Menten constant) value.</li> <li>In contrast to competitive inhibition, non-competitive inhibition cannot be overcome by increasing substrate concentration.</li> <li>Fig. 4.9 illustrates non-competitive inhibition, and Fig. 4.10 shows the Michaelis-Menten plot for non-competitive inhibition.</li> </ul> <p>======</p> <h5 id="iii-uncompetitive-inhibition">(iii) Uncompetitive inhibition</h5> <ul> <li>Uncompetitive inhibition occurs when the inhibitor binds only to the enzyme-substrate (ES) complex, not to the free enzyme.</li> <li>The inhibitor does not compete with the substrate for the same binding site.</li> <li>This type of inhibition cannot be overcome by increasing substrate concentration.</li> <li>Both $\mathrm{K} _{\mathrm{m}}$ and $\mathrm{V} _{\text {max }}$ values are altered in uncompetitive inhibition.</li> <li>The binding of the inhibitor to the ES complex is either direct or facilitated by the conformational change after the substrate binds to the enzyme.</li> </ul> <p>======</p> <h5 id="417-allosteric-enzymes">4.1.7 Allosteric enzymes</h5> <ul> <li>Allosteric enzymes do not follow Michaelis-Menten kinetics, resulting in sigmoidal graphs when v0 is plotted against substrate concentration [S].</li> <li>These enzymes are made up of multiple protein subunits, each with an active site and a regulatory site.</li> <li>Regulatory molecules can bind to the regulatory site, altering the enzyme’s affinity for the substrate.</li> <li>Most enzymes that obey Michaelis-Menten kinetics are normal enzymes, while allosteric enzymes are key regulators of metabolic pathways in cells.</li> <li>The kinetics of allosteric enzymes are different from those of normal enzymes, as shown in Fig. 4.12.</li> </ul> <p>======</p> <h5 id="42-brief-introduction-to-bioenergetics">4.2 Brief Introduction to Bioenergetics</h5> <ul> <li>Bioenergetics focuses on the transformation and use of energy by living cells.</li> <li>Energy is released in biological reactions as they move from a higher to a lower energy level, often in the form of heat.</li> <li>The conversion of metabolites (e.g., A to B) is accompanied by the release of energy.</li> <li>This release of energy is coupled with another reaction that requires energy for a metabolic conversion (e.g., X to Y).</li> <li>The coupling of these reactions ensures energy is released and utilized effectively within the cell.</li> </ul> <p>======</p> <h5 id="421-the-laws-of-thermodynamics">4.2.1 The laws of thermodynamics</h5> <ul> <li>Energy is the capacity to do work, existing in various forms such as electrical, mechanical, chemical, heat, and light, which are interconvertible.</li> <li>Bioenergetics focuses on energy changes in biochemical processes, excluding mechanisms or speeds.</li> <li>Thermodynamics, a branch of physical chemistry, studies energy changes during interconversions.</li> <li>The two fundamental laws of thermodynamics: <ul> <li>Law 1: Explains the direction of a reaction (forward or reverse)</li> <li>Law 2: Explains the accomplishment of work (useful or non-useful)</li> <li>Law 3: Determines if external energy is required for a reaction</li> </ul> </li> </ul> <p>======</p> <h5 id="the-first-law-of-thermodynamics">The first law of thermodynamics</h5> <ul> <li>The first law of thermodynamics involves energy exchange between a system and its surroundings.</li> <li>A system is a defined region of matter, while surroundings constitute the rest of the universe.</li> <li>The law states that energy can’t be created or destroyed, only converted into other forms.</li> <li>The equation for the first law is E=E_B - E_A = Q - W, where: <ul> <li>E = change in internal energy</li> <li>E_A = initial energy of the system</li> <li>E_B = final energy of the system</li> <li>Q = heat absorbed by the system</li> <li>W = work done by the system</li> </ul> </li> <li>The change in a system’s energy depends on the initial and final stages, not the transformation path.</li> </ul> <p>======</p> <h5 id="the-second-law-of-thermodynamics">The second law of thermodynamics</h5> <ul> <li>The second law of thermodynamics introduces the term ’entropy’ (S), representing the degree of randomness or disorder in a system.</li> <li>Entropy increases when a system becomes more disordered, reaching its maximum as the system nears equilibrium.</li> <li>Biological systems maintain a highly ordered form of life by consuming chemical or light energy, converting it to a less organized form (heat) or utilizing it to perform work.</li> <li>The second law states that the entropy or disorder of the universe always increases, and a process can occur spontaneously only if the sum of the entropies of the system and its surroundings increases.</li> <li>The second law is concerned with the availability of energy for doing work, while the first law focuses on the transformation of various kinds of energy in a given process.</li> </ul> <p>======</p> <h5 id="combining-the-two-laws">Combining the two laws</h5> <ul> <li>Entropy is not a reliable criteria for the spontaneity of biochemical reactions as changes in entropy cannot be measured.</li> <li>The concept of ‘free energy’ was introduced by Gibbs in 1878 to overcome this problem, by combining the first and second laws of thermodynamics.</li> <li>The free energy equation, G=H-TS, shows the relationship between the change in free energy (G), heat (H), and entropy (S) in chemical reactions at constant temperature (T) and pressure (P).</li> <li>In a closed system at constant temperature and pressure, if a process involves a transfer of heat and a change in volume, the first law of thermodynamics can be expressed as E=H-PV.</li> <li>Here, E represents the change in internal energy, H the change in enthalpy, and PV the work done on the surroundings by increasing the volume of the system at constant pressure (P) and temperature (T).</li> </ul> <p>======</p> <h5 id="atp-the-universal-currency-of-free-energy">ATP: the universal currency of free energy</h5> <ul> <li>Free energy for cellular processes is derived from sunlight (photosynthetic organisms) or oxidation of food stuff (chemotrophs).</li> <li>This free energy is partly converted into ATP, a special form that serves as the universal currency of free energy.</li> <li>ATP plays a central role in transferring free energy from exergonic processes to endergonic processes in cells.</li> <li>It is synthesized from ADP and inorganic phosphate, and donates energy to energy-requiring processes by converting itself into ADP and Pi.</li> <li>ATP can be converted into AMP and pyrophosphate while donating its energy in various processes, such as the production of light flashes by fireflies.</li> </ul> <p>======</p>