The Behavior of Proteins:

Enzymes kinetics

2.0 Factors affecting rate of reaction

2.1 Michaelis-Menten & Lineweaver-Burke
2.2 Enzyme inhibition & regulation
 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.

• Catalysis? proteins

- speed up the reaction; the catalysts that serve

this function called enzymes. specific
Free
Energy
Diagram

Fig. 6-1a,
Fig. 6-1b,
Catalyzed Reaction
Table 6-1, p.133
p.143
 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
 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
Fig. 6-2,
 • The induced-fit model: shape of
substrate is not exactly like the
• The Lock-and-key model: high
active site of enzyme
degree of similarity/exactly
between the shape of substrate • the binding of the substrate
and the geometry of the binding induces the conformational
site on the enzyme. change in the enzyme.
• The substrate binds to the active • The shape of the active site
site of enzyme whose shape becomes complementary to the
compliments to its own. shape of the substrate only after
the substrate binds to the enzyme.
• Form enzyme-substrate complex
• Mimics the transition state.
• Produce product and release
• Form enzyme-substrate complex
• eg. Like a key in lock or the
correct piece in jigsaw puzzle. • Produce product and release
 Michaelis-Menten model
• Devised in 1913 by Leonor Michaelis 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.
E + S ↔ ES → E + P
k1 k2

k-1
Enzyme Saturation
plateau
• The rate (velocity) of an
enzymatic reaction
depends on the [S].
• Fig. 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)
• The [S] at which the
reaction proceeds at
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,
• When experimental conditions are adjusted so
that [S] = KM,

and

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].
 Linearizing the
Michaelis-Menten
Equation
• 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,
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.
• Vmax is related to the turnover number of an E, a quantity equal to the
catalytic constant,k2. ( Vmax / [ET]) = turnover number = kcat or kp
- no. of moles of S that react to form P/mole E/unit time.

Illustrate the
efficiency of
enzymatic catalysis
 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,
 • Mechanism: inhibitor
is shaped similar to
the substrate and
temporarily competes
with it for binding sites.
• Reversed: by
increasing [S]
• KM: higher [I] increases
KM – simulating
decreasing E-S affinity
• Vmax: not changed

Fig. 6-11b,
p.146
• Mechanism: binds
temporarily to enzyme
somewhere other than
active site but halts
catalysis. Has the same
effect as reducing [E].
• Reversed: by increasing
[E]
• KM: not changed
• Vmax: lowered

The inhibitor binds to both; Fig. 6-11c,

enzyme and enzyme- p.146
substrate complex
More-Subtle Inhibition of Active Sites (1/2)
More-Subtle Inhibition (2/2)
Multi-Subunit Enzymes (1/2)

�
Recall that a Multi-Subunit Enzyme is a
catalytic Protein that consists of more

�
than one Polypeptide

This is a description of Allosteric Regulation

Multisubunit Enzymes (2/2) This is
Cooperativity

This also is a form of Allosteric Regulation

(activation)
 Kinetics of competitive inhibition
In the presence of competitive inhibitor, the equation for an enzymatic
reaction becomes
EI +↔I
E↔ +S
ES → E + P

The dissociation constant for the E-I complex can be written:

EI ↔ E + I KI = [E] [I] / [EI]

Fig. 6-12,
Important: substrate or inhibitor can bind the
 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.

Fig. 6-13,
 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 bound to the complex, it

will remain there. The enzymes loss their biology function →
reaction STOP.

•e.g. drugs, heavy metal (Boron), iodoacetic acid

• Practice session
The following data obtained for the hydrolysis of
carbobenzoxyglycyl-L-tryptophan catalyzed by the enzyme
carboxypeptidase. Plot these results using the Lineweaver-
Burk method, and determine values for KM and Vmax. The
symbol mM represents millimoles per liter; 1 mM = 1 x 10-3
mol L-1. (the [enzyme] is the same in all experiments).

[S] Velocity
(mM) (mM sec-1)
2.5 0.024
5.0 0.036
10.0 0.053
15.0 0.060
20.0 0.064
• Solution
The reciprocal of substrate conc and of velocity gives the
following results:

1/[S] 1/V
(mM-1) (mM sec-1) -1
0.400 41.667
0.200 27.778
0.100 18.868
0.067 16.667
0.050 15.625

Plotting the results gives a straight line; 1/V = 75.431 (1/[S]) +

11.8. The reciprocal of y intercept is Vmax, and the slope is
KM/Vmax.
Hence, Vmax = 0.0847 mM sec-1; KM = 6.39 mM.
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.
 Practice session
• Sucrose is hydrolyzed to glucose and fructose in a classic
experiment in kinetics. The reaction is catalyzed by enzyme
invertase.using the following data, determine by the Lineweaver-
Burk method, whether the inhibition of this reaction by 2 M urea is
competitive or non-competitive.
[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