1. The Behavior of Proteins:
Enzymes
Chapter 02 : Enzyme kinetics
2.0 Factors affecting rate of reaction
2.1 Michelis-Menten & Lineweaver-Burke
2.2 Enzyme inhibition & regulation

2. Recall…
• Activation energy?
- the energy that used to initiate the reaction,
where this energy is needed to break the
chemical bonding so that the reaction can occur.
- this energy is low with the usage of enzyme; do
not influence the product (P), path of reaction
and final concentration of molecules.
proteins
• Catalysis?
- speed up the reaction; the catalysts that serve
this function called enzymes. specific

3. Fig. 6-1a, p.132

4. Fig. 6-1b, p.132

5. Table 6-1, p.133

6. p.133

7. Enzyme characteristics
 Protein
 Catalysts
 Specific reaction
 Reacts at optimal pH and temperature
 Regulate/control the metabolism processes
 Need in a low amount
 Reversible reaction
 Reaction may be inhibited by inhibitor

8. Factors affecting rate of reaction
i. Enzyme concentration [E] – with a constant [S],
the rate of reaction increased with the increasing
[E]
ii. Substrate concentration [S] – the rate of reaction
increased until the amount of S = E
iii. pH – depends on functional group (R); -COOH or
-NH2
iv. Effect of temperature – increment of temperature
will increase the rate of reaction
v. Effect of inhibitor – chemical substance that binds
on the active site/other site on the enzyme
(allosteric site) → competitive and non-competitive inhibition

9. Fig. 6-2, p.134

10. • The Lock-and-key model: high • The induced-fit model: the binding
degree of similarity between the of the substrate induces the
shape of substrate and the conformational change in the
geometry of the binding site on enzyme.
the enzyme. • The binding site has a different 3-
• The substrate binds to a site D shape before the substrate is
whose shape compliments to its bound.
own. • The shape of the active site
• eg. Like a key in lock or the becomes complementary to the
correct piece in jigsaw puzzle. shape of the substrate only after
• Weakness? the substrate binds to the enzyme.
• Mimics the transition state.

12. Michaelis-Menten model
• Devised in 1913 by Leonor Michelis and Maud
Menten.
• Basic model for nonallosteric enzyme.
• The main feature of this model for enzymatic
reaction is the formation of an E-S complex.
• The [E-S] is low but remains unchanged to any
appreciable extent over the course of the
reaction.
• The S → P; released from the E.
• The E is regenerated at the end of the reaction.
k1 k2
E + S ↔ ES → E + P
k-1

13. • The rate (velocity) of an
enzymatic reaction
depends on the [S].
• Fig. 6-8 shows the rate
and the observed kinetics
of an enzymatic reaction.
• In lower region of the
curve (at low level of S) –
V0 depends on S.
• In upper portion of the
curve (at higher levels of
S), the reaction is zero.
• At infinite [S], the reaction
would proceed at its max
velocity (Vmax)

14. Vmax [ S ]
• The [S] at which the V =
reaction proceeds at KM +[S ]
one-half its Vmax has a
special significance.
• It’s given the symbol KM
(Michaelis constant)
which considered an
inverse measure of the
affinity of the E for the S.
• The lower the KM, the
higher the affinity.
• The Vmax for the E can be
estimated from the
graph. Thus, the value
of KM also can be
estimated from the
Fig. 6-9, p.142

15. Vmax [S ]
V =
K M +[S ]
• When experimental conditions are adjusted so
that [S] = KM,
Vmax [ S ] Vmax
V = V =
[S ] +[S ] and 2
Note : Michaelis-Menten model is the simplest
enzyme equation, where it’s considered the
reaction of one single S to a single P.
: the term KM only appropriate for E that
exhibit a hyperbolic curve of V vs [S].

16. 1 K +[ S ]
= M
V Vmax [ S ] Linearizing the
1
=
KM
+
[S ] Michaelis-Menten
V Vmax [ S ] Vmax [ S ]
1 K 1
= M × +
1 Equation
V Vmax S Vmax • The curve that describe
the rate of nonallosteric
enzymatic reaction is
hyperbolic.
• It is considerably easier to
work with straight line than
a curve.
• The equation for a
hyperbola transformed into
an equation for a straight
line by taking the
reciprocal of both sides:
Lineweaver-Burk double
reciprocal plot
Fig. 6-10, p.143

17. Significance of KM and Vmax
• When V = Vmax / 2, then KM = [S] → interpret that KM is equals the
concentration of S at which 50% of the enzyme’s active sites are occupied
by S.
• Another interpretation of KM relies on the assumptions of the original
Michaelis-Menten model of enzyme kinetics.
• The KM is a measure of how tightly the S is bound to the E. KM >>, the less
tightly the S bound to the E.
Illustrate the
• Vmax is related to the turnover number of an E, a quantity equal of the catalytic
efficiency to
constant,k2. ( Vmax / [ET]) = turnover number = kcat or kp
enzymatic catalysis
- no. of moles of S that react to form P/mole E/unit time.

18. How Do Enzymatics
Reactions Respond to
Inhibitors?
Inhibitor – a substance that interferes with the
action of an enzyme and slows the rate of a
reaction.
2 ways in which inhibitors can affect an
enzymatic reaction:
i.A reversible inhibitor
ii.An irreversible inhibitor
There 2 major classes of reversible inhibitors
which can be distinguished on the basis of
the sites on the E to which they bind:
i.Competitive inhibition
ii.Noncompetitive inhibition
Fig. 6-11, p.146

19. Fig. 6-11b, p.146

20. Fig. 6-11c, p.146

21. Kinetics of competitive inhibition
In the presence of competitive inhibitor, the equation for an enzymatic
Important: substrate or inhibitor can bind the
reaction becomes
enzyme, not both. Because both are vying for the
EI + I E ↔ ES → E + P
↔ +S
same location, sufficiently high substrate will
“outcompete” the inhibitor. This is why the Vmax
The dissociation constant for the E-I complex can be written:
does not change.
EI ↔ E + I KI = [E] [I] / [EI]
1 KM [I ] 1 1
= (1 + )× +
V Vmax K I [ S ] Vmax
y = m× x + b
Fig. 6-12, p.148

22. Kinetics of noncompetitive inhibition
In the presence of noncompetitive inhibitor, the reaction pathway has
become more complicated+S
E ↔ ES → E + P
+I ↕ ↕ +I
+S
EI ↔ ESI
•The value of Vmax decreases, but KM remains the same; the inhibitor
doesn’t interfere with the binding of S to the active site.
1 KM [I ] 1 1 [I ]
= (1 + )× + (1 + ) Fig. 6-13, p.149

23. Kinetics of uncompetitive inhibition
•The inhibitor can bind to the ES complex but not to free E.
•The Vmax decreases and KM decreases as well.
•Once the uncompetitive inhibitor biond to the complex, it will
remain there. The enzymes loss their biology function →
reaction STOP.
•e.g. drugs, heavy metal (Boron), iodoacetic acid

24. • Practice session
Sucrose is hydrolyzed to glucose and fructose. The
reaction is catalyzed by the enzyme invertase.
Using the following data, by the Lineweaver-Burk
method, whether the inhibition of this reaction by 2
M is competitive or noncompetitive.
[Sucrose] V, no inhibitor V, Inhibitor
(mol L-1) present
0.0292 0.182 0.083
0.0584 0.265 0.119
0.0876 0.311 0.154
0.117 0.330 0.167
0.175 0.372 0.192
p.151a

25. Enzyme inhibition in
the treatment of AIDS –
important target is HIV
protease that essential
to the production of
new virus particles in
infected cells.
Treatment is most
effective when
combination of drug
therapies is used and
HIV protease inhibitors
play an important role.