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Consortium for Educational Communication Frequently Asked Questions: 1. What is the importance of ES complex in enzyme catalyzed reactions? Answer: The formation of an enzyme-substrate complex is indispensable for enzymatic catalysis. It is only when substrate binds to the active site of enzyme to form ES complex that the reaction will proceed. The substrate in ES complex is enclosed and sequestered completely from solution, getting a specific environment and surface for the reaction to occur more readily. The weak non-covalent (binding energy) and transient covalent interactions between the enzyme and substrate are possible only in enzyme-substrate (ES) complexes that promote the formation and stabilization of the transition states in enzyme-substrate (ES) complexes and result in lowering of the energy of activation through multiple mechanisms. 2. What do you mean by activation energy? How does activation energy of catalyzed reactions differ from that of uncatalyzed reactions? Answer: Activation energies are energy barriers to chemical reactions. The difference between the energy levels of the ground states (substrates or products) and the transition state is called the Gibbs free energy of activation or simply the activation energy, ∆G‡. For uncatalyzed forward reaction, activation energy is the difference in free energy between the substrate and the transition state: ∆G‡uncat = Gs‡ - Gs. However, the activation energy in the enzyme-catalyzed reaction is the energy difference between ES complex and ES‡ transition state complex: ∆G‡cat = GES‡ - GES. The energy barrier between ES and ES‡ is less than the energy barrier between S and S‡, thereby meaning that the activation energy for the catalyzed reaction is lower than the activation energy for the uncatalyzed reaction (∆G‡cat < ∆G‡Uncat). Consortium for Educational Communication 3. What is the relationship between activation energy and the reaction rate? Answer: The relationship between the activation energy and the reaction rate is given by the equation: where V is reaction rate; k, rate constant; ∆G‡, activation energy; [S], substrate concentration; T, absolute temperature; k, the Boltzmann constant; h, Planck’s constant and R, gas constant. The relationship between the activation energy and the reaction rate is inverse and exponential, thereby meaning that the rate at which a molecule undergoes a particular reaction decreases as the activation energy for that reaction increases. In simplified terms, the lower activation energy means a faster reaction rate. 4. What is the source of the energy for the dramatic lowering of the activation energies for enzyme catalyzed reactions? Answer: The sources of the energy for the dramatic lowering of the activation energies for enzyme catalyzed reactions are: A)Noncovalent interactions between enzyme and substrate: Formation of a large number of weak, noncovalent bonds and interactions (hydrogen bonds and hydrophobic and ionic interactions) between substrate and enzyme in an ES complex is accompanied by a release of free energy called binding energy. Binding energy is a major source of free energy used by enzymes to lower the activation energies. B)Transient covalent interactions of enzyme with a substrate or group transfers to or from a substrate: Besides binding energy, other sources of energy for the lowering of the activation energies are transient covalent interaction of enzyme with a substrate or group transfer to or from a substrate. 5. Do enzymes affect reaction equilibria? Do they make nonspontaneous reactions to occur spontaneously? Consortium for Educational Communication Answer: Enzymes, like other catalysts, do not affect reaction equilibria. They do not change the position of reaction equilibria. The enzyme catalyzes and accelerates the reaction in both the directions to same extent. However, the reaction reaches equilibrium much faster when the appropriate enzyme is present. In other words, enzymes provide a reaction pathway whose transition-state energy is lower than that of the uncatalyzed reaction. Reaction equilibria (spontaneity) depend on free-energy change for the reaction, ∆G; while reaction rates depend on activation energies, ∆G‡. ∆G of the reaction, which tells about the spontaneity of the reaction, is independent of the activation energy. Since enzymes lower only the activation energy, therefore, they enhance reaction rates without altering the free-energy change for the reaction. Thus, enzymes do not make non-spontaneous reactions to occur spontaneously. 6. What is the role of binding energy in enzyme catalyzed reactions? Answer: The binding energy contributes to specificity as well as to catalysis of reactions. Binding energy is a major source of free energy used by enzymes to lower the activation energies of reactions. Under conditions commonly found in cells, a 10-fold rate enhancement requires a ∆∆G‡cat of only 5.71 kj/mol, while a million-fold rate enhancement requires a ∆∆G‡cat of ≈ 34 kj/ mol. The binding energy available from formation of a single weak interaction is generally estimated to be 4 to 30 kJ/mol. Considering a number of such interactions in ES and ES‡ complex, the overall binding energy available is therefore sufficient to lower the activation energies by 60 to 100 kJ/mol, thereby showing such a large rate enhancements. Besides, specificity is also derived from the formation of many weak interactions (binding energy) between the enzyme and its specific substrate molecule. The binding energy is used to lower the activation energy through following mechanisms: Stabilization of transition state, Entropy Loss and destabilization of the ES complex, and Proximity and orientation effects. Consortium for Educational Communication 7. Mention the various mechanisms employed by enzymes to catalyze the reactions? Answer: The mechanisms employed by enzymes to catalyze the reactions are: 1 Stabilization of transition state 2 Entropy Loss and destabilization of the ES complex 3 Proximity and orientation effects 4 General Acid–base catalysis 5 Covalent catalysis 6 Metal ion catalysis 8. Name the various mechanisms through which binding energy lowers the activation energy? Answer: The binding energy is used to lower the activation energy through following mechanisms: Stabilization of transition state, Entropy Loss and destabilization of the ES complex and Proximity and orientation effects. 9. What do you mean by Stabilization of transition state? Answer: Stabilization of transition state means lowering of its free energy. Enzymes are “designed” by nature to bind the transition-state structure more tightly than the substrate (or the product). In other words, weak interactions are optimized in the reaction transition state; enzyme active sites are complementary not to the substrates per se but to the transition states through which substrates pass as they are converted to products during Consortium for Educational Communication an enzymatic reaction. Since these weak interactions occur preferentially in the reaction transition state, the transition state has more binding energy and gets therefore stabilized, thereby lowering the activation energy. 10. How does entropy loss occur? How does it raise the free energy of ES complex that lowers the activation energy? Answer: The loss of entropy occurs due to the binding of substrate to enzyme. The entropy loss arises from the fact that the ES complex is a highly organized (low-entropy) entity compared to enzyme and substrate in solution (a disordered, high-entropy situation). Because ΔS is negative for this process, the term -TΔS is a positive quantity, and, therefore, the free energy content of ES complex increases. In other words, entropy reduction raises the free energy of ES complex that in turn lowers the activation energy. 11. How does destabilization of the ES complex occur? How does it lower the activation energy? Answer: The destabilization of ES complex occurs by strain, distortion, desolvation, or other similar effects. The strain or distortion facilitates the conversion of substrate to the transition state by weakening critical bonds and by permitting formation of new weak bonding interactions. The facilitation of additional weak bonding interactions in the transition state stabilizes it, thereby lowering the activation energy. In aqueous solution, most biomolecules (charged groups) are stabilized by the solvation shell of hydrogen-bonded water molecules. Upon binding in the active site, enzyme-substrate interactions replace most or all of these hydrogen bonds, thereby desolvating and destabilizing them. Besides, there occurs electrostatic destabilization as the charged groups on Consortium for Educational Communication substrate may be forced to interact (unfavorably) with charges of like sign on active site. The total destabilization energy (ΔGd) raises the free energy of ES complex, thereby lowering the activation energy. 12. How do proximity and orientation effects help in lowering of the activation energy? Answer: Enzymes take the reactants out of dilute solution and hold them close to each other or to catalytic residues. This proximity of reactants is said to raise the “effective” concentration over that of the substrates in solution, and leads to an increased reaction rate. Enzymes not only bring substrates and catalytic groups close together, but they also orient them in a manner suitable for catalysis as well. Proximity and orientation may also lead to electrostatic interactions that result in the stabilization of transition state (electrostatic catalysis). 13. Name the various mechanisms through which transient covalent interactions or group transfers lower the activation energy? Answer: The various mechanisms that generally involve transient covalent interaction with a substrate or group transfer to or from a substrate are: General Acid–base catalysis, Covalent catalysis and Metal ion catalysis. 14. How does General Acid–base catalysis result in rate enhancement? Answer: Transition states can also be stabilized by the transfer of protons to or from the proton acceptors or donors. Nonenzymatic reactions involve either the constituents of water (H+ or OH-) alone or other weak proton donors or acceptors for the proton transfers to/from transition state. Since the protons are transferred slowly, Consortium for Educational Communication the transition state is not effectively stabilized every time it forms, thus rendering the catalysis slow. However, a number of amino acid side chains in the active site of an enzyme act as acids or bases, readily donating or accepting a proton from transition state, thereby effectively stabilizing it. This results in lowering of the activation energy and hence rate enhancement. 15. What do you mean by Covalent catalysis? Answer: In Covalent catalysis, a powerful nucleophile in the active site forms a transient covalent bond between the enzyme and the substrate that results in lowering of the activation energy. The nucleophilic groups readily attack electrophilic centers of substrates, forming covalently bonded enzyme-substrate intermediates which undergo further reaction to give the desired products. 16. What is Metal ion catalysis? Answer: The catalysis by metals, bound to the enzyme, is called Metal ion catalysis. These metals participate in catalysis in several ways: by binding to substrates and orienting them, by mediating oxidation-reduction reactions, by electrostatically or electrophilically stabilizing or shielding negative charges. 17. Does a particular enzyme employ just one mechanism or all of these mechanisms simultaneously during catalysis? Answer: Whether based on noncovalent interactions (binding energy), transient covalent interactions or group transfers, these mechanisms are not mutually exclusive. A given enzyme might employ a combination of several catalytic strategies in its overall mechanism of action to bring about a rate enhancement. For most Consortium for Educational Communication enzymes, it is difficult to quantify the contribution of any one catalytic mechanism to the rate and/or specificity of a particular enzyme-catalyzed reaction. However, binding energy is a major source of free energy used by enzymes to lower the activation energies.