Enzyme catalysis, effect of organic solvent
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INTRODUCTION Enzyme catalysis Mechanisms of catalysis Organic solvent Classes of Organic Solvent Biocatalysis in organic solvents Enzyme Reactions in Organic solvents Effect of the organic solvent in enzyme catalysis Conclusion References
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Enzyme catalysis, effect of organic solvent
- 1. ENZYME CATALYSIS EFFECT OF ORGANIC SOLVENT By KAUSHAL KUMAR SAHU Assistant Professor (Ad Hoc) Department of Biotechnology Govt. Digvijay Autonomous P. G. College Raj-Nandgaon ( C. G. )
- 2. SYNOPSIS INTRODUCTION Enzyme catalysis Mechanisms of catalysis Organic solvent Classes of Organic Solvent Biocatalysis in organic solvents Enzyme Reactions in Organic solvents Effect of the organic solvent in enzyme catalysis Conclusion References
- 3. Enzyme catalysis Enzyme catalysis is the catalysis of chemical reactions by specialized proteins known as enzymes 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 mechanism of enzyme catalysis is similar in principle to other types of chemical catalysis.
- 4. Mechanisms of catalysis •Enzymes are very specific. Each enzyme will catalyze only one type of reactions and often will only work with a specific substrate. Electrostatic catalysis Acid-base catalysis Covalent catalysis Metal ion catalysis
- 5. The Active Site Enzymes are typically HUGE proteins, yet only a small part are actually involved in reaction. The active site has two basic components. catalytic site binding site Model of trios-p-isomerase
- 6. Catalytic site Where reaction occurs Binding Site holds substrate in place Substrate Enzyme The Active Site
- 8. Lock and Key Theory Enzyme is “lock” and Substrate is the “key”. Substrate structure must fit into enzyme’s structure.
- 9. Induced Fit Theory Active site may not fit substrate. Site must change in order to form the complex.
- 10. Enzymes have optimal operating temperatures and pHs. These graphs show how enzyme catalysis is affected by changes in temperature and pH for enzymes that operate most effectively at physiological conditions. Temperature and pH Effects on Enzyme Catalysis 10
- 12. •Common Classes of Organic Solvents Chlorinated Aliphatic hydrocarbons Aromatic hydrocarbons trichloroethane trichloroethylene methylene chloride chloroform carbon tetrachloride tetrachlorethylene n - hexane n - heptane pentane cyclohexene cyclohexane petroleum ether benzene toluene xylenes naphthalenes Alcohols/Glycols/Ethers Esters/Ketones/Aldehydes Others methanol ethanol propanol butanol ethylene glycol diethyl ether ethyl acetate acetone methyl ethyl ketone methyl isobutyl ketone methyl n-butyl ketone formaldehyde glutaraldehyde carbon disulphid pyridine amides amines
- 13. Biocatalysis in organic solvents •Systems with organic solvents •Water and a water miscible organic solvent •Two-phase systems •PEG-modified enzymes in organic solvents •Reversed micelles •Monophasic organic solvents
- 14. EFFECT OF ORGANIC SOLVENT ON ENZYME CATALYSIS Effects on enzyme stability Hydrophobic solvent: small redistribution of water: conservation of native protein structure Polar solvent: stronger effect Interaction of solvent with protein surface Strip tightly bound water Destruction of hydrogen bond network Lowering of surface tension Onset of protein unfolding Extreme thermostability in inert solvents Fewer side reactions (hydrolysis) Conformational rigidity in dehydrated state Ex.Chymotrypsin,
- 15. Water-water miscible organic solvents Polar solvents Reactant, inhibitor, increase of flexibility (rate) Operational stability Change in product pattern Substrate solubility Presence of organic solvent can have a large effect on substrate solubility A substrate with a low affinity for solvent binds strongly to the enzyme Polar substrates have high Km in polar solvent
- 16. Two-phase systems About equal volumes of an aqueous solution and an immiscible organic solvent Catalysis takes place in the aqueous phase or at the interface [S] low, limits rate of catalysis Traces of solvent can influence activity and stability Enzyme recovery is difficult
- 17. PEG-modified enzymes Modification of lysine residues with PEG molecules of different size Synthesis of organic solvent soluble enzymes 10 - 20 PEG chains per enzyme molecule Increase in molecular mass Protects enzyme from surrounding organic solvent and prevents stripping of essential water High enzymatic activity with water immiscible solvents
- 18. Reversed micelles Effects on enzyme stability - dependent on protein properties low water content increases stability Effects on enzyme activity Water content too low pH different from stock buffer solution due to binding of protons or hydroxyl groups with surfactant head groups Effects on enzyme kinetics effects substrates Increase in apparent Km
- 19. Effect of solvent on the water associated with the enzyme water-stripping in the hydrophilic solvents Since enzyme and solvent compete for water, optimal water content is related to the amount of enzyme and the concentration of substrate hydrophilization of enzyme surface minimize removal of the H2O from enzyme
- 20. Effect of solvent on the enzyme Protein dynamics Protein conformation Enzyme active center Effect of solvent on substrates and products •change the conce. of substrates or products in the aqueous layer around enzyme Effect of substrate solvation on kinetics increase Km app in organic solvent solvent affects enzyme activity and specificity by altering the partitioning of substrates and products
- 21. CONCLUSION The technological utility of enzymes can be enhanced greatly by using them in organic solvents rather than in their natural aqueous reaction media. Strong acids and bases, organic solvents, mechanical action, and high temperature decrease an enzyme- catalyzed rate of reaction. Even slight changes in the pH can have profound effects on enzyme catalysis
- 22. REFERENCES •Principle of Biochemistry (fifth edition) by Nelson & Cox •Biochemistry (fourth edition) by Donald voet and Judth G. Voet •Klijn, J and Engberts, J. Organic chemistry: Fast reactions 'on water'. Nature 435, 746-747 (9 June 2005) •www.wikipedia.com
Editor's Notes
- Catalytic site = Where the reaction actually occurs. Binding site = Area that holds substrate in proper place. Enzymes uses weak, non-covalent interactions to hold the substrate in place based on R groups of amino acids. Shape is complementary to the substrate and determines the specificity of the enzyme. Sites are pockets or clefts on the enzyme surface.
- As the conformation of the enzyme changes in response to the binding substrate, a strain is induced in the substrate. This strain makes it less stable and thereby reduces the energy of activation required for conversion of substrate to product.