HOW ENZYMES

WORK
 ENZYMES SPEED UP CHEMICAL REACTIONS

Enzymes are biological catalysts – substances that speed a reaction

without being altered in the reaction.

Most enzymes are proteins.

Enzymes are essential for life.

Model of the surface of an enzyme.

 Enzymes

� Cofactors
� Coenzymes
� Holoenzyme
� Apoenzyme
 How Enzymes Work?

• Body conditions(temperature, pressure etc.) not

good for reaction

• Only enzymes can catalyse the reactions

in this conditions

• A special environment inside enzymes for

reaction ACTIVE SITE

• Molecule binds active site SUBSTRATE

Enzymes
Lower a
Reaction’s
Activation
Energy
Each reaction has a transition state where the
substrate is in an unstable, short-lived
chemical/structural state.

Free Energy of Activation

is symbolized by ΔG‡.

Enzymes act
by lowering the free
energy of the transition
state
 Enzymes speed up metabolic
reactions by lowering energy barriers

 Enzyme speed reactions by lowering EA.

– The transition state
can be reached at
moderate temperatures.

 Enzymes do not change

delta G.
– It speed-up reactions
that would occur eventually.

 Because enzymes are

so selective, they determine which
chemical processes will occur
at any time
 Enzymes lower the free energy of activation by
binding the transition state of the reaction better
than the substrate

 The enzyme must bind the substrate in the correct

orientation otherwise there would be no reaction

 Not a lock & key but induced fit – the enzyme

and/or the substrate distort towards the transition
state
 Induced Fit

A change in the shape

of an enzyme’s active site

Induced by the
substrate
 Lock and Key Model
 An enzyme binds a substrate in a region called the active site

 Only certain substrates can fit the active site

 Amino acid R groups in the active site help substrate bind

 Enzyme-substrate complex forms

 Substrate reacts to form product

 Product is released
 Enzyme Kinetics

- Kinetics The study of the rate of change.

- Enzyme Kinetics Rate of chemical

reactions mediated by enzymes. Enzymes
can increase reaction rate by favoring or
enabling a different reaction pathway with
a lower activation energy, making it easier
for the reaction to occur.
 Michaelis-Menten kinetics
Vmax
V0 varies with [S] approached
asymptoticall
V0 is ymoles of
product
formed per sec.
when [P]
Eis + SESE
low (close to + P
zero
time)
Michaelis-Menten
Model
V0 = Vmax x[S]/([S] + Km)
Michaelis-Menten Equatio
Determining initial velocity (when
[P] is low)
 Steady-state & pre-steady-state
conditions

At pre-steady-state,
At equilibrium, [P] is low (close to zero
no net change of [S] & [P] time), hence, V0 for initial
or of [ES] & [E] reaction velocity
At pre-steady state, we can ignore the back reactions
 Michaelis-Menten kinetics
(summary)
Enzyme kinetics (Michaelis-Menten Graph) :
At fixed concentration of enzyme, V0 is almost linearly proportional to
[S] when [S] is small, but is nearly independent of [S] when [S] is large
Proposed Model: E + S  ES  E + P
ES complex is a necessary intermedi
Objective: find an expression that relates rate of catalysis to the
concentrations of S & E, and the rates of individual steps
Start with: V0 = k2[ES], and derive, V0 = Vmax x[S]/([S] + Km)
This equation accounts for graph data.
At low [S] ([S] < Km), V0 = (Vmax/Km)[S]
At high [S] ([S] > Km), V0 = Vmax
When [S] = Km, V0 = Vmax/2.
Thus,
 Range of Km values

Km provides approximation of [S] in vivo for many enzyme

Lineweaver-Burk plot (double-
reciprocal)
Eadie-Hofstee plot
Hanes-Woolf Plot
 Allosteric enzymes
 Allosteric enzymes tend to be
multi-sub unit proteins

 The reversible binding of an

allosteric modulator (here a
positive modulator M) affects
the substrate binding site
 Mechanism and Example of Allosteric Effect
Kinetics Models Cooperation
Allosteric site
R = Relax
R Homotropic
(active) vo (+) S
S (+)
S
R Concerted
Allosteric site
[S]
R
(+) A
T Heterotropic
vo (+)
S S (+)
R Sequential
X [S]
T
T = Tense I Heterotropic
vo T
(inactive) (-)
(-) X
(-)
X
T Concerted
[S]
 Enzyme Inhibitors
• Specific enzyme inhibitors regulate enzyme activity and help us
understand mechanism of enzyme action. (Denaturing agents are not
inhibitors)

• Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive. 3 classes:
groupspecific reagents, substrate analogs, suicide inhibitors

• Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor. 3 groups based on their mechanism of
action: competitive, non-competitive and uncompetitive.
Enzyme Inhibition
 Competitive inhibitors
• Compete with substrate for binding to enzyme

• E + S = ES or E + I = EI . Both S and I cannot bind enzyme at the same

time

• In presence of I, the equilibrium of E + S = ES is shifted to the left

causing dissociation of ES.

• This can be reversed / corrected by increasing [S]

• Vmax is not changed, KM is increased by (1 + I/Ki)

• Eg: AZT, antibacterial sulfonamides, the anticancer agent methotrexate

etc
Competitive Inhibition
 Kinetics of competitive inhibitor
Increase [S] to
Ki = overcome
dissociation inhibition
constant for
inhibitor Vmax attainable,
Km is increased
V max unaltered, Km increased
 Non-competitive Inhibitors
• Inhibitor binding site is distinct from substrate binding site. Can bind
to free enzyme E and to ES

• E + I = EI, ES + I = ESI or EI + S = ESI

• Both EI and ESI are enzymatically inactive

• The effective functional [E] (and [S]) is reduced

• Reaction of unaffected ES proceeds normally

• Inhibition cannot be reversed by increasing [S]

• KM is not changed, Vmax is decreased by (1 + I/Ki)

Mixed (Noncompetitive) Inhibition
Kinetics of non-competitive inhibitor
Increasing [S] cannot
overcome inhibition

Less E available,
V max is lower,
Km remains the same
for available E
Km unaltered, V max decreased
 Uncompetitive Inhibitors

• The inhibitor cannot bind to the enzyme directly, but

can only bind to the enzyme-substrate complex.

• ES + I = ESI

• Both Vmax and KM are decreased by (1+I/Ki).

Uncompetitive Inhibition
 Substrate Inhibition
 Caused by high substrate concentrations

[ S ][ ES ] ' [ S ][ E ]
E+S
Km’
ES
k2
E+P K Si  , Km 
+ [ ES 2 ] [ ES ]
S
Vm [ S ]
v 2
KS1 [ S ]
K m'  [ S ] 
ES2
K S1
 Substrate Inhibition
 At low substrate concentrations [S]2/Ks1<<1 and
inhibition is not observed

 Plot of 1/v vs. 1/[S] gives a line Vm

v
 Slope = K’m/Vm  Km  '
 Intercept = 1/Vm 1  [ S ] 
 
'
1 1 K 1
  m
v Vm Vm [ S ]
 Substrate Inhibition
 At high substrate concentrations, K’m/[S]<<1, and
inhibition is dominant

 Plot of 1/v vs. [S] gives a straight line

 Slope = 1/KS1 · Vm Vm
v
 Intercept = 1/Vm  [S ] 
1  
dv / d [ S ]  0  K S1 
1 1 [S ]
[ S ]max  K K S 1  
'
m
v Vm K S 1Vm
 1/V I>0 1/V
I>0
I=0 I=0
1/Vm,app
1/Vm
1/Vm
-1/Km -1/Km,app 1/[S] -1/Km,app-1/Km 1/[S]
Competitive Uncompetitive

1/V 1/V
I>0
I=0

1/Vm,app
1/Vm 1/Vm

-1/Km 1/[S] -1/Km 1/[S]

Non-Competitive Substrate Inhibition

 Enzyme Inhibition (Mechanism)

I Competitive I Non-competitive I Uncompetitive

Substrate E
E S X
Cartoon Guide

S S I
E S I I
I
Compete for S I
Inhibitor active site Different site
Equation and Description

E + S→
← ES → E + P E + S←→ ES → E + P E + S←
→ ES → E + P
+ + + +
I I I I
↓↑ ↓↑ ↓ ↑ ↓↑
EI EI + S →EIS EIS
[I] binds to free [E] only, [I] binds to free [E] or [ES] [I] binds to [ES] complex
and competes with [S]; complex; Increasing [S] can only, increasing [S] favors
increasing [S] overcomes
not overcome [I] inhibition. the inhibition by [I].
Inhibition by [I].
 Enzyme Inhibition (Plots)

I Competitive I Non-competitive I Uncompetitive

Vmax Vmax Vmax
vo vo
Direct Plots

Vmax’ Vmax’
I I I

Km Km’ [S], mM Km = Km’ [S], mM Km’ Km [S], mM

Vmax unchanged Vmax decreased
Both Vmax & Km decreased
Km increased Km unchanged
Double Reciprocal

1/vo I 1/vo I 1/vo

I
Two parallel
Intersect lines
at Y axis 1/ Vmax Intersect 1/ Vmax 1/ Vmax
at X axis

1/Km 1/[S] 1/Km 1/[S] 1/Km 1/[S]

Factors Affecting Enzyme
Kinetics
 Effects of pH
- on enzymes

- enzymes have ionic groups on their active sites.

- Variation of pH changes the ionic form of the active sites.

- pH changes the three-Dimensional structure of enzymes.

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme.

 Effects of Temperature
 Reaction rate increases with temperature up to a limit
 Above a certain temperature, activity decreases with temperature
due to denaturation
 Denaturation is much faster than activation
 Rate varies according to the Arrhenius equation
v  k2 [ E ]
Where Ea is the activation energy
k 2  Ae  Ea / RT
(kcal/mol)
[ E ]  [ E0 ]e  k d t [E] is active enzyme concentration

k d  Ad e  Ea / RT
v  Ae  Ea / RT E0 e  k d t
 Factors Affecting Enzyme Kinetics
 Temperature
- on the rate of enzyme catalyzed reaction
d [ P]
v  k 2 [ ES ]
dt
k2=A*exp(-Ea/R*T)

T k2 v
- enzyme denaturation d[E ]
  kd [E]
T Denaturation rate: dt
kd=Ad*exp(-Ea/R*T)
kd: enzyme denaturation rate constant;
Ea: deactivation energy
 REFERENCES
 Michael L. Shuler and Fikret Kargı, Bioprocess
Engineering: Basic Concepts (2 nd
Edition),Prentice Hall, New York, 2002.

 1. James E. Bailey and David F. Ollis,

Biochemical Engineering Fundementals (2 nd
Edition), McGraw-Hill, New York, 1986.

 www.biochem.umass.edu/courses/420/lectu
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 class.fst.ohio-
state.edu/fst605/605p/Enzymes.pdf –
 www.horton.ednet.ns.ca/staff/selig/powerpoint
s/bio12/biochem/enzymes.pdf
 www.siu.edu/departments/biochem/som_pbl/S
SB/powerpoint/enzymes.ppt
 www.associazioneasia.it/adon/files/2005_luisi_
05_why_are_enzymes.pdf
 www.fatih.edu.tr/~abasiyanik/Chapter6_enzym
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 http://www.authorstream.com/presentation/kkoz
ar-14001-enzymes-enzyme-ppt-education-powerpo
int/

 http://highered.mcgraw-hill.com/sites/0072495
855/student_view0/chapter2/animation__how_enz
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 http://www.wiley.com/college/pratt/0471393878/s
tudent/animations/enzyme_kinetics/index.html