LSM1101 Lecture 2 Enzyme Kinetics / Enzyme inhibition

Dr Deng Lih Wen Dept of Biochemistry

Quick review
T

Free energy, G

Free energy of activation (activation energy)

G
A+B P+Q

Greaction

Gibbs Free Energy G = H TS (H: enthalpy; S: entropy). Greaction = Gproducts - Greactants

Enzyme Alter Only the Reaction RATE But Not the Reaction Equilibrium
A P
Keq =
[ P] [ A] = 100

The equilibrium concentration of P is 100 times of A, whether or not the enzyme is present. It may takes much longer to approach this equilibrium without enzymes The Equilibrium position is a function only of Greaction The greater the activation energy, the slower the reaction Higher temperature, higher reaction rate (but typically enzymes are denatured above 50 C) 1
Reaction rate
e -G /RT

Greaction= - RT ln Keq

R: gas constant; T: absolute temperature

e G /RT

Rate-determining Step
A ) I P
intermediate; stable chemical structure which can be isolated

In a multi-step reaction, the step with the highest activation energy is the rate-determining step The overall rate of the reaction can only be as fast as the slowest step

Transition state diagram for a two-step reaction

Definition
Kinetics : Study the rate of change of reactants and products from the initial to final state Velocity : change of concentration of product/substrate per unit time initial velocity (v )

S
d [P] v = dt
or

P
d [S] v = - dt

[P]

Time (sec)

Rate law: the mathematical relationship between reaction rate and concentration of reactants. k is the proportionality constant, or rate constant v=k[A]

Chemical Kinetics
A P A I 1 I 2 P

d [P] d [A] First-order reaction v = = - = k [ A ] Unimolecular, k (s-1) dt dt Rate Law

d [A] d [P] 2A P v = - = =k [ A ] 2 dt dt d [A] d [B] d [Q] d [P] A + B P + Q v = - = - = = = k [ A ] [ B ] dt dt dt dt

Second-order reaction bimolecular, k (M-1 s-1)

Third-order reaction is rarely found. Simultaneous collision of three molecules is very, very small.

Sample Calculation 1
Determine the velocity of the elementary reaction A+ B C when the sample contains 5M A and 3M B and k for the reaction is 500 M-1 s-1 Make sure all units are consistent when you calculate.
v = k [A ][B] = (500M -1 s -1 )(5M)(3M) = (500M -1 s -1 )(5 10 6 M)(3 10 6 M) = 7.5 10 9 M s -1 = 7.5nM s -1
For short assay questions, please provide the answer with units.

Enzyme-catalyzed reaction
Chemical reaction Enzyme-catalyzed reaction

Low [S], vo is linear to [S] Rectangular hyperbola curve As [S] is increased, vo approaches the limiting value Vmax At high [S], vo is independent of [S]. The velocity is limited only by conditions (Temp, pH) and by the amount of enzyme present.

How to Determine VelocitySubstrate curve by experiments?

Set up a series of tubes containing graded concentrations of [S] * only one substrate is varied while the concentration of other substrates are held constant. Obtain the initial velocity Vo for each reaction.

[P]

time

Progression Curves for a Simple Enzyme-Catalyzed Reaction

[S0]>>[E]

The slopes for [E] and [ES] are essentially zero

Steady State
E+S
k1 k-1

ES

k2

E+P

The velocity of the back reaction (E + P ES) is negligible when we observe only the initial velocity for the reaction

At steady state of enzyme reaction, the concentration of the enzyme-substrate complex ES quickly reaches a constant value. The rate of formation of ES is equal to the breakdown of ES. [ES] remains approximately constant.

d [ES] =0 dt

Michaelis-Menten Equation
E+S

k1 k-1

V max [S] V0 = KM + [S]

d [P ] V0 = = k 2 [ES] dt
V 0 = k2

ES

k2

E+P

Steady state

Formation rate of ES equals to breakdown rate of ES

k 1 [E ][S ] = k

[ES ] + k 2 [ES ]
k1([E ]T [ES]) [S] k 1[ES]+ k 2[ES] = (k1)[ES] (k1)[ES]

Letting [E ] = [E ]T [ES] and rearranging yields

[E]T [S] KM + [S]

k 1 + k 2 ([E]T [ES ]) [S ] = [ES] k1

KM (Michaelis constant)

Maximal velocity of a reaction, Vmax occurs at high substrate concentration when enzyme is saturated or when it is entirely in the ES form

Vmax = k 2[E ]T

K M [ES
[ES ] = [E ] T [S ] (K M + [S ])

( [E ] T [ES ] ) [S ]
Solving for [ES]

K M [ES ] = [E ] T [S ] [ES ][S ] [ES ](K M + [S ]) = [E ] T [S ]

[ES] = [E ]T [S] KM + [S]

V max [S] V0 = KM + [S]

Sample Calculation 2
When S = Km or 3 Km, what would the Vo be?

V max [S] V0 = KM + [S]

Vmax Km When S = Km, V0 = Km + Km Vmax Km Vmax = = 2 Km 2

Vmax 3Km When S = 3Km, V0 = Km + 3Km

Vmax 3Km 3 Vmax = = 4 Km 4

Plots of v Versus [S] illustrate the relationships between Vmax, KM and reaction orders

vo

Vmax

Vmax/2

V0=

V max [S ] K M + [S ]
[S] = KM [S] >> KM
V0 V
max

Km

[S]

[S] < KM
V0 V
max

Vmax: Theoretical value, occurs at very high [S]; when enzyme is saturated with substrate Km: the substrate concentration at which the reaction velocity is half-maximal.

KM

[S ]

First order

V0 =V

Zero order
max

/2

Disadvantage of MichaelisMenton Plot

Difficult to assess Vmax and Km Large [S] is required

Vo

???

[S]

Double-Reciprocal Plot (Lineweaver-Burk Plot)

V max [S] V0 = KM + [S]
1 KM + [S] = V 0 V max [S]

1 KM 1 1 = + V 0 V max [S] V max

Y = aX + b

Hanes-Woolf Plot (Reference)

1 KM 1 1 = + V 0 V max [S] V max
Multiplying both sides by [S]

V0

[S] =

[S] KM + V max V max

V0

[S] =

1 KM [S] + V max V max

Michaelis Constant KM
KM is the substrate concentration that gives maximal velocity. If an enzyme has a smaller value of KM it achieves maximal
velocity at lower substrate concentrations.

KM is unique for each enzyme-substrate pair.

KM is a measure of the affinity of the enzyme for its substrate when k2/k1 is small compared to Kd .
k 1 + k2 k2 KM = = Kd + k1 k1
k2 << k1

1 1 K M Kd Ka affinity

kcat (turnover number)

kcat : the number of substrate molecules converted into
product per enzyme molecule per unit time when the enzyme is saturated with substrate. When enzyme is saturated with substrate, the turnover number (k2) becomes rate limiting. E+S k1 k-1 ES k2 E+P

v = k 2[ES]
When enzyme is fully saturated

V max = kcat[E ]T
Principles of Biochemistry 4th Edition

The Ratio, kcat / KM Defines the Catalytic Efficiency of an Enzyme

E+S k1 k-1 ES k2 E+P

kcat/Km catalytic efficiency

However, kcat itself is not particularly informative to represent Catalytic Efficiency. Under physiological conditions, [S] is seldom saturating. The in vivo ratio of [S] / KM usually falls in the range of 0.01-1.0, so active sites often are not filled with substrate. kcat saturation condition

When [S] << Km

V0

max

KM

k c at [S ] = [ E ]total [ S ] KM

To compare enzymes, both kcat and Km are needed

Sample Calculation 3
Measurement of the rate constants for a simple enzymatic reaction obeying Michaelis-Menten kinetics gave the following results: k1 = 2 x 108 M-1sec-1 k-1 = 1 x 103 sec-1 k2 = 5 x 103 sec-1 a. b. c. d. e. What is Kd, the dissociation constant for the enzyme-substrate complex? What is Km, the Michaelis constant for this enzyme? What is kcat (the turnover number) for this enzyme? What is the catalytic efficiency for this enzyme? What is the Vmax if the total concentration of enzyme in the reaction is 2 nM?

Inhibition of Enzymatic Reaction

Important pharmaceutical reagents Two types of enzyme inhibition: Irreversible inhibition Reversible inhibition Competitive Uncompetitive Mixed Inhibition (non competitive inhibition)

Irreversible inhibition
Forms covalent (extremely tight) bonds with functional groups at the active site of the enzyme. Dilution or dialysis of the Enzyme: Inhibitor solution neither dissociate the EI complex nor restore enzyme activity Effectively decreases the amount of active enzymes, hence decreases Vmax Also called suicide inhibition

Penicillin A suicide substrate

Essential Serine in the active site

Biochemistry, 3rd Ed, Garrett & Grisham. Fig 13.18

Bacterial cell wall peptidoglycans Glycopeptide transpeptidase cross-linking of different peptidoglycan strands

Neurotoxin Sarin
Sarin acetylcholine acetylcholinesterase choline
Acetylcholine: Neurotransmitters it transmits nerve impulses across junctions between nerve cells (called synapse)

Inhibition of Enzymatic Reaction

Important pharmaceutical agents Two types of enzyme inhibition: Irreversible inhibition Reversible inhibition Competitive Uncompetitive Mixed Inhibition (Non competitive)

Competitive Inhibition

Inhibitor binds to free enzyme and competes with substrate binding Structural analogs of substrate Increase Km Can be overcome by increasing substrate

Competitive Inhibition
v0 = Vmax [S] KM + [S] [I] where = 1 + KI

Reference (self-study): Derive an expression for v as a function of [S] for competitive inhibition. d [ES] k ES + k 2 ES = k E S =0
1

k1

k2

dt

k-1

ES =

k 1[E ][S] [E ][S] = k - 1+ k 2 KM

k-3

k3

Ki =

v = k 2 ES =

k 2[E ][S] KM
T

See left on how to solve for [E]

k3

When it reaches equilibrium

k 3 E I = k 3 E I

v =

1 + KM

[E ] x [S ] + [I ]
Ki

k 2 [S] KM

EI =

k3 [E][I] = [E][I] k 3 Ki

Knowing

[E ]

= [E ] + [ES ] + [EI ]
Solving for [E]
T

v=

[E ]

k 2 S

KM [I ] K M + [S ] + Ki

[E ]

= [E ] +

[E ][S ] + [E ][I ]
KM Ki

[E ] =

1 + KM

[E ] [S ] + [I ]
Ki

v=

[S ] +

[I ] K M 1 + Ki

V max S

KM (KMapp)

Liver alcohol dehydrogenase (ADH) can oxidize ethanol or other alcohols, including methanol. Methanol oxidation yields formaldehyde, which is very toxic, causing blindness, brain damage and even death.

Lovastatin: a Competitive Inhibitor for HMG CoA Reductase

Structural analog of the natural substrate for Hydroxymethylglutaryl CoA reductase

Inhibit cholesterol synthesis

Lowering cholesterol levels

Sample Calculation 4
The Km for an enzyme at the absence and presence of 3 M of competitive inhibitor is 8 M and 12 M, respectively. Calculate Ki.
= 12 M 8 M = 1.5

[I] =1+
Ki

3 uM 1.5 = 1 + Ki

Ki =6 M

Uncompetitive Inhibition

[S] Vmax v0 = ' KM / ' + [S]

Binds to Enzyme-Substrate complex The 3-D structure of the enzyme has been altered, thus affects the catalytic capacity of the enzyme Km (of the real substrate) decreases because inhibitor binding to ES shifts the binding equilibrium of S and E toward ES binding (inhibitor is removing ES) Vmax decreases because inhibitor binding affects formation of product and increasing substrate cannot help Ratio of Km/Vmax remains the same

Uncompetitive Inhibition
1/V

S Vmax is decreased Km is decreased Ratio of Km/Vmax remains the same

1/S

* Uncompetitive inhibition affects the catalytic function of the enzyme but not its substrate binding to free enzyme.

Mixed Inhibition (Non competitive inhibition)

[S] Vmax v0 = ' ( / ') KM + [S]

Inhibitor binds to the free enzyme and the ES complex Km (of the real substrate) will increase, decrease, or remain unchanged depending on whether the inhibitor prefers to bind the E, the ES, or bind both the same (in other words, depend on the relative values of Ki and Ki) Vmax decreases because the inhibitor binding affects formation of product

Mixed Inhibition (Non competitive inhibition)

Inhibitor binds to enzyme sites that participate in both substrate binding and catalysis. It can bind to both E and ES. It includes two types: Pure non competitive: Vmax decreases; Km remains the same. Mixed non competitive: Vmax decreases; Km may increase or decrease. *The concept you have learned in your Junior College belongs to Pure Non Competitive Inhibition

Double-reciprocal plot: Mixed Inhibition

Ki = Ki

Ki < Ki

Ki > Ki

Reference

Sample Calculation 5
Determine the type of inhibition of an enzymatic reaction from the following data collected in the presence and absence of the inhibitor. Assume total enzyme concentration is the same in each experiment.
[S] (mM) 1 2 4 5 12

(mMsec-1)
12 20 29 35 40

with I present
(mMsec-1) 4.3 8 14 21 26