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
V0 varies with [S]
Vmax
approached
asymptotically
V0 is moles of product
formed per sec. when [P]
is low (close to zero time)
E + SESE + P
Michaelis-Menten Model
V0 = Vmax x[S]/([S] + Km)
Michaelis-Menten Equation
Determining initial velocity (when [P] is low)
Steady-state & pre-steady-state conditions
At equilibrium,
no net change of [S] & [P]
or of [ES] & [E]
At pre-steady-state,
[P] is low (close to zero
time), hence, V0 for initial
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 intermediate
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,
Km = substrate concentration at which the reaction rate (V0) is half max.
Range of Km values
Km provides approximation of [S] in vivo for many enzymes
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
R = Relax
(active)
Models
Cooperation
Allosteric site
R
vo
(+)
R
S
[S]
R
S
S
A
(+)
vo
S
Allosteric site
S
Heterotropic
(+)
Sequential
X
Heterotropic
(-)
Concerted
T
(+)
R
X
[S]
T
T = Tense
(inactive)
Homotropic
(+)
Concerted
I
vo
(-)
(-)
X
T
[S]
T
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
Ki =
dissociation
constant for
inhibitor
Increase [S] to
overcome
inhibition
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
E+S
Km’
ES
+
S
KS1
ES2
k2
E+P
[ S ][ ES ] ' [ S ][ E ]
K Si 
, Km 
[ ES2 ]
[ ES ]
v
Vm [ S ]
2
[
S
]
K m'  [ S ] 
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


Slope = K’m/Vm
Intercept = 1/Vm
Vm
v
'
 Km 
1  [ S ] 


'
m
1 1 K 1


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
Intercept = 1/Vm
dv / d [ S ]  0
[ S ]max  K m' K S 1
v
Vm
 [S ] 
1 

 K S1 
1 1
[S ]


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]
Competitive
1/V
-1/Km,app-1/Km
1/[S]
Uncompetitive
I>0
1/V
I=0
1/Vm,app
-1/Km
1/Vm
1/Vm
1/[S]
Non-Competitive
-1/Km
1/[S]
Substrate Inhibition
Enzyme Inhibition (Mechanism)
I
Competitive
I
Non-competitive
Equation and Description
Cartoon Guide
Substrate
E
I
Inhibitor
E
S
S
Compete for
active site
E + S←
→ ES → E + P
+
I
↓↑
EI
[I] binds to free [E] only,
and competes with [S];
increasing [S] overcomes
Inhibition by [I].
S
I
I
Uncompetitive
S
E
I
I
Different site
E + S←
→ ES → E + P
+
+
I
I
↓↑
↓↑
EI + S →EIS
[I] binds to free [E] or [ES]
complex; Increasing [S] can
not overcome [I] inhibition.
S
I
E + S←
→ ES → E + P
+
I
↓↑
EIS
[I] binds to [ES] complex
only, increasing [S] favors
the inhibition by [I].
Enzyme Inhibition (Plots)
I
Competitive
Non-competitive
I
Direct Plots
Vmax
vo
I
Double Reciprocal
I
[S], mM
Km = Km’
Vmax decreased
Km unchanged
1/vo
1/vo
1/Km
I
Vmax
Vmax’
[S], mM
Vmax unchanged
Km increased
Intersect
at Y axis
Uncompetitive
Vmax
vo
Km Km’
I
I
Km’ Km
Vmax’
[S], mM
Both Vmax & Km decreased
1/vo
I
I
Two parallel
lines
1/ Vmax
1/[S]
Intersect
at X axis
1/Km
1/ Vmax
1/[S]
1/ Vmax
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 ]
k 2  Ae  Ea / RT
Where Ea is the activation energy
(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
v
k2
- enzyme denaturation
T
Denaturation rate:
d[ E]

 kd [E]
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/le
ctures/Ch08B.ppt -

class.fst.ohiostate.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
es.pdf -

http://www.authorstream.com/presentation/kkoza
r-14001-enzymes-enzyme-ppt-educationpowerpoint/

http://highered.mcgrawhill.com/sites/0072495855/student_view0/chapte
r2/animation__how_enzymes_work.html

http://www.wiley.com/college/pratt/0471393878/s
tudent/animations/enzyme_kinetics/index.html