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Learn about the role of prosthetic groups, cofactors, and coenzymes in enzymatic reactions. Understand how these small molecules and metal ions contribute to substrate binding and catalysis. Explore the theories of enzyme action, enzyme kinetics, and the factors that affect enzymatic activity.
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Departement of Biochemistry Faculty of Medicine USU ENZYMOLOGY BIOCHEMISTRY MEDICAL FACULTY USU
Prosthetic Groups, Cofactors, & Coenzymes • Many enzymes contain small nonprotein molecules and metal ions that participate directly in substrate binding or catalysis. • Prosthetic groups are tightly bound or attached via a covalent or non covalent bond to an enzyme that are required for many enzyme-catalyzed reactions pyridoxal phosphate,flavin mononucleotide (FMN), flavin dinucleotide (FAD), thiamin pyrophosphate, biotin, and the metal ions of Co, Cu, Mg, Mn, Se, & Zn
Prosthetic Groups, Cofactors, & Coenzymes The heme group in hemoglobin is a prosthetic group • Cofactors serve functions similar to those of prosthetic groups but bind in a transient, dissociable manner either to the enzyme or to a substrate such as ATP • Coenzymes are organic substances that directly participate as substrates in an enzyme reaction
Prosthetic Groups, Cofactors, & Coenzymes • Derivatives of B vitamins (and AMP) precursors of cofactors, coenzymes & prosthetic groups for enzymatic reactions • Others are involved in the visual process and regulation of transcription (vitamin A), redox reactions (vitamins C and E), bone formation (vitamin D), blood coagulation (vitamin K) • Ascorbic acid has specific roles in the copper-containing hydroxylases and the α-ketoglutarate-linked iron-containing hydroxylases
Prosthetic Groups, Cofactors, & Coenzymes. • The enzymes, require Prosthetic Groups, Cofactors, Coenzymes: Oxidoreductases Transferases Isomerases Ligases
Hypothesis of Enzyme Action Collision Theory • In reaction A + B C + D, reactants A and B are surrounded by the hydration shell • Before conversion into the products C + D,thecollision complex A-B has to pass through a transition state • The formation of which usually requires a large amount of activation energy • Since only a few A–B complexes can produce this amount of energy, a productive transition state arises even less often than a collision complex.
Hypothesis of Enzyme Action Collision Theory • In solution, a large proportion of the activation energy is required for the removal of the hydration shells between A and B. • As a result of these limitations, conversion only happens occasionally in the absenceof a catalyst, and the reaction rate very is low
Hypothesis of Enzyme Action Lock and Key Theory • Emil Fischer compared the highly specific fit between enzymes and their substrates to that of a lock and its key. • The tertiary and quaternary structures of an enzyme have the substrate binding sites (active site), which have exactly the complementary shape of the substrate molecules. • This helps in the binding of the appropriate substrate to the active centers just like a key fits in the keyhole
Hypothesis of Enzyme Action Induced Fit Theory • Some enzymes change the shape of the active center slightly to accommodate the substrate molecules, a process known as induced fit. • The characteristic functional groups of the amino acid residues present in the active sites of the enzyme molecule can interact with the various bonds of the substrate molecule. • The induced fit model has been amply confirmed by biophysical studies of enzyme motion during substrate binding
Enzyme Kinetics • Leonor Michaelis and Maud Leonora Mentenproposed a quantitative theory of enzyme kinetics • In the theory, enzyme reactions in two stages. • The first, the substrate binds reversibly to the enzyme, forming the enzyme-substrate complex. →the Michaelis complex. • The enzyme then catalyzes the chemical step in the reaction and releases the product
Enzyme Kinetics • The reaction of an enzyme (E) with a substrate (S) to form a product (P) can be described as: • ES is the enzyme-substrate complex (Michaelis complex) • The constant k is the rate constant of the uncatalyzed reaction
Enzyme Kinetics • The velocity of an enzyme-mediated reaction or the enzyme activity is closely controlled by a number of factors: • Substrate Concentration • Enzyme Concentration • Temperature • pH • Ion/inhibitor
Enzyme Kinetics • Change in any particularly factors such as temperature, pH, and ionic strength can destroy the enzyme conformation or the three-dimensional structure. • This change ofconformation in the three-dimensional structure of an enzyme followed by the loss of activity is called denaturation
Effect of Substrate Concentration • Enzyme rates depend on solution conditions and substrate concentration • Raising substrate concentration tends to increase activity. • 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 the enzyme active sites are bound to substrate, and the amount of ES complex is the same as the total amount of enzyme.
Effect of Substrate Concentration • The amount of substrate needed to achieve a given rate of reaction is also important. • 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
Effect of Substrate Concentration • If the Km value of an enzyme is low it indicates its high affinity toward the substrate. • The relationship between the V, Km, Vmax, and substrate concentration [S] can be represented by an equation, the Michaelis–Menten equation
Effect of Enzyme Concentration • Increase enzyme concentration there is a corresponding increase in the rate of reaction • It’s only up to a particular point. • After this point there is no further increase in the rate of reaction corresponding to the increase of enzyme concentration.
Effect of Temperature • Raising the temperature increases the kinetic energy of molecules. • With increasing temperature, the increased thermal movement of the molecules initially leads to a rate acceleration • Mammalian enzymes, for example, always show temperature optima around 37°C • Most enzymes are denatured at 50°C
Effects of pH • The effect of enzymes is strongly dependent on the pH value • With animal enzymes,the pH optimum (the pH value at which enzyme activity is at its maximum) is often close to the pH value of the cells (pH 7). • However, there are also exceptions to this, ex: the proteinase pepsin (active in the acidic gastric lumen)has a pH optimum of 2
Inhibitor • Enzyme reaction rates can be decreased by various types of enzyme inhibitors. • Inhibitors,can inactivate the enzyme permanently or temporarily. • There are many types of inhibitors. • A large proportion of medicines act as enzyme inhibitors. • Natural metabolites are also involved in regulatory processes as inhibitors • Enzymes are often inactivated by some molecule (an inhibitor) that changes their shape or blocks the active site.
Types of Inhibitor Competitive Inhibitors • These are molecules that bind reversibly or irreversibly to the active site. • They compete with the substrate for space in the active site. • For example, methotrexate is a competitive inhibitor of the enzyme dihydrofolate reductase, which catalyzes the reduction of dihydrofolate to tetrahydrofolate
Types of Inhibitor Competitive Inhibitors • Most naturally occurring competitive inhibitors are irreversible.
Competitive Inhibitors Reversible Competitor. • These competitors are not the substrate molecules but molecules similar to the substrate molecules. • They compete with the substrate to occupy the active center of the enzyme molecule. • The competition is reversible and the competitor can be overwhelmed by high concentration of substrate. • For example, fumaric acid is the competitive inhibitor of the enzyme, succinicdehydrogenase. • The structure of fumaric acid is similar to that of succinicacid,the actual substrate of the enzyme.
Competitive Inhibition C-OO- C-H C-H C-OO- C-OO- H-C-H H-C-H C-OO- C-OO- H-C-H H-C-H H-C-H C-OO- C-OO- H-C-H C-OO- C-OO- C-OO- Product Competitive Inhibitor Substrate Fumaric Succinic Glutarate Malonate Oxalate Succinic Dehydrogenase
Competitive Inhibitors Irreversible Competitor. • The competitor and substrate both compete for the active site but the competitor occupies the active site permanently → deactivating the enzyme. • Non-classical competitive inhibition is the binding of substrate at the active site and prevents the binding of inhibitor at a different site • Carbon monoxide is an irreversible competitive inhibitor of hemoglobin. • Oxygen is the substrate.
Types of Inhibitor Inhibitors Non-competitive • In non-competitive inhibition the inhibitor and substrate do not compete for space in the active site. • The substrate enters the active site but the inhibitor reacts with some other part of the enzyme molecule. • It may be reversible or non-reversible. • Reversible non-competitive inhibition is a major metabolic control mechanism
The Lineweaver-Burk plot • The Lineweaver-Burk plot is a linear transformation of the Michaelis-Menten equation, and is useful for determining Km and Vmax graphically. • If 1/v0 is plotted VS. 1/[S], then the intersections of this line with abscissa and ordinate allow the determination of Km and Vmax.
Enzymatic Analysis • Inbody fluids—even tiny quantities of an enzyme can be detected by measuring its catalytic activity. • Enzymes are also used as reagents to determine the concentrations of metabolites—e. g., the blood glucose level • Most enzymatic analysis procedures use the method of spectrophotometry
