biochem chapter 7 LOs

enzyme kinetics and inhibition

Describe how an enzyme’s activity is measured.

• Enzyme kinetics is the quantitative study of reaction rates

• Reaction rate (velocity, v) is measured by:

  • Disappearance of substrate (S)

  • Appearance of product (P)

• Expressed as:

  • v=−d[S]dt=d[P]dtv = -\frac{d[S]}{dt} = \frac{d[P]}{dt}v=−dtd[S]​=dtd[P]​

• Initial rate (v₀) is measured shortly after ES reaches equilibrium when:

  • [ES] is constant

  • [P] is negligible

Depict enzyme saturation in graphical form.

• When [E] is fixed and [S] is varied, the velocity vs. substrate concentration plot is hyperbolic

• At high [S], the enzyme becomes saturated, and velocity approaches Vmax

Distinguish first-order and second-order reactions.

• First-order (unimolecular):

  • Rate depends on the concentration of one substrate

  • v=k[A]v = k[A]v=k[A], units of k = sec⁻¹

• Second-order (bimolecular):

  • Rate depends on two substrate concentrations

  • v=k[A][B]v = k[A][B]v=k[A][B], units of k = M⁻¹·sec⁻¹

Define Km and kcat.

• Km:

  • Substrate concentration at ½ Vmax

  • Inverse measure of enzyme–substrate affinity

  • Lower Km = higher affinity

• kcat (turnover number):

  • kcat=k2k_{cat} = k_2kcat​=k2​

  • kcat=Vmax[E]totalk_{cat} = \frac{V_{max}}{[E]_{total}}kcat​=[E]total​Vmax​​

Derive the values of Km and Vmax from graphical data.

• From a Michaelis–Menten plot:

  • Vmax = plateau of the curve

  • Km = [S] at ½ Vmax

• From a Lineweaver–Burk plot:

  • y-intercept = 1/Vmax

  • x-intercept = −1/Km

List the limitations of the Michaelis–Menten model.

• Does not apply to all enzymes

• Enzymes with multiple active sites often show sigmoidal, not hyperbolic, kinetics

• Allosteric enzymes show cooperative behavior and do not follow Michaelis–Menten assumptions

Compare the action of reversible and irreversible inhibitors.

• Reversible inhibitors:

  • Bind non-covalently

  • Continuously dissociate and reassociate

• Irreversible inhibitors:

  • Covalently bind enzyme

  • Permanently inactivate enzyme

  • Includes suicide inhibitors

Describe the effects of competitive, noncompetitive, mixed, and uncompetitive inhibitors on a reaction’s apparent Km and Vmax.

• Competitive inhibitors:

  • Increase Km

  • Do not affect Vmax

  • Prevent substrate from reaching the active site

• Noncompetitive inhibitors:

  • Decrease Vmax

  • Km unchanged

  • Bind E and ES, not at substrate site

• Uncompetitive inhibitors:

  • Decrease both Km and Vmax

  • Bind only to ES complex

• Mixed inhibitors:

  • Affect both Km and Vmax (shown graphically)

Explain why transition state analogs often act as competitive inhibitors.

• Transition state analogs resemble the transition state

• Bind more tightly than substrate analogs

• Compete for the active site

Explain why allosteric enzymes can be activated or inhibited

• Regulatory molecules bind at a site different from the active site

• Binding changes enzyme conformation and activity

Summarize the ways that cells regulate enzyme activity.

• Allosteric activation and inhibition

• Feedback (end-product) inhibition

• Regulation of first committed step in a pathway