Medical Biochemistry Enzyme Kinetics & Regulation PDF

Medical Biochemistry ENZYME KINETICS & REGULATION  Enzymes are protein catalysts that increase the rate of reactions without being changed in the overall process Virtually all reactions in the body are mediated by enzymes Kinetics is the study of the factors which influence reacti...

Medical Biochemistry ENZYME KINETICS & REGULATION  Enzymes are protein catalysts that increase the rate of reactions without being changed in the overall process Virtually all reactions in the body are mediated by enzymes Kinetics is the study of the factors which influence reaction rates Ef + S ↔ ES→ E + P Where, E- enzyme S- substrate [ES] - enzyme-substrate complex P - product  ATP „universal energy currency“ r p  p r Not spontaneous ENERGY CHANGES OCCURRING DURING THE REACTION Virtually all chemical reactions have an energy barrier separating the reactants and the products. This barrier, called the free energy of activation, is the energy difference between that of the reactants and a high-energy intermediate that occurs during the formation of product. The enzyme does not change the free energies of the reactants or products and, therefore, however, accelerate the rate by which equilibrium is reached. A number of factors are responsible for the catalytic efficiency of enzymes, including the following: 1. Transition-state stabilization: By stabilizing the transition state, the enzyme greatly increases the concentration of the reactive intermediate that can be converted to product and, thus, accelerates the reaction. 2. Other mechanisms: The active site can provide catalytic groups that enhance the probability that the transition state is formed. (Acid-base; covalent catalysis.) 3. Visualization of the transition state: The enzyme- catalyzed conversion of substrate to product can be visualized as being similar to removing a sweater from an uncooperative infant. A parent acting as an enzyme, first coming in contact with the baby (forming ES), then guiding the baby’s arms into an extended, vertical position, analogous to the ES transition state.  RATE OF REACTION For molecules to react, they must contain sufficient energy to overcome the energy barrier of the transition state. In the absence of an enzyme, only a small proportion of a population of molecules may possess enough energy to achieve the transition state between reactant and product The lower the free energy of activation, the more molecules have sufficient energy to pass through the transition state, and, therefore, the faster the rate of the reaction. Rate of reaction at fixed enzyme [E] but varying substrate [S]  SUBSTRATE CONCENTRATION AFFECTS THE REACTION RATE For a typical enzyme, as substrate concentration is increased, velocity (vi) increases until it reaches a maximum value, Vmax. When substrate concentration further increases the velocity (vi) fails to increase. Enzyme is said to be “saturated” with the substrate.  SUBSTRATE CONCENTRATION AFFECTS THE REACTION RATE At point C all the enzyme is present as the ES complex. Since no free enzyme remains available for forming ES, further increase in [S] cannot increase the rate of the reaction. Under these saturating conditions, vi depends solely on—and thus is limited by—the rapidity with which product dissociates from the enzyme so that it may combine with more substrate. The concentration of substrate ([S]) is much greater than the concentration of enzyme ([E]), so that the percentage of total substrate bound by the enzyme at any one time is small.  STEADY-STATE ASSUMPTION: [ES] does not change with time (the steady-state assumption), that is, the rate of formation of ES is equal to that of the breakdown of ES (to E + S and to E + P). In general, an intermediate in a series of reactions is said to be in steady state when its rate of ES synthesis is equal to its rate of ES degradation k k E + S ↔ ES→ E + P 1 3 f The relative rates of formation and dissociation of [ES] is denoted as Km, the Michaelis constant. Each enzyme/substrate combination has a Km value under defined conditions. Numerically, the Km is the substrate concentration required to achieve 50% of the maximum velocity of the enzyme; the unit for Km is therefore the same as the unit for substrate concentration, typically mmol/L. The maximum velocity the enzyme-catalysed reaction can achieve is expressed by the Vmax typical unit mmol/min.  MICHAELIS-MENTEN EQUATION Vmax [S] Vo = Km+ [S] The Michaelis constant Km is the substrate concentration at which velocity (vi) is half the maximal velocity (Vmax/2) attainable at a particular concentration of the enzyme. The Michaelis constant (Km) is a measure of the substrate concentration at which an enzyme achieves half of its maximum reaction velocity (Vmax) when operating at saturating substrate concentrations.  Significance of Km Affinity of enzyme-substrate interaction: The Km value reflects the affinity of an enzyme for its substrate. A low Km value indicates high affinity, meaning the enzyme can achieve half-maximal velocity at relatively low substrate concentrations. Conversely, a high Km value indicates low affinity, requiring higher substrate concentrations to reach half-maximal velocity. Substrate specificity: Km values can provide insights into the substrate specificity of enzymes. Enzymes with low Km values tend to be highly specific for their substrates, whereas enzymes with higher Km values may exhibit broader substrate specificity.  Km MEANING FOR ISOEMZYMES Hexokinase Km 0,1 mM, (Brain) Glucokinase Km 5 mM , (Liver) Glucose + ATP→ Glucose 6-phosphate + ADP Differing Km values of hexokinase and glucokinase reflect their distinct roles in glucose metabolism and homeostasis. Glucokinase acts as a glucose sensor, primarily in the liver and pancreatic β-cells, while Hexokinase functions to maintain basal glucose metabolism in various tissues (e.g. brain). These differences allow for precise regulation of glucose utilization and storage in different tissues FACTORS AFFECTING ENZYME FUCNTION TEMPERATURE The reaction velocity increases with temperature until a peak velocity is reached Further elevation results in a decrease in reaction velocity as a result of temperature- induced denaturation of the enzyme  FACTORS AFFECTING ENZYME FUCNTION pH the catalytic process usually requires that the enzyme and substrate have specific chemical groups in either an ionized or un-ionized state in order to interact Extremes of pH can also lead to denaturation of the enzyme, because the structure of the catalytically active protein molecule depends on the ionic character of the amino acid side chains.  THE pH OPTIMUM VARIES FOR DIFFERENT ENZYMES The pH at which maximal enzyme activity is achieved is different for different enzymes, and reflects the [H+] at which the enzyme functions in the body. For example, pepsin, a digestive enzyme in the stomach, is maximally active at pH 2, whereas other enzymes, designed to work at neutral pH, are denatured by such an acidic environment  Enzyme Inhibition Types 1. Irreversible inhibition: bind to enzymes through covalent bonds. Combine with the functional groups of the amino acids in the active site, irreversibly and destroy enzymes. Examples: nerve gases and pesticides, containing organophosphorus, combine with serine residues in the enzyme acetylcholine esterase. 2. Reversible inhibition: typically bind to enzymes through noncovalent bonds. Can be washed out of the solution of enzyme by dialysis and enzymes can be recycled.  The effect of enzyme inhibition Competitive: They compete with the substrate molecules for the active site The inhibitor’s action is proportional to its concentration. Competitive inhibitor Resembles the substrate’s structure closely. The effect of a competitive inhibitor is reversed by increasing SUBSTRATE E+I EI Reversible Enzyme inhibitor reaction complex COMPETITIVE INHIBITION  Competitive Inhibition Succinate Fumarate + 2H++ 2e- Succinate CH2COOH dehydrogenase CHCOOH COOH CH2COOH CH2 CHCOOH COOH Malonate Malonate, an Inhibitor resembles the substrate  The effect of enzyme inhibition Non-competitive: Not influenced by the concentration of the substrate. Inhibits by binding irreversibly to the enzyme but not at the active site Examples Cyanide combines with the iron in the enzymes cytochrome oxidase Heavy metals, Ag or Hg, combine with –SH groups. These can be removed by using a chelating agent such as EDTA. NONCOMPETITIVE INHIBITION  REGULATION Enzyme activity can be regulated, that is, increased or decreased, so that the rate of product formation responds to cellular need. Covalent Control on Enzyme Activity by Phosphorylation/Dephosphorylation Allosteric regulation  INDUCTION AND REPRESSION OF ENZYME SYNTHESIS The regulatory mechanisms described above modify the activity of existing enzyme molecules. However, cells can also regulate the amount of enzyme present by altering the rate of enzyme degradation or, more typically, the rate of enzyme synthesis. The increase (induction) or decrease (repression) of enzyme synthesis leads to an alteration in the total population of active sites.  INDUCTION AND REPRESSION OF ENZYME SYNTHESIS Enzymes subject to regulation of synthesis are often those that are needed at only one stage of development or under selected physiologic conditions. For example, elevated levels of insulin as a result of high blood glucose levels cause an increase in the synthesis of key enzymes involved in glucose metabolism In contrast, enzymes that are in constant use are usually not regulated by altering the rate of enzyme synthesis. Alterations in enzyme levels as a result of induction or repression of protein synthesis are slow (hours to days), compared with allosterically or covalently regulated changes in enzyme activity, which occur in seconds to minutes.