Advanced Topics in Biochemistry and Molecular Biology Spring 2003

1
Advanced Topics in Biochemistry and Molecular
Biology Spring 2003

• Kinetics and Mechanism Session 4 1/22/03

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kapp (or k) for first order reactions and kapp
vs Bo plots

• -kapp is the rate constant for the transition
  from the chemical state at t 0 to that at t 8
  under the particular initial reactant
  concentrations chosen
• - equilibrium approached from both sides

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Exploring enzyme inhibitors

• Back to basics.. Michaelis-Menten kinetics

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Reversible competitive inhibitor
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Effect of preincubation time on reversible
competitive inhibitor e.g., ibuprofen and
cyclooxygenase (cox) activity of PGH synthase-1
or 2

• rapid initial inhibition, proportional to I
• preincubation with I makes no difference

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Some inhibitors behave very differently when
preincubated with enzyme e.g., aspirin and
cyclooxygenase (cox) activity of PGH synthase-1
or 2

• Two aspects to such inhibition
• instantaneous (t 0) inhibition
• proportional to I
• fast reversible I binding
• competing with S
• inhibition during preincubation
• increases with time
• first order process
• rate increases with I

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Accounting for progressive inhibition during
preincubation

• k (or kapp) is a saturable function of aspirin
• reminiscent of saturable binding of ligand ( E
  I ? EI )
• suggests a first order process beginning with EI
  complex ( EI gt X )
• need to modify competitive inhibition mechanism

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Modifying competitive inhibitor mechanism

• Two aspects to inhibition by inhibitor such as
  aspirin
• initial rapid reversible binding to form EI
• slower, irreversible conversion of EI to EI2
• examine the case of reversible second stage later
• Second step behaves like slow, very tight binding
• Such inhibitors called slow-binding or
  time-dependent inhibitors
• slow here refers to process requiring several
  seconds or minutes

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Slow-binding (time-dependent) inhibitors

• Slow-binding inhibitors not restricted to aspirin
  and PGHS
• many other so-bindng inhibitors of PGHS
  cyclooxygenases known
• indomethacin, flurbiprofen, Celebrex,Vioxx
• slow-binding inhibitors known for wide variety of
  other enzymes
• Review Morrison JF Walsh CT (1988) Adv Enzymol
  61, 201-301
• Advantages of slow-binding inhibitors over
  reversible competitive inhibitors
• inhibition persists long after inhibitor cleared
  from circulation
• accumulation of S (i.e., by decreased conversion
  to P) does not displace bound I
• S below saturation for most enzymes in cell
• competitive I blocks pathway, increases S,
  tends to overcome inhibition
• EI readily dissociates to E, binds increased S
• increased S has little effect on EI

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Cyclooxygenase substrate and inhibitors

• Slow-binding inhibitors specific for
  cyclooxygenase activity of PGHS-2 (cox-2)
  developed recently
• PGHS-1 levels generally constitutive, associated
  with housekeeping functions
• hemostasis, vascular tone, gastric mucosa
  protection
• PGHS-2 inducible by mitogens, cytokines,
  associated with inflammation, cell proliferation
• macrophage activation, colon cancer progression

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Mechanism of selective inhibition vs. cox-2 known

• Assigned Paper 2
• Time-dependent action against cox-2, simple
  reversible inhibitors of cox-1
• E I ? EI gt EI or E I ? EI ? EI
  cox-2 mechanims
• E I ? EI cox-1 mchanim

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DuP697 and NS398 actions as slow-binding
inhibitors of cox-2

• Second step assumed to be irreversible in this
  analysis
• Do reported Ki values fit data in figures?

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Simulations today explore inhibitor actions

• Time-dependent inhibition of cox-2 by NS398
• two-stage simulation
• preincubation with inhibitor
• cox started with substrate
• Reversible inhibition of cox-1 by NS398
• two-stage simulation
• preincubation with inhibitor
• cox started with substrate
• Control cox reaction under Vmax conditions
• single stage simulation

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Structural determinants of cox-2 inhibitor
selectivity

• Assigned Paper 4
• PGHS-2 Val509 mutated to Ile (as in PGHS-1)
• Effect on inhibitor kinetics analyzed
• Discuss more in class Friday

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Reminder

• Homework due at beginning of class Friday