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Enzyme Inhibition: Mechanisms and Analysis, Study notes of Biochemistry

McMurry University (McM)Biochemistry

The molecular mechanisms of competitive and non-competitive enzyme inhibition, including how inhibitors bind to enzymes and the effects on steady-state kinetics. It also covers the analysis of inhibition and the determination of inhibition constants (ki and ki').

What you will learn

  • How can the inhibition constants (KI and KI') be determined?
  • How does the binding of an inhibitor affect the enzyme's active site?
  • What is the difference between competitive and non-competitive inhibition?

Typology: Study notes

2021/2022

Uploaded on 09/12/2022

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Biochemistry I Lecture 19 Oct 14, 2005
1
Lecture 19: Enzyme Inhibition & Analysis of Inhibition
Reading in Campbell: Chapter 6.7
19.1 Molecular Mechanisms of Inhibition:
A: Competitive Inhibition
1. Inhibitor binds to the same site on the enzyme as the substrate.
2. Inhibitor binds ONLY the free enzyme.
3. Inhibitor is usually structurally very similar to the substrate.
• For example, succinate is the
normal substrate for the enzyme
succinate dehydrogenase.
Malonate is an effective
competitive inhibitor of this
enzyme.
In another example, p-aminobenzoate (PABA) is the normal substrate for the bacterial enzyme
dihydrofolate synthetase. The sulfa drug sulfanilamide is an effective competitive inhibitor of this
enzyme. Because the conversion of p-aminobenzoate to folic acid by dihydrofolate synthetase is
essential for the survival of certain bacteria, the sulfa drug is an effective antibiotic.
In yet another example, methanol and ethanol compete for the same binding site in alcohol
dehydrogenase:
[Note: Ethanol is not a
competitive inhibitor in
the
classical
sense
because it is converted
to acetaldehyde
by the enzyme.]
O
HO
O
HO
O
HO
O
HO
Succinate
OH
Alcohol
Dehydrogenase
Alcohol
Dehydrogenase
O
O
O-
Methanol
Ethanol
OH
Formaldehyde
O
Acetaldehyde
Acetate
pf3
pf4

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Download Enzyme Inhibition: Mechanisms and Analysis and more Study notes Biochemistry in PDF only on Docsity!

Lecture 19: Enzyme Inhibition & Analysis of Inhibition

Reading in Campbell: Chapter 6. 19.1 Molecular Mechanisms of Inhibition: A: Competitive Inhibition

  1. Inhibitor binds to the same site on the enzyme as the substrate.
  2. Inhibitor binds ONLY the free enzyme.
  3. Inhibitor is usually structurally very similar to the substrate.
    • For example, succinate is the normal substrate for the enzyme succinate dehydrogenase. Malonate is an effective competitive inhibitor of this enzyme. In another example, p-aminobenzoate (PABA) is the normal substrate for the bacterial enzyme dihydrofolate synthetase. The sulfa drug sulfanilamide is an effective competitive inhibitor of this enzyme. Because the conversion of p-aminobenzoate to folic acid by dihydrofolate synthetase is essential for the survival of certain bacteria, the sulfa drug is an effective antibiotic. In yet another example, methanol and ethanol compete for the same binding site in alcohol dehydrogenase: [Note: Ethanol is not a competitive inhibitor in the classical sense because it is converted to acetaldehyde by the enzyme.] HO O HO^ O HO O O HO Succinate Malonate OH Alcohol Dehydrogenase Alcohol Dehydrogenase O

O

O-

Methanol Ethanol

OH

Formaldehyde

O

Acetaldehyde (^) Acetate

A competitive inhibitor reduces the amount of [E] by the formation of an [EI] complex. The inhibitor cannot affect the [ES] complex after it has formed since the inhibitor can no longer bind. There are two anticipated consequences of this binding mode on the steady-state kinetics:

  1. Vmax is unchanged: At high levels of substrate all of the inhibitor can be displaced by substrate.

2. The apparent Km is increased: It requires more substrate to reach 1/2 maximal velocity

because some of the enzyme is complexed with inhibitor. B: Non-competitive Inhibition: In this case the inhibitor binds to both [E] and [ES]. The binding site of the inhibitor is not at the active site. However, the inhibitor binding causes a change in the conformation of the protein that affects either substrate binding (Km), the chemical step (Kcat), or both. Both Vmax and Km can be altered by non-competitive inhibitors since the precise geometry of the active site is altered when the inhibitor is bound.

E + S ES E + P

EI

Kcat

ESI

K 1

K-

K I K I’

E + S ES E + P

EI

K 1 Kcat

K-

K I

1/vo

1/[S]

- inh

+ inh

1/vo

1/[S]

- inh

+ inh

Obtaining KI and KI’ The values of a and a’ can be easily found from the slope and intercept of double reciprocal plots. The five easy steps are:

  1. Obtain v versus [S] in the absence of the inhibitor.
  2. Obtain v versus [S] in the presence of a fixed and known concentration of inhibitor.
  3. Plot both data sets on a double reciprocal plot.
  4. a = ratio of the slopes.
  5. a’ = ratio of y-intercepts. Example: [I]=10 μM Obtain a = slope (+inh) / slope (-inh): Slope (+inh): Slope (-inh): a = Obtain KI:

KI =

[ I ]

( a - 1 )

Obtain a ’ = y-int (+inh) / y-int (-inh): Summary of Kinetic Parameters: Parameter Information content Vmax Information on Kcat – fundamental mechanism Km Information on how tight the substrate binds (but not quite its KD) KI Information on how tight the inhibitor binds to the free enzyme [E] (this is a KD) KI’ Information on how tight the inhibitor binds to the [ES] complex (this is also a KD)

[S]

mM v ([I]=0) v ([I]= 1/[S] 1/v ([I]=0) 1/v ([I=10]) 1 16.67 6.25 1.00 0.0600 0. 5 50.00 25.00 0.20 0.0200 0. 20 80.00 57.14 0.05 0.0125 0.