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  • 1. BT-202 Netaji Subhas Institute of Technology, Dwarka, New Delhi. Dr. Amita Pandey Sept 5, 2011
  • 2.
    • Enzyme kinetics
    • Catalytic mechanisms
    • Regulation of enzyme activity
    • Enzyme inhibition
  • 3.
    • Louis Pasteur (1850s): ferments, vitalism
    • Eduard Buchner (1897): fermentation is done by molecules
    • Frederick W. Kuhne: coined the name enzyme
    • James Sumner (1926): isolated urease and said enzymes are made up of proteins.
    • J.B.S. Haldane: weak interactions between enzyme and substrate are important for catalysis
  • 4. Enzymes
    • Definition;
    • Chemical reactions in cells require
    • specific catalysis.
    • Enzymes are proteins which perform
    • this function.
    • - 12kDa - 1,000kDa or more
    • - larger than their substrate
  • 5. Enzymes
    • Metabolite acted upon is called the enzyme ’s substrate.
    • Active site
    • - some residues involved in binding substrate
    • -others catalyze reaction
  • 6.
    • Cofactors
    • for some reactions, the amino acids are not powerful enough for catalysis. Enzymes overcome this by incorporate additional factors.
    • - metal ions as cofactors Zn 2+ , Fe 2+ , Cu 2+
    • -coenzymes are organic cofactors
    • Prosthetic Group
    • Coenzyme or metal ion cofactor bound to enzyme either tightly or covalently.
    • - holoenzyme
    • -apoenzyme
  • 7. Cofactor and Coenzymes
  • 8. Properties of Enzymes
    • Enzymes are excellent catalysts
    • - speeding up reactions 10 8 to 10 20 fold
    • Not used up during a reactions
    • Specificity for substrate
    • - absolute (eg., DNA polymerase)
    • - broad range (eg., synthesis of secondary metabolites)
    • Regulated- some enzymes can sense
    • metabolic signals.
  • 9. Classification of Enzymes
    • named and classified according to the substrate acted upon and the reaction catalyzed.
    • trivial names- end in –ase . (eg., urease, hexokinase.
    • based on a formal systemic catalog (IUB) with six major classifications.
  • 10.  
  • 11. Reaction Rates and the Transition State
    • In order to react, the molecules involved
    • are distorted, strained or forced to have
    • an unlikely electronic arrangement.
    • That is the molecules must pass through a
    • high energy state.
    • This high energy state is called the transition state .
  • 12.
    • The energy required to achieve it is called
    • the activation energy for the reaction.
    The higher the free energy change for the transition barrier, the slower the reaction rate.
  • 13. Enzymes lower energy barrier by forcing the reacting molecules through a different transition state.
  • 14. Eg., C 12 H 22 O 11 + 12O 2 12CO 2 + 11H 2 O Reaction Intermediates Transient chemical species formed during a rxn Rate limiting step When many steps are involved the overall rate of the Reaction is determined by the step which has the Highest activation-energy
  • 15. Relationship between reaction equilibrium and free energy Equilibrium constant is directly related to free energy Large negative free energy favors reaction
  • 16. Rate of a reaction Unimolecular or first order reaction; V=k[S] Units are s -1 Rate for second order reaction; V= k[S 1 ][S 2 ] for transition state k = k T / h e - Δ G/RT Lower activation energy means faster rxn rates
  • 17. How enzymes do it? Binding energy ( Δ G B ) Energy derived from enzyme substrate interaction. -transient covalent bond in the active site -non-covalent interactions to form ES
  • 18.  
  • 19. Role of binding energy in catalysis k = k T / h e - Δ G/RT Δ G + must be lowered by 5.7kJ/mol to accelerate first order reaction.
  • 20.
    • Specificity is derived from the formation of many weak interactions between the enzyme and its specific substrate.
    • Entropy reduction
  • 21.
    • Desolvation of substrate
    • ES complex
    • Enzyme undergoes conformation changes i.e. induced fit, postulated by Daniel Koshland (1958)
  • 22. Modes of Enzymatic Enhancement of Rates
    • Involve transient covalent interactions with a substrate or group transfer to or from a substrate
    • General acid-base catalysis
    • -good proton donors & acceptors positioned just right.
  • 23.  
  • 24.
    • covalent catalysis
    • -unstable intermediate
    • metal ion catalysis
    • -electron donor or acceptor
  • 25.
    • At low concentrations of [S] V o increases almost linearly
    • At higher [S] concentration V o increases by smaller amounts in response to increase in [S]
    • Finally increase in V o is negligible as [S] increases
    Enzyme kinetics
  • 26. Steady state kinetics
    • rate stops increasing or plateaus because the complex ES becomes filled at high [S]
    • Pre-steady state and steady state
    • Introduced by Briggs and Haldane
  • 27. Michaelis-Menten Model
    • Rate equation
  • 28. Lineweaver-Burk equation Double reciprocal plot
  • 29. Interpreting V max and K m Steady state kinetics is the means by which biochemists can compare and characterize The catalytic efficiencies of enzymes.
  • 30. K cat it is the limiting rate of any enzyme catalyzed reaction. Eg. , K cat is first order rate constant has units s -1 . It is also called Turnover number ( the number of substrate molecules converted to product) .
  • 31.
    • Specificity constant compares the catalytic
    • efficiencies of different enzymes or the turnover
    • of different substrates by the same enzyme.
            • V o = k cat / K m [E t ][S]
            • -V 0 depends upon [Et] and [S]
            • -k cat /K m is a second order rate constant
            • -units M -1 s -1
  • 32. Reactions with more than one substrate
  • 33. Learning check!
    • In a simple enzyme catalyzed rxn., the sum of
    • which two chemical species is strictly constant?
    • [E] + [ES]
    • [E] + [P]
    • [S] + [P]
    • [ES] + [P]
    • 2. For a simple enzymatic rxn., what is the value of
    • The initial velocity when S=5km?
    • 5 Vmax
    • 5/2 Vmax
    • 4/5 Vmax
    • 5/6 Vmax
  • 34.
    • Match the following
    • Michaelis complex The velocity at t=0
    • Michaelis constant Km Also known as turnover number
    • Initial velocity The enzyme substrate complex
    • Maximal velocity The velocity when the enzyme is saturated with substrate
    • Catalytic velocity The conc. of substrate @ which the velocity is half maximal
    • rxns. are enzyme reactions with two substrates.
    • Bisubstrate
    • In Lineweaver-Burk plot of a simple enzymatic rxn. , what is the value of the y-intercept @ 1/Vo axis?
    • Vmax
    • 1/Vmax
    • Km
    • 1/Km
  • 35. Regulatory enzymes Exhibit increased or decreased catalytic activity in response to certain signals. -Reversible non-covalent modulation - allosteric modulators -small metabolites or cofactors -Reversible covalent modification -inhibition with separate regulatory protein -proteolytic cleavage
  • 36. Allosteric enzymes -Homotropic -Heterotropic
  • 37. Heterotropic Allosteric Modulation In multienzyme pathways the regulatory enzyme is inhibited by the end product. Eg., L-Threonine to L-Isoleucine in bacteria.
  • 38. Kinetics of Allosteric enzymes -sigmoid curve is observed -substrate concentration is represented by [S] 0.5 or K 0.5 homotropic allosteric enzymes
  • 39. -reflects cooperative interaction between protein molecules -small changes in conc. of modulator can be associated with large changes in activity
  • 40. Heterotropic Allosteric enzymes -Activation may cause increased velocity for fixed K 0.5 -Negative modulator cause decreased velocity for fixed K 0.5
  • 41. -increased V max with little change in substrate
  • 42. Learning check! Match the following Competitive Inhibiton decreases Km and Vmax Uncompetitive inhibition decreases [E]T Mixed inhibition increases Km Inactivation decreases Vmax, while Km may increase or decrease Mixed inhibition is characterized by two dissociation constants for the inhibition. True False
  • 43. Reversible covalent modification
  • 44.  
  • 45. How do the modulators act? -oxygen atom of phosphoryl group undergo H-bond formation -repulsion of neighboring residues with negative charges phosphatase kinase
  • 46. Glucose-6-phosphate ATP synthesis in muscles Free glucose in liver
  • 47.  
  • 48. -phosphorylation occurs in structural motif called consensus sequence -AA sequence is not the only factor which determines phosphorylation
  • 49. Regulation by proteolytic cleavage -zymogen an inactive precursor is cleaved to form the active enzyme
  • 50. -precursors are also called proprotein eg., collagen, fibrin, and thrombin
  • 51.  
  • 52. Enzyme inhibitors Interfere with catalysis, slowing or halting enzymatic reactions. -Reversible Inhibition -competitive Since it is reversible so increasing the concentration of substrate remove inhibition.
  • 53. Uncompetitive inhibition Lower the measured V max Apparent K m also decreases
  • 54. Mixed Inhibition
  • 55. Irreversible Inhibition DIFP Diisopropylfluorophosphate Suicide inactivators such compounds are inactive untill they bind active site of a specific enzyme.