10/18/23

ENZYMES: Binding & Catalysis

A. Binding • Reading: Ch6; 191, 197-200 Lecture 15
•
B.
C.
Catalysis
Nomenclature
NEXT
Homework #15

• Reading: Ch6; 181-186, 195-196

(10/18/23)
D. Catalysis • Homework #16 a. [S] = Km
b. [S] >> Km
1. Transition State Theory
c. [S] << Km
2. Catalytic strategies (What)
7. Collection and manipulation of data
3. Mechanistic strategies (How) a. Lineweaver-Burk; double reciprocal;
E. Quantifying the Catalytic Power: 1/v0 vs. 1/[S]
Kinetics b. Eadie-Hofstee; v0 vs. v0/[S]
1. Review c. Hanes-Woolf; [S]/v0 vs. 1/[S]
2. Enzyme Kinetics 8. Inhibition
3. Rate vs. [S] for enzyme catalyzed a. Irreversible: protein modification
reaction; initial rate (!0) b. Reversible
i. Competitive; like substrate; Km affected
4. ES complex by (1 + [I]/KI) = a
a. Reaction ii. Uncompetitive; binds only ES; both Km
i. Binding reaction and Vmax affected in opposite ways
ii. Catalytic reaction iii. Noncompetitive; binds both E & ES
b. Meaning of rate curve: hyperbolic (mixed, non-equal binding); Vmax
curve affected
iv. Mixed inhibition if I binds E differently
5. Rate expression; Michaelis-Menten than it binds ES
Kinetics (M-M) v. Using Steady-state kinetics: Active
a. Assumptions
Site
b. M-M equation derivation
6. Meaning of rate expression (M-M eqn) vi. Energetics of Catalysis

Enzyme Kinetics
Determination of Kinetic Parameters

Slopes = v0 (rates)

A nonlinear Michaelis-Menten plot could be

used to calculate parameters Km and Vmax.
Lineweaver-Burk derived a linear form of the M-M equation
by taking the reciprocal of both sides. This is called the
linearized double-reciprocal plot. Its good for analysis of
enzyme kinetic data to get these kinetic parameters.

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Enzyme Kinetics
Lineweaver-Burk Plot:
Linearized, Double-Reciprocal
The Michaelis-Menten equation can be manipulated
into one that yields a straight-line plot.

Vmax [S] y = bx + a
!0 =
Km + [S] y=
1
x= 1
!0 [S]
1 K + [S]
!0 = m
Vmax [S]
This double-reciprocal
1 equation is called the
Km [S]
!0 = + Lineweaver-Burk
Vmax [S] Vmax [S] equation.

1 Km 1
!0 = +
Vmax [S] Vmax

Enzyme Kinetics
Other Linearized Derivations of the M-M Equation
Hanes-Woolf Eadie-Hofstee
!0 = Vmax – Km !0
[S]

[S] vs. [S]

!0 !0 vs. !0 [S]
[S]/v0
Vmax

v0
–Km

Vmax/Km
[S] v0/[S]

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Enzyme Kinetics
Linearized Derivations of the M-M Equation
Lineweaver-Burk Hanes-Woolf Eadie-Hofstee
!0 = Vmax – Km !0
[S]

[S] vs. [S]

!0 vs. !0 [S]
!0
[S]/v0
Vmax

v0
–Km

Vmax/Km
[S] v0/[S]

Vmax [S]
!0 =
Km + [S]

Enzyme Kinetics

ENZYME
INHIBITION

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Enzyme Kinetics

What is Enzyme Inhibition?

This is the action of a small molecule that results in
loss of enzyme activity

This is not regulation by the action of another enzyme

or protein
This is not loss of enzyme activity due to
denaturation/unfolding of the enzyme.

Two major kinds of inhibition

1) Irreversible
2) Reversible

Enzyme Kinetics
Reversible Enzyme Inhibition:
There are THREE-to-FOUR types of
reversible inhibition:

1) Competitive
2) Un-Competitive
3) Non-Competitive
These are closely related
4) Mixed

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Enzyme Kinetics
Reversible Enzyme Inhibition:
Km (Binding) Vmax (Catalysis)

k1 k2
E+S⇌
k
ES ⇀ E + P
-1

No Inhibition

Enzyme Kinetics
Reversible Enzyme Inhibition:
Km (Binding) Vmax (Catalysis)

k1 k2
E+S⇌
k
ES ⇀ E + P
+
-1

I
⇌

E•I
Competitive

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Enzyme Kinetics
Reversible Enzyme Inhibition:
Km (Binding) Vmax (Catalysis)

k1 k2
E+S⇌
k
ES ⇀ E + P
+
-1

⇌
ES•I
Un-Competitive

Enzyme Kinetics
Reversible Enzyme Inhibition:
Km (Binding) Vmax (Catalysis)

k1 k2
E+S⇌
k
ES ⇀ E + P
+ +
-1

I I
⇌

E•I ES•I

Non-Competitive

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Enzyme Kinetics
Reversible Enzyme Inhibition:
Km (Binding) Vmax (Catalysis)

k1 k2
E+S⇌
k
ES ⇀ E + P
-1

+ +
I I
⥄
⇌

⇌
⥄
E•I ES•I

Mixed

Enzyme Kinetics
Reversible Enzyme Inhibition:
There are THREE-to-FOUR types of
reversible inhibition:

1) Competitive
2) Un-Competitive
3) Non-Competitive
These are closely related
4) Mixed

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Competitive Inhibition
•Competes with substrate for binding
–easiest to remember
–binds active site
–does not affect catalysis (e.g., once ES is formed, catalysis occurs)
How does this inhibition affect the rate expression?
It pulls on the binding reaction (competing with
S for free E)

Which kinetic constant will be affected?

Km becomes an “Apparent” Km (in the
presence of inhibitor): AppKm
Vmax [S]
!0 =
Km + [S]
⇓
Vmax [S]
!0 =
Km (1+ [KII] ) + [S]
Derivation on line

Competitive Inhibition (1+ [KII] ) = a

[E][I] Vmax [S]

KI =
[EI]
!0 =
Km (1+ [KII] ) + [S]

Vmax [S]
!0 =
Km (1+ [KI ] ) + [S]
I

Km App
Km

•No change in Vmax; apparent increase in Km

•Lineweaver-Burk: lines intersect at the y-axis.

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Uncompetitive Inhibition
• Only binds to ES complex; AFTER S
– The binding of S causes a conformational change and creates the site
for inhibitor
– affects substrate binding by pulling binding equilibrium (makes it look
better!)
– affects catalytic function by pulling catalysis equilibrium (depleting [ES])

How does this inhibition affect the rate expression?

Affects both Binding and Catalysis, but in
opposite ways

Which kinetic constant will be

affected?
Apparent Km gets smaller
Apparent Vmax gets smaller
Vmax[S] (1+ [K’I ]I )
!0 =
Km (1+ [K’I ]I ) + [S]
⇓ V [S]
max
!0 =
Km + [S] (1+ [K’I ]I )

Uncompetitive Inhibition (1+ [K’I ]I ) = a’

K’I =
[ES][I] Vmax[S] a’
[ESI] !0 = [I]
Km + [S] ( 1+
KI )
a’
Vmax a’
Vmax (1+ [ I ] )
K I a’
App
Apparent Maximal Velocity Vmax

a’
a’

AppK
m Km

Km (1+ [KI ] )
I

• Decrease in Vmax & decrease in Km (but to same extent!)

• No change in Vmax/Km
• Lineweaver-Burk: lines are parallel (recall slope is 1/Vmax/Km)

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Non-competitive Inhibition
• Binds BOTH free enzyme (E) and enzyme bound to substrate
(ES)
― binds to a entirely different site from the active site
(regulatory/inhibitory site)
― Essentially just titrates out the [enzyme]
― inhibits both substrate binding and catalysis equally (rare)
Non-competitive inhibition
How does this inhibition affect the rate expression?
Affects Catalysis; Binding is affected but to the
same degree in opposite ways, so it cancels.

Which kinetic constant will be

affected?
Apparent Km stays the same as
its pulled and pushed the
same.
Apparent Vmax gets smaller due
to loss of [E]T:
([E]+[ES]+[EI]+[ESI]).

Non-competitive Inhibition
Non-competitive inhibition
Vmax [S]
!0 =
Km + [S]

Vmax [S] (1+ [ I ] )

KI
!0 =
Km + [S]

Vmax [S]
[E]K =
[E][I] !0 =
I
[EI] (Km + [S]) •(1+ [ KI I] )
[ES]K =
[ES][I]
I
[ESI]

[E]K = [ES]KI
I

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Non-competitive Inhibition (1+ [KII] ) = a

[E][I] Vmax [S]
[E] KI =
[EI] [E]
KI = [ES]KI !0 =
[ES]K =
[ES][I] (Km + [S])•(1+ [ KI I] )
I
[ESI]

App
Apparent Maximal Velocity Vmax

Vmax (1+ [ I ] )
K I

Km

•Decrease in Vmax; no change in Km

–pulls binding reaction to both directions equally
–the effect is to effectively decrease the [E]
•Lineweaver-Burk: lines intersect on the x-axis.

Enzyme Kinetics
Reversible Enzyme Inhibition:
There are THREE-to-FOUR types of
reversible inhibition:

1) Competitive
2) Un-Competitive
3) Non-Competitive
These are closely related
4) Mixed

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Mixed Inhibition
• Variation of Non-competitive*
– binds to regulatory/inhibitory site differently Vmax [S]
in free enzyme (E) versus enzyme- !0 =
substrate complex (ES) Km + [S]

Vmax [S] (1+ [ I ] )

K’I
!0 =
(1+ [KII] ) Km (1+ [ I ] ) + [S]
K’I

KI KI’ Vmax [S]

!0 =
Km (1+ [KII] ) + [S] ( 1+ [K’I ]I )

[E][I]
[E]KI =
[EI] [E]K ≠ [ES]KI
I
[ES]K =[ES][I]
I
[ESI]

*Noncompetitive inhibitors are mixed inhibitors such that there is no change in Km.

(1+ [KII] ) = a
Mixed Inhibition (1+ [K’I ]I ) = a’
[E][I]
KI =
[E]
[EI]
Vmax [S]
[ES][I]
[E]K
I ≠ [ES]KI !0 =
[ES]K =
I
[ESI]
Km (1+ [KII] ) + [S] ( 1+ [K’I ]I )

App
Apparent Maximal Velocity Vmax

Km (1+ [KI ] )I
Vmax
( 1+ [K’I ] ) ( 1+ [K’I ] )
I
I

Km App
Km

• Decrease in Vmax; change in Km

• Lineweaver-Burk: lines intersect left from the y-axis.

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SUMMARY: Reversible Inhibition = got worse = got better

App App
Competitive Km Vmax
Km (Binding) Vmax (Catalysis)

k1 k2
E+S ES E+P Km (1+ [KI ] ) I
Vmax
k-1
+ +
I I Un-Competitive

E•I ES•I Km Vmax

( 1+ [K’I ] )I
( 1+ [K’I ] )
I

Non-Competitive
(1+
[I]
KI )=a
Km Vmax
(1+ [K’I ]I ) = a’ ( 1+ [K’I ] )
I

Mixed

Km (1+ [KI ] ) I
Vmax
( 1+ [K’I ] )
I
( 1+ [K’I ] )
I

Enzymes

ACTIVE
SUMMARY SO FAR: SITE
We have described enzymes in general terms such as:
catalytic cycle
binding, even stereo-specific binding
catalysis, turnover number & proficiency
nomenclature
transition state theory
catalytic strategies (what to do)
mechanistic strategies (how to do)
enyzme kinetics and inhibition

ALL of this happens at the ACTIVE SITE

Now, we want to ask what all happens here & how do we determine what happens?

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Enzymes
How do your determine what is
going on at the active site?
We will discuss FOUR methods for study of the active site

1. Enzyme kinetics
2. pH studies
3. Protein modification
4. Structural studies

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