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Lecture 16. Inhibition Quiz on Friday, Oct. 14 (MM derivation (assume no E+P -> ES) and 6 enzyme classes-what types of reactions do they catalyze?). X. Y. X. Y. X. Y. Enzymes. Oxidoreductases:A - + B  A + B - Transferases:A-B + C  A + B-C Hydrolase:A-B + H 2 O  A-H + B-OH

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Lecture 16

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Lecture 16 l.jpg

Lecture 16

  • Inhibition

  • Quiz on Friday, Oct. 14 (MM derivation (assume no E+P -> ES) and 6 enzyme classes-what types of reactions do they catalyze?)


Enzymes l.jpg

X

Y

X

Y

X

Y

Enzymes

Oxidoreductases:A- + B  A + B-

Transferases:A-B + C  A + B-C

Hydrolase:A-B + H2O  A-H + B-OH

Lyases:A-B  A=B + X-Y

Isomerases:A-B  A-B

Ligases (synthases)A + B  A-B

  • The 6 enzyme classes can be illustrated by the general reactions catalyzed


Enzyme reactions can be slowed by the presence of inhibitors l.jpg

Enzyme reactions can be slowed by the presence of inhibitors

  • Other inhibitors bind noncovalently and reversibly to their target enzymes.

  • These are usually divided into three broad classes, competitive, noncompetitive, and uncompetitive, depending on their manner of binding.

  • Kinetic analysis can distinguish among these inhibitors if the reaction rate is measured against substrate concentration at different inhibitor concentrations.


Figure u2 4 1 competitive noncompetitive and uncompetitive inhibition l.jpg

Figure U2-4.1 Competitive, noncompetitive and uncompetitive inhibition


Figure u2 4 1a competitive inhibition l.jpg

E

Figure U2-4.1a Competitive inhibition

ES

(a) A competitive inhibitor (I) binds to the same site as does the substrate (S; top).

The inhibitor changes the apparent Km for the reaction, but not the Vmax because enough substrate can keep any inhibitor from binding.

A Lineweaver-Burk plot (bottom) for the reaction at various concentrations of the inhibitor reflects this behavior.

EI


Slide6 l.jpg

v0 = Vmax[S]

KM +[S] +[I] (KM/KI)

KI = [E][I]

[EI]

Competitive Inhibition

ES

P

S + E

+ I

EI

Vmax

w/o I

w/ I

v (µmol/min)

[S]


Slide7 l.jpg

v0 = Vmax[S]

KM +[S] +[I] (KM/KI)

KM

1/V0 =

1

1

(1+ ) +

[I]

Vmax

Vmax

[S]

KI

KM

1/V0 =

1

() +

1

Vmax

[S]

Vmax

I

w/o I

1/v0

-1/KM

1/Vmax

-1

-1

1/[S]

[I]

KM

KM

(1+ )

KI

Competitive Inhibition

ES

P

S + E

+ I

EI

-1/KM’ or apparent KM


Competitive inhibition l.jpg

Competitive inhibition

  •  = 1 +[I]/KI

  • As [I] increases, v decreases (1/v increases)

  • As [I] increases, KM decreases (1/KM increases)

  • Vmax is the same and the inhibition can be overcome by high [S]


Figure u2 4 1b noncompetitive inhibition l.jpg

Figure U2-4.1b Noncompetitive inhibition

E

ES

(b) A noncompetitive inhibitor (I) (green) does not bind to the substrate binding site and can bind to both the free enzyme or the ES complex.

Usually a noncompetitive inhibitor resembles one substrate (S2) in a two-substrate reaction, as shown here, where both substrates are present on the enzyme at the same time.

In the simplest cases, noncompetitive inhibitors don't change the Km for the first substrate (S), because they don't affect its binding.

But providing the concentration of S2 is not high enough to out-compete all the inhibitor, the inhibitor does reduce the Vmax for the reaction.

EI


Slide10 l.jpg

ES

P

S + E

KI’

+ I

KI

+ I

EIS

EI + S

KI = [E][I]

KI’ = [ES][I]

[EI]

[ESI]

Noncompetitive Inhibition (Mixed Inhibition)

v0 = Vmax[S]

KM +’[S]

 = 1 +[I]/KI

’ = 1 +[I]/KI’

Vmax

w/o I

v (µmol/min)

w/ I

[S]


Slide11 l.jpg

ES

P

S + E

KI’

+ I

KI

+ I

EIS

EI + S

KM

1/V0 =

’

1

() +

Vmax

[S]

Vmax

I

w/o I

1/v0

-1/KM

1/Vmax

1/[S]

Noncompetitive Inhibition (Mixed Inhibition)

v0 = Vmax[S]

KM +’[S]


Noncompetitive inhibition l.jpg

Noncompetitive inhibition

  •  = 1 +[I]/KI, ’ = 1 +[I]/KI’

  • As [I] increases, v decreases (1/v increases)

  • As [I] increases, KM decreases (1/KM increases), but in the simplest case does not change (very slight).

  • Cannot be overcome by increasing [S]

  • May intersect above, blow or even on the line (x or y axis)

    • x-axis - no affect on KM (KM = KM’)

    • y-axis - no affect on Vmax


Figure u2 4 1c uncompetitive inhibition l.jpg

Figure U2-4.1c Uncompetitive inhibition

(c) An uncompetitive inhibitor (blue) binds only to the enzyme-substrate (ES) complex and slows down the reaction probably by inducing a conformational change in the enzyme.

Both the apparent Km and Vmax are affected proportionally by such an inhibitor, leading to parallel Lineweaver-Burk plots for different inhibitor concentrations.


Slide14 l.jpg

KI’ = [ES][I]

[ESI]

KM

1/V0 =

’

1

+

Vmax

[S]

Vmax

I

Uncompetitive Inhibition

ES

P

S + E

v0 = Vmax[S]

KI’

+ I

KM +’[S]

EIS

’ = 1 +[I]/KI’

w/o I

1/v0

1/Vmax

1/[S]


Uncompetitive inhibition l.jpg

Uncompetitive inhibition

  • ’ = 1 +[I]/KI’

  • Reacts only with ES complex

  • As [I] increases, v decreases (1/v increases)

  • As [I] increases, apparent KM increases (apparent 1/KM decreases), but there is no effect on binding of E to S.

  • Cannot be overcome by increasing [S]

  • Relatively rare in single substrate reactions but can be more common in complex cases.


Enzyme reactions can be slowed by the presence of inhibitors16 l.jpg

Enzyme reactions can be slowed by the presence of inhibitors

  • A key parameter that can be obtained from such an analysis is the affinity of the inhibitor for the enzyme, the inhibition constant Ki.

  • By convention, Ki is given as the dissociation constant for the enzyme-inhibitor equilibrium:

Ki’ = [ES][I]

Ki= [E][I]

[ESI]

[EI]

  • The lower the value of Ki the tighter the inhibitor binds. In pharmacology, the value of Ki is often used as a measure of the effectiveness of a drug.

  • A compound with a very low Ki, say 10-9 M (nanomolar) or less, can be given at very low doses and will still be able to bind its target.


Slide17 l.jpg

Enzyme Inhibitor Classification

  • Competitive inhibitors:

  • Most common class of reversible inhibitor consists of compounds that resemble the substrate.

  • Such molecules can fit into the substrate binding site, thereby blocking access from substrate molecules. These inhibitors compete with the substrate for the active site.

  • Example: Many HIV protease inhibitors that have proven to be effective in treatment of AIDS are competitive inhibitors that were designed to resemble the peptide substrate of the HIV protease.


Slide18 l.jpg

Enzyme Inhibitor Classification

  • Not all inhibitors compete with the substrate for the active site of the enzyme; other inhibitors bind to a separate site.

  • Noncompetitive inhibitors bind to both the free enzyme and the enzyme-substrate complex.

  • If an enzyme has two substrates that must bind simultaneously for the reaction to occur, an inhibitor might compete with one substrate but not the other.


Slide19 l.jpg

Enzyme Inhibitor Classification

  • Not all inhibitors compete with the substrate for the active site of the enzyme; other inhibitors bind to a separate site.

  • Uncompetitive inhibitors bind only to the enzyme-substrate complex.

  • In effect, the inhibitor reduces the amount of ES that can go on to form product.

  • Lineweaver-Burk plots characteristically show a series of parallel lines (Fig. U2-4.1c).

  • Uncompetitive inhibitors often stabilize an alternative conformation of the protein, and in this case they are called allosteric inhibitors.

  • There are also allosteric activators: molecules that activate enzymes by stabilizing a conformation of the enzyme that is more active than the conformation that exists in their absence.


Slide20 l.jpg

Figure 3-10 Ligand-induced conformational change activates aspartate transcarbamoylase

Binding of the allosteric activator ATP to its intersubunit binding sites on the regulatory subunits (that between R1, outlined in purple, and R6 is arrowed) of the T state of ATCase (top) causes a massive conformational change of the enzyme to the R state (bottom).

In this state the structure of the enzyme is opened up, making the active sites on the catalytic subunits (C) accessible to substrate.

Al and Zn in the lower diagram indicate the allosteric regions and the zinc-binding region, respectively; cp and asp indicate the binding sites for the substrates carbamoyl phosphate and aspartate, respectively.

The red and yellow regions are the intersubunit interfaces that are disrupted by this allosteric transition.


Allostery l.jpg

Allostery

  • Does not follow Michalis-Menton kinetics!

  • EIS goes to products at the same rate as ES but with lower affinity for [S]

  • Doesn’t affect Vmax.

  • Does affect KM

  • Can operate in both directions and involves a second binding site.

  • Positive-activator, cooperative

  • Negative-inhibitor, antagonistic

  • Enzyme controlled by binding at second site

    homotrophic (modifier is related to the substrate)

    heterotrophic (related to substrate).


Enzyme catalyzed reactions can have multiple steps with several intermediates l.jpg

Enzyme-catalyzed reactions can have multiple steps with several intermediates

  • Multi-step reactions can have very complicated kinetics, e.g., “double-displacement” or “ping-pong” enzymes.

  • These are enzymes that use two or more substrates but catalyze reactions that are strictly ordered in the sequence in which substrates bind and products are released (Figure U2-3.3a).

  • Lineweaver-Burk plots of the velocity against one substrate concentration at a series of fixed concentrations of other substrate give a family of parallel lines (Fig. U2-3.3b).

Insert: double reciprocal plot of observed initial

velocities versus CO2 concentration for CA at different concentrations of

TAPS buffer: 5 mM, 10 mM, 20 mM, 50 mM.

Biochemistry 1999, 38, 13119-13128


Enzyme catalyzed reactions can have multiple steps with several intermediates23 l.jpg

Enzyme-catalyzed reactions can have multiple steps with several intermediates

Example of Ping-Pong Enzyme: Aspartate aminotransferase: Catalyzes the conversion of aspartate to glutamate with production of oxaloacetate and consumption of alpha-ketoglutarate.

The reaction sequence starts with the binding of aspartate to the enzyme followed by its conversion to oxaloacetate, in the process of which the aspartate leaves behind its amino group bound to a cofactor in the active site.

After oxaloacetate departs (the so-called ping step), alpha-ketoglutarate reacts with the amino group and is converted to glutamate (the so-called pong step), bringing the enzyme back to its original state.

The two substrates, aspartate and alpha-ketoglutarate, never encounter each other on the enzyme. The kinetics are characteristically simple for this kind of reaction:

Lineweaver-Burk plots of the velocity against aspartate concentration at a series of fixed concentrations of alpha-ketoglutarate, give a family of parallel lines (Fig. U2-3.3b).


Figure u2 3 3 ping pong or double displacement kinetic behavior l.jpg

Figure U2-3.3 Ping-pong or double-displacement kinetic behavior

  • (a) In ping-pong reactions, two substrates bind sequentially to an enzyme. In this example, a chemical group (green) is transferred from substrate A (red) to substrate B (blue).

  • (b) A LineweaverBurk plot of 1/v against 1/[substrate A] at various fixed concentrations of substrate B shows a set of parallel lines which are diagnostic for the ping-pong reaction mechanism.


Enzyme reactions can be slowed by the presence of inhibitors25 l.jpg

Enzyme reactions can be slowed by the presence of inhibitors

  • The rate of an enzyme-catalyzed reaction can be affected by molecules that do not themselves participate in the chemical reaction.

  • Activators increase the reaction rate and inhibitors decrease the rate.

  • Many drugs, including aspirin, penicillin, statins and Viagra are enzyme inhibitors:

  • they achieve their pharmacological effects by reducing the rate of a key enzyme-catalyzed reaction.

  • In some cases the effect is achieved by forming a dead-end covalent complex between the inhibitor and enzyme.

  • Penicillin and aspirin work this way: they form stable chemical bonds with residues in the active sites of the enzymes they inhibit.

  • Such “suicide inhibitors” permanently inactivate their target enzyme molecules, and cells can only overcome their effects by synthesizing fresh enzyme.


Figure u2 3 4 effect of temperature on reaction rate l.jpg

Figure U2-3.4 Effect of temperature on reaction rate

Reaction rates depend on collisions between reacting species, which in turn depend on concentrations and temperature.

The temperature dependence of a reaction thus relates a thermodynamic quantity (the free energy of the transition state) to kinetics (the rate of the reaction). The equation that expresses this relationship is called the Arrhenius Equation.

As the temperature increases, the rate of an enzyme-catalyzed reactionincreasesuntil the protein unfoldsand the rate thenrapidly drops.

Predicts a two-fold increase in rate for every 10 °C rise in temperature


How do kinetics relate to biochemistry l.jpg

How do kinetics relate to biochemistry?

  • Classic collision theory - to increase the rate of P

    • Increase the concentration of A or B

    • Increase the temp.

    • Decrease the concentration of the products

  • Catalysis is a process that increases the rate at which a reaction approaches equilibrium.


Proximity and orientation l.jpg

Proximity and Orientation

  • By simply positioning 2 molecules in such a way that they are close together, the probability of reaction is increased.

  • Example is anhydride formation with different degrees of rotational freedom.

  • By limiting motions, intramolecular reactions are greatly increased


Proximity and orientation29 l.jpg

Proximity and Orientation

  • Probably the least important by itself

  • Uses binding energy to position correctly.

  • Koshland figures only 2-3-fold increase, but can see increases up to 107.


Strain or distortion l.jpg

CO2-

CO2-

H

NH

NH

H

H

N

H

N+

NH

NH

CO2-

CO2-

CO2-

CO2-

Strain or Distortion

  • Binding of substrate or conformational change in enzyme may induce strain in bond.

  • This lowers the required energy to reach EA.

  • Stabilize or force bonds closer to the transition state.

    Ex. Proline racemase

sp2 planar

D-pro

L-pro

+ H+

- H

If we look at the binding of inhibitors to active site:

>>


Strain or distortion31 l.jpg

Vitamin B1

O

-O

CH3

O

TPP

S

S

R’

R’

pyruvate

R

+N

HO

O

C

C

-O

CH3

Strain or Distortion

  • Model reaction - Vitamin B1 (thiamine)

  • Part of thiamine pyrophosphate (TPP)

  • Mechanism for pyruvate decarboxylase

TPP

R

+N

CH3

-


Strain or distortion32 l.jpg

R

HO

O

N

C

C

O

CH3

S

S

S

R’

R’

R’

R

HO

O

N

II

+

C

C

CH3

O

R

+N

HO

Strain or Distortion

O

  • Compound I is stable in H2O, so decarboxylation is slow.

  • Compound II is stable in DMSO, so decarboxylation is fast

  • Pyruvate decarboxylase puts TPP in very hydrophobic region of enzyme.

I

C

C

-O

CH3


General acid base catalysis l.jpg

General Acid-Base Catalysis

  • General acid catalysis- a process in which partial proton transfer from a Brønstead acid (a species that can donate protons) lowers the free energy of a reaction’s transition state.

  • General base catalysis - process in which partial proton abstraction by a Brønstead base (a species that can combine with a proton) lowers the free energy of a reaction’s transition state.

  • General acid-base catalysis-a combination of both.


Figure 15 1a mechanisms of keto enol tautomerization a uncatalyzed l.jpg

Figure 15-1aMechanisms of keto–enol tautomerization.(a) Uncatalyzed.

Page 497


Figure 15 1b mechanisms of keto enol tautomerization b general acid catalyzed l.jpg

Figure 15-1bMechanisms of keto–enol tautomerization.(b) General acid catalyzed.

Page 497


Figure 15 1c mechanisms of keto enol tautomerization c general base catalyzed l.jpg

Figure 15-1cMechanisms of keto–enol tautomerization.(c) General base catalyzed.

Page 497


General acid base catalysis37 l.jpg

H

H

d

O

O

O

+ H+

H2O

OR

C

OR

C

OR

C

d

d

+

O

H

H

H

H

O

O

- H+

OH

C

OR

C

H+

+ ROH

O

General Acid Base Catalysis

  • Ex. Ester hydrolysis

+


General acid base catalysis38 l.jpg

General Acid-Base Catalysis

  • Large number of possible amino acids

    • Requires that they can accept and donate a proton

  • Glu, Asp

  • Lys, His, Arg

  • Cys, Ser, Thr

  • Also can include metal cofactors

  • Example can be observed in carboxypeptidase A (both acid and base catalysis)


General acid base catalysis39 l.jpg

R

CO2-

H-C

H

N

d

d

C

O

H-C-R

O

NH

Glu270

C-O-

C

O

General Acid-Base Catalysis

  • Ex. Carboxypeptidase A

Zn plays role of acid (4th ligand is normally H2O, but it is displaced by peptide binding)

+ Arg145

Glu72

Key aas that holds molecule in place

His196

Zn++

HO-Tyr248

His69

Tyr also plays role as 2nd acid catalyst

O

H

d

d

H

+ Arg

Glu acts as base catalyst to polarize water and form nucleophile


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