Enzyme Inhibition Notes

Enzyme Inhibition

Enzyme Inhibition

• Inhibitors are compounds/ions that decrease an enzyme’s activity; they alter enzyme’s KM$and/or$V{max} values.

1. Irreversible inhibitors (inactivators) react with the enzyme.

• Chemical modification of the enzyme results in inactivation.

• One inhibitor molecule can permanently shut off one enzyme molecule.

• Usually bind covalently, but sometimes by non-covalent binding with very high affinity.

• They are often powerful toxins, but also may be used as drugs (e.g., penicillin, amoxicillin)

• Labeling used to determine catalytic serines for serprotease

• Covalently modify bacteria that synthesize cell wall

2. 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 (competitive).

    • The enzyme-substrate complex and prevent the reaction (uncompetitive).

    • The free enzyme or the enzyme-substrate complex (non-competitive)

  • Noncovalent Interaction.

Classes of Reversible Inhibitors

• All can utilize Michaelis-Menten Kinetics!

  • Competitive: Inhibitor binds to the enzyme, preventing substrate binding.

  • Uncompetitive: Inhibitor binds to the enzyme-substrate complex.

  • Noncompetitive: Inhibitor can bind to either the free enzyme or the enzyme-substrate complex.

    • Can act like both competitive and uncompetitive inhibitors.

Reversible Inhibitors – Competitive

• Competitive inhibition: The inhibitor is structurally similar to the substrate and can bind to the active site, preventing the actual substrate from binding.

• The inhibitor can bind but it can't react like substrate

• Can be relieved by increase substrate concentration, outcompete the inhibitors for the active site

Competitive Inhibitors

• Role: reduce

• Dihydrofolate reductase

• To synthesize purines/pyrimidines.

Substrate

• Dihydrofolate

Inhibitor

• Methotrexate

  • Struct. analog

  • bind 1000x better

Competitive Inhibitors

• Product inhibition – negative feedback loops

• Product accumulates and competes for active site

• How cell controls activities of its enzymes

Transition State Analogs

• $K_M = 3 × 10^{−5} M$

• $K_I = 3 × 10^{−4} M$

• $K_I = 1.5 × 10^{−13} M$

• $K_I$ = Dissociation constant for enzyme-inhibitor binding

• Effective catalysis often depends on an enzyme’s ability to bind to and stabilize its reaction’s transition state.

• Example: 1,6-Dihydroinosine inhibits adenosine deaminase

• Transition State Analogs are Strong Competitive Enzyme Inhibitors

• Inhibitor bind better to enz

Human Immunodeficiency Virus Protease inhibitors

• HIV protease substrate

• Bond to be cleaved

  • Saquinavir: $K_I= 0.40 nM$

  • Ritonavir: $K_I= 0.015 nM$

Competitive Inhibition

• A competitive inhibitor is typically a structural analog of the substrate which binds at the active site as the substrate does, and thus prevents the substrate from binding.

• Competitive Inhibitor Binding at Active Site of Enzyme

Competitive Inhibition: Mechanism

• Competes with substrate for binding

  • Binds active site

  • Does not affect catalysis

• The dissociation constant of the inhibitor: $K_I = \frac{[E][I]}{[EI]}$

• A competitive inhibitor reduces the [free enzyme] available for substrate binding.

• Enz still can bind substrate then produce products

• $K_{cat}$

• Inhibitor binds eat well

Competitive Enzyme Inhibition: Elucidation of ATP- Binding

• Comparing the $K_I$ values of competitive inhibitors with different structures

• Provide information about the binding properties of an enzyme’s active site and hence its catalytic mechanism.

Competitive Inhibition: Kinetic Parameters

• No change in $V_{max}$; substrate can outcompete inhibitor

• Apparent increase in $K<em>M$ ($K</em>{M}^{app} = αK_M$)

  • α: a function of the inhibitor’s concentration and its affinity for the enzyme (cannot be less than 1)

  • $α = 1 + \frac{[I]}{K_I}$

• $K_M$ increase by the factor

  • Which ran reaches its max velocity

  • Max rate of rxn when all enz active sites saturated

  • If two inhibitor then $K_M$ with inhibitor

Competitive Inhibition: Kinetic Curves

• Michaelis-Menten resulting equation that has been modified by a factor α

• No change in $V_{max}$

• Bound inhibitor does not inactivate the enzyme.

• Inhibition can be overcome by sufficiently high [S]

• Does not affect turnover number ($k_{cat}$)

• Apparent increase in KM$by the factor α ($K{M}^{app} = αK_M)

• Presence of [I], [S] appear to be less

• Having inhibitors, enz rxn needs higher [S] to reach half its maximum velocity

Competitive Inhibition: Lineweaver Burk

• Equation for a double-reciprocal plot in the presence of a competitive inhibitor is:

• lines intersect at the y-axis:

• Inhibitor has no effect on V{max}$but increases$KM

Uncompetitive Inhibition

• An uncompetitive inhibitor binds only to the enzyme–substrate complex.

• No bind free Enz

• Can't be overcome by Is

Uncompetitive Inhibition

• An uncompetitive inhibitor binds only to the enzyme-substrate complex.

Uncompetitive Inhibition: Mechanism

• Only binds to ES complex

• Does not affect substrate binding

• Inhibits catalytic function

• The dissociation constant of the inhibitor: $K_I’ = \frac{[ES][I]}{[ESI]}$

• An uncompetitive inhibitor bind at a site distinct from the active site, distorting the active site to make enzyme catalytical inactive

• No need for inhibitor to be structurally similar to substrate.

• Lowering $K_S$ stronger Inhibition

Uncompetitive Inhibition: Kinetic Parameters

• Enzyme-inhibitor-substrate complex doesn’t form product.

• The apparent $V<em>{max}$ ($V</em>{max}^{app} = \frac{V<em>{max}}{α’}$) is lower in the presence of inhibitor; $K</em>M$ ($K<em>{M}^{app} = \frac{K</em>M}{α’}$) is also lower.

• No change in $K<em>M/V</em>{max}$

• Inhibition cannot be overcome by sufficiently high [S]

  • Binding substrate => No interference with inhibitor

• $α’ = 1 + \frac{[I]}{K’_I}$

• $K_M$ Kind by factor

• Catalytic function of ESI enz affected

• $V_{max}$ by factor of

Uncompetitive Inhibition: Kinetic Curves

• Adding [S] does not reverse the uncompetitive inhibition affect

• [S] approaches infinity, Vo can no longer reach $V_{max}$ for any value of α

• Reduced k{cat}$(Note:$k{cat} = V{max}/[Enzyme]{total}

Uncompetitive Inhibition: Lineweaver Burk

• lines are parallel:

Noncompetitive Inhibition

• A noncompetitive inhibitor binds to either the free enzyme or the enzyme–substrate complex and does not prevent the substrate from binding to the enzyme

Mixed Inhibition (Noncompetitive)

• Noncompetitive inhibition: The inhibitor binds either the enzyme or enzyme-substrate complex.

Noncompetitive Inhibition: Mechanism

• Binds enzyme with or without substrate

  • Binds to regulatory site (distinct from the active site)

  • Inhibits both substrate binding and catalysis

• The dissociation constant of the inhibitor: KI = \frac{[E][I]}{[EI]} & KI’ = \frac{[ES][I]}{[ESI]}

• Ex metal ions Tithe and ES

Noncompetitive Inhibition: Mechanism

• Decrease in $V<em>{max}$ ($V</em>{max}^{app} = \frac{V_{max}}{α’}$

• $K<em>M$ can increase or decrease ($K</em>{M}^{app} = K_m(\frac{α}{α’})$) or remain unchanged for pure noncompetitive inhibition (where inhibitor binds equally well to E and ES so α = α’)

• Inhibition cannot be overcome by sufficiently high [S]

• Either bind prevent the formation of$K_{I prop}$

• $K_M$ to

Noncompetitive Inhibition: Kinetic Curves

• Adding [S] does not reverse the uncompetitive inhibition affect

• [S] approaches infinity, Vo can no longer reach Vmax for any value of α

• Reduced kcat

• KM may or may not change

• If depend on us is

Noncompetitive Inhibition: Lineweaver Burk

• lines intersect left from the y-axis:

Summary of Enzyme Inhibitor Effects

• Effects of Inhibitors on Michaelis-Menten Reactions

• Effects of Reversible Inhibitors on $V<em>{max}$ and Apparent $K</em>M$

Enzyme-Catalyzed Reaction

• If the red line represents the rate of an enzyme-catalyzed reaction in the presence of a competitive inhibitor, which line represents the activity of the same enzyme in the absence of the inhibitor?

Enzyme Inhibition Summary

• comp Inh No $V_{max}$

• Is will overcome inhibition

• Km THEY

• Kmt for comp inh US kind w o inhibitor