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Biochemistry

Biochemistry. Lecture 8 Enzyme Kinetics. Why Enzymes?. Higher reaction rates Greater reaction specificity Milder reaction conditions Capacity for regulation. Metabolites have many potential pathways of decomposition Enzymes make the desired one most favorable.

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Biochemistry

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  1. Biochemistry Lecture 8 Enzyme Kinetics

  2. Why Enzymes? • Higher reaction rates • Greater reaction specificity • Milder reaction conditions • Capacity for regulation • Metabolites have many potential pathways of decomposition • Enzymes make the desired one most favorable

  3. Enzymatic Substrate Selectivity No binding Binding but no reaction Example: Phenylalanine hydroxylase

  4. Enzymes Affect Reaction Rates • Catalyst • Protein (globular) or RNA • Classified based on the reaction catalyzed

  5. Ea ' H2O + CO2HOCO2– + H+ Ea Potential Energy Reaction

  6. How to Lower G? Enzymes bind transition states best

  7. How Do Enzymes Stabilize the Transition State and Increase Reaction Rate? Conserved active site amino acid residuesin the enzyme can help with orientation of the substrate and stabilizing transition state

  8. Serine Protease Mechanism

  9. How Do Enzymes Stabilize the Transition State and Increase Reaction Rate? Conserved active site amino acid residues,metal ions and organic molecules in the enzyme can help with orientation of the substrate and stabilizing transition state

  10. How is TS Stabilization Achieved? • acid-base catalysis: give and take protons • covalent catalysis: change reaction paths • metal ion catalysis: use redox cofactors, pKa shifters • electrostatic catalysis: preferential interactions with TS End result? Rate enhancements of 105 to 1017!

  11. How is TS Stabilization Achieved? • covalent catalysis: change reaction paths

  12. How to Lower G? Enzymes organizes reactive groups into proximity

  13. Enzyme Kinetics • Kinetics is the study of the rate at which compounds react • Rate of enzymatic reaction is affected by • Enzyme • Substrate • Effectors • Temperature

  14. How to Do Kinetic Measurements

  15. Steady-State Assumption

  16. What equation models this behavior? Michaelis-Menten Equation

  17. Michaelis-Menten Kinetics k2 k1 Derived using a few assumptions: • steady state assumption: formation of ES = breakdown of ES (until a significant amount of S has been consumed). • consider initial velocity at early time-points, [P] = 0: rate of reaction depends exclusively on the breakdown of ES (k-2 can be ignored). • free ligand assumption: [S] is in such excess that its decrease in concentration when forming ES is negligible (total [S] = free [S] + [ES]). E + S  ES  E + P k-1

  18. Simple Enzyme Kinetics • The final form in case of a single substrate is • kcat (turnover number): how many substrate molecules can one enzyme molecule convert per second • Km (Michaelis constant): an approximate measure of substrate’s affinity for enzyme • Microscopic meaning of Km and kcat depends on the details of the mechanism

  19. Calculating Vmax and Km: The Double Reciprocal Plot

  20. Enzyme Inhibition • Inhibitors are compounds that decrease enzyme’s activity • Irreversible inhibitors (inactivators) react with the enzyme • one inhibitor molecule can permanently shut off one enzyme molecule • they are often powerful toxins but also may be used as drugs • Reversible inhibitors bind to, and can dissociate from the enzyme • - they are often structural analogs of substrates or products • - they are often used as drugs to slow down a specific enzyme • Reversible inhibitor can bind: • To the free enzyme and prevent the binding of the substrate • To the enzyme-substrate complex and prevent the reaction

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