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The mechanism of enzyme catalysis is similar in principle to other types of Chemical Catalysis . By providing an alternative reaction route and by stabilizing intermediates the enzyme reduces the energy required to reach the highest energy Transition State of the reaction. The reduction of activation energy (ΔG) increases the number of reactant molecules with enough energy to reach the activation energy and form the product. INDUCED FIT The favored model for the enzyme- Substrate interaction is the induced fit model.1 This model proposes that the initial interaction between enzyme and substrate is relatively weak, but that these weak interactions rapidly induce Conformational Change s in the enzyme that strengthen binding. Catalysis by induced fit The advantages of the induced fit mechanism arise due to the stabilising effect of strong enzyme binding. There are two different mechanisms of substrate binding; uniform binding which has strong substrate binding, and differential binding which has strong transition state binding. The stabilizing effect of uniform binding increases both substrate and transition state binding affinity and differential binding increases only transition state binding affinity. Both are used by enzymes and have been evolutionarily chosen to minimize the ΔG of the reaction. Enzymes which are saturated, ie. have a high affinity substrate binding, require differential binding to reduce the ΔG, whereas largely substrate unbound enzymes may use either differential or uniform binding. These effects have lead to most proteins using the differential binding mechanism to reduce the ΔG, so most proteins have high affinity of the enzyme to the transition state. Differential binding is carried out by the induced fit mechanism - the substrate first binds weakly, then the enzyme changes conformation increasing the affinity to the transition state and stabilizing it, so reducing the activation energy to reach it. It is important to clarify, however, that the induced fit concept cannot be used to rationalize catalysis. That is, the chemical catalysis is defined as the reduction of ΔG‡ (when the system is already in the ES‡) relative to ΔG‡ in the uncatalyzed reaction in water (without the enzyme). The induced fit only suggests that the barrier is lower in the closed form of the enzyme but does not tell us what the reason for the barrier reduction is. MECHANISMS OF TRANSITION STATE STABILIZATION These conformational changes also bring catalytic residues in the Active Site close to the chemical bonds in the substrate that will be altered in the reaction. After binding takes place, one or more mechanisms of catalysis lowers the energy of the reaction's Transition State , by providing an alternative chemical pathway for the reaction. There are six possible mechanisms of "over the barrier" catalysis as well as a "through the barrier" mechanism: Catalysis by bond strain This is the principal effect of induced fit binding, where the affinity of the enzyme to the transition state is greater than to the substrate itself. This induces structural rearrangements which strain substrate bonds into a position closer to the conformation of the transition state, so lowering the energy difference between the substrate and transition state and helping catalyze the reaction. However, the strain effect is, in fact, a ground state destabilization effect, rather than transition state stabilization effect Jencks W.P. "Catalysis in Chemistry and Enzymology" 1987, Dover, New York Warshel A., Sharma P.K., Kato M., Xiang Y., Liu H. and Olsson M.H.M. "Electrostatic Basis of Enzyme Catalysis", Chem. Rev., 2006, 106: 3210-3235. Furthermore, enzymes are very flexible and they cannot apply large strain effect Warshel A. and Levitt M. "Theoretical Studies of Enzymatic Reactions: Dielectric Electrostatic and Steric Stabilization of the Carbonium Ion in the Reaction of Lysozyme" J. Mol. Biol., 1976, 103: 227 . In addition to bond strain in the substrate, bond strain may also be induced within the enzyme itself to activate residues in the active site. Catalysis by proximity and orientation This increases the rate of the reaction as enzyme-substrate interactions align reactive chemical groups and hold them close together. This reduces the Entropy of the reactants and thus makes reactions such as ligations or addition reactions more favorable, there is a reduction in the overall loss of entropy when two reactants become a single product. This effect is analogous to an effective increase in concentration of the reagents. The binding of the reagents to the enzyme gives the reaction Intramolecular character, which gives a massive rate increase. However, the situation might be more complex, since modern computational studies have established that traditional examples of proximity effects cannot be related directly to enzyme entropic effects Stanton R.V., Perakyla M., Bakowies D. and Kollman P.A. "Combined ab initio and Free Energy Calculations To Study Reactions in Enzymes and Solution: Amide Hydrolysis in Trypsin and Aqueous Solution" J. Am. Chem. Soc. 1998; 120:3448-3457 Kuhn B.; Kollman P.A. "QM-FE and Molecular Dynamics Calculations on Catechol O-Methyltransferase: Free Energy of Activation in the Enzyme and in Aqueous Solution and Regioselectivity of the Enzyme-Catalyzed Reaction" J. Am. Chem. Soc. 2000; 122:2586-2596 Bruice T.C. and Lightstone F.C. "Ground State and Transition State Contributions to the Rates of Intramolecular and Enzymatic Reactions" Acc. Chem. Res. 1999; 32:127-136. Also, the original entropic proposal Page M.I. and Jencks W.P. "Entropic Contributions to Rate Accelerations in Enzymic and Intramolecular Reactions and the Chelate Effect" Proc. Natl. Acad. Sci. USA 1971, 68:1678-1683 has been found to largely overestimate the contribution of orientation entropy to catalysis Warshel A. and Parson W.W. "Dynamics of Biochemical and Biophysical Reactions: Insight from Computer Simulations" Quart. Rev. Biophys. 2001 34: 563-679 . Catalysis Involving Proton donors or acceptors See also: Protein PKa Calculations Proton donors and acceptors, i.e. Acid s and Base s, may donate and accept protons in order to stabilize developing charges in the transition state. This typically has the effect of activating Nucleophile and Electrophile groups, or stabilizing leaving groups. Histidine is often the residue involved in these acid/base reactions, since it has a PKa close to neutral PH and can therefore both accept and donate protons. Many reaction mechanisms involving acid/base catalysis assume a substantially altered pKa. This alteration of pKa is possible through the local environment of the residue. The pKa is can be modified significantly by the environment, to the extent that residues which are basic in solution may act as proton donors, and vice versa. It is important to clarify that the modification of the pKa’s is a pure part of the electrostatic mechanism Warshel A., Sharma P.K., Kato M., Xiang Y., Liu H., and Olsson M.H.M. "Electrostatic Basis of Enzyme Catalysis", Chem. Rev. 2006, 106: 3210-3235. Furthermore, the catalytic effect of the above example is mainly associated with the reduction of the pKa of the oxy anion and the increase in the pKa of the histidine, while the proton transfer from the serine to the histidine is not catalyzed significantly since, it is not the rate determining barrier Warshel A., Naray-Szabo G.,Sussman F.and Hwang J.-K. "How do Serine Proteases Really Work?" Biochemistry, 1989, 28: 3629 . Electrostatic catalysis Stabilization of charged transition states can also be by residues in the active site forming Ionic Bonds (or partial ionic charge interactions) with the intermediate. These bonds can either come from Acid ic or Basic side chains found on Amino Acid s such as Lysine , Arginine , Aspartic Acid or Glutamic Acid or come from metal Cofactors such as Zinc . Metal ions are particularly effective and can reduce the pKa of water enough to make it an effective nucleophile. Systematic computer simulation studies established that electrostatic effects give, by far, the largest contribution to catalysis Warshel A., Sharma P.K., Kato M., Xiang Y., Liu H., and Olsson M.H.M. "Electrostatic Basis of Enzyme Catalysis", Chem. Rev. 2006, 106: 3210-3235.. In particular, it has been found that enzyme provides an environment which is more polar than water, and that the ionic transition states are stabilized by fixed dipoles. This is very different from transition state stabilization in water, where the water molecules must pay with "reorganization energy" Marcus R. A. "On the Theory of Electron-Transfer Reactions. VI. Unified Treatment for Homogeneous and Electrode Reactions" J. Chem. Phys. 1965 43:679-701 in order to stabilize ionic and charged states. Thus, the catalysis is associated with the fact that the enzyme polar groups are preorganized Warshel A. "Energetics of Enzyme Catalysis", Proc. Natl. Acad. Sci. USA, 1978 75: 5250 . Covalent Catalysis Covalent catalysis involves the substrate forming a transient covalent bond with residues in the active site. This adds an additional covalent intermediate to the reaction, and helps to reduce the energy of later transition states of the reaction. The covalent bond must, at a later stage in the reaction, be broken to regenerate the enzyme. This mechanism is found in enzymes such as Protease s like Chymotrypsin and Trypsin , where an acyl-enzyme intermediate is formed. Schiff Base formation using the free Amine from a Lysine residue is another mechanism, as seen in the enzyme Aldolase during Glycolysis . It is important to clarify that covalent catalysis does correspond in most cases to simply the use of a specific mechanism rather than to true catalysis Warshel A., Sharma P.K., Kato M., Xiang Y., Liu H., and Olsson M.H.M. "Electrostatic Basis of Enzyme Catalysis", Chem. Rev. 2006, 106: 3210-3235.. For example, the energetics of the covalent bond to the serine molecule in chymotrypsin should be compared to the well-understood covalent bond to the nucleophile in the uncatalyzed solution reaction. A true proposal of a covalent catalysis (where the barrier is lower than the corresponding barrier in solution) would require, for example, a partial covalent bond to the transition state by an enzyme group (e.g., a very strong hydrogen bond), and such effects do not contribute significantly to catalysis. Quantum Tunneling These traditional "over the barrier" mechanisms have been challenged in some cases by models and observations of "through the barrier" mechanisms ( oxidation by aromatic amine dehydrogenase.Masgrau L, Roujeinikova A, Johannissen LO, Hothi P, Basran J, Ranaghan KE, Mulholland AJ, Sutcliffe MJ, Scrutton NS, Leys D. ''Atomic Description of an Enzyme Reaction Dominated by Proton Tunneling.'' Science. 2006 April 14;312(5771):237-41. PMID 16614214 Interestingly, quantum tunneling does not appear to provide a major catalytic advantage, since the tunneling contributions are similar in the catalyzed and the uncatalyzed reactions in solution Olsson M.H.M, Siegbahn P.E.M and Warshel A. "Simulations of the Large Kinetic Isotope Effect and the Temperature Dependence of the Hydrogen Atom Transfer in Lipoxygenase" J. Am. Chem. Soc., 2004, 126: 2820-2828 Ball, P. " Enzymes: By chance, or by design?" Nature, 2004, 431: 396 Olsson M.H.M., Parson W.W. and Warshel A. "Dynamical Contributions to Enzyme Catalysis: Critical Tests of A Popular Hypothesis" Chem. Rev. 2006, 105: 1737-1756. EXAMPLES OF CATALYTIC MECHANISMS In reality, most enzyme mechanisms involve a combination of several different types of catalysis. Triose phosphate isomerase Triose Phosphate Isomerase () catalyses the reversible interconvertion of the two Triose phosphates Isomer s Dihydroxyacetone Phosphate and D- Glyceraldehyde 3-phosphate . Trypsin Trypsin () is a Serine Protease s that cleaves Protein substrates at Lysine and Arginine amino acid residues. Aldolase Aldolase () catalyses the breakdown of Fructose 1,6-bisphosphate (F-1,6-BP) into Glyceraldehyde 3-phosphate and Dihydroxyacetone Phosphate ( DHAP ). REFERENCES FURTHER READING
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