ENZYME PART 1-5

NAMA
NPM

VIKNESWARAN MUTAYAH
260110132004

HARI/TANGGAL

14 OCTOBER 2014

NAMA DOSEN
KULIAH

PAK MUTAKIN
BIOCHEMISTRY

FAKULTAS FARMASI
UNIVERSITAS PADJADJARAN
JATINANGOR
2014

Part 1

Enzymes are macromolecules that act as catalysts in most of the organism's biochemical
reactions that have functions indispensable to maintenance and activity of life. It is crucial to
note that reaction rates of certain chemical conversions occurring in living organisms are
extremely low, and catalysis is necessary to maintain reasonable time of cell development and
division. Majority of functional enzymes are proteins, like trypsin, fumarase or papain.
Biological catalyst is also the enzyme which will increase the rate of reaction in biochemistry
reaction. Enzyme increases the rate of reaction by lowering the activation energy without
altering the thermodynamic of the reaction. Thus this will result as the product to be formed
rapidly and reaction reaches their equilibrium state more rapidly. On the other hand, enzyme
also increase the rate of reaction when rate of concentration increases. Whereby it will be
designed as the

G of a reaction is not altered or changed by an enzyme. Another thing that

it does is also it increases the rate constants, Rate = k [A]x[B]y.

Enzymes are also referred to the specific lock and

key model.

Whereby, it is associated with the substrate bind at the active sites. The way of lowering
activation energy is to stabilize the transition state which is known as a free energy. In the
diagram below, it shows that co-relation of the free energy against reaction process. The
diagram shows the high energy intermediate between reactant and product. Transition state of
catalyzed reaction and un-catalyzed reaction is stabilized.

Part 2
The primary function of enzymes is to enhance rates of reactions so that they are compatible
with the needs. For many enzymes, the rate of catalysis V0, which is defined as the number of
moles of product formed per second, varies with the substrate concentration [S] in a manner
shown in figure below. The rate of catalysis rises linearly as substrate concentration increases
and then begins to level off and approach a maximum at higher substrate concentrations.
Consider an enzyme that catalyzes the S to P by the following pathway:

A plot of the reaction velocity (V0) as a function of the substrate concentration [S] for an
enzyme that obeys Michaelis-Menten kinetics shows that the maximal velocity (Vmax) is
approached asymptotically. The Michaelis constant (KM) is the substrate concentration
yielding a velocity of Vmax/2. KM, called the Michaelis constant:

, KM has the

units of concentration. KM is an important characteristic of enzyme-substrate interactions and

is independent of enzyme and substrate concentrations. At very low [S] ([S] << Km), Vo
approaches (Vo)[S].Vmax and Km max/K are constants, so linear relationship between Vm and
[S] at low [S]. When [S] = Km, Vo = 1/2 V. At very high [S], ([S] >> Kmax m), Vo approaches V
(velocity independent of [S]).

Part 3
The determination of KM and Vmax values required algebraic manipulation of the basic
Michaelis-Menten equation. Because Vmax is approached asymptotically (see Figure below) it
is impossible to obtain a definitive value from a typical Michaelis-Menten plot.
Because KM is the concentration of substrate atVmax/2, it is likewise impossible to determine
an accurate value of KM. However, Vmax can be accurately determined if the Michaelis-Menten
equation is transformed into one that gives a straight-line plot.

A plot of 1/V0 versus 1/[S], called a Lineweaver-Burk or double-reciprocal plot, yields a

straight line with an intercept of 1/Vmax and a slope of KM/Vmax (Figure below). The intercept
on the x-axis is -1/KM.

Figure ABOVE represents Double-Reciprocal or Lineweaver-Burk Plot

A double-reciprocal plot of enzyme kinetics is generated by plotting 1/V0 as a function 1/[S].
The slope is the KM/Vmax, the intercept on the vertical axis is 1/Vmax, and the intercept on the
horizontal axis is -1/KM.

Part 4
Competitive Inhibition
A competitive inhibitor binds only to free enzyme. Often this binding event occurs on the
active site of the target, precisely where substrate also binds. Although this is the case for a
majority of competitive inhibitors, it is a misleading oversimplification. It is more appropriate
to state that the binding of a competitive inhibitor and the binding of substrate are mutually
exclusive events. Figure below provides illustrations of some possible mutually exclusive
binding events.

Examples of Competitive Inhibition where Substrate (S) and Inhibitor (I) binding events are
mutually exclusive. (a) Classical model for competitive inhibition where S and I compete for
the same precise region of the active site. (b) It does not bind to the active site, but sterically
hinders S binding. (c) S and I binding sites are overlapping. (d) S and I share a common
binding pocket on the enzyme. (e) I binding can result in a conformational change that
prevents S binding (and vice versa). All competitive inhibitors have the same effects on
substrate binding and catalysis. A competitive inhibitor will raise the apparent Km value for its
substrate with no change in the apparent Vmax value. As a result, it is often stated that
competitive inhibition can be overcome, observed by an increase in the apparent Ki value, at
higher concentrations of substrate. This characteristic will have physiological consequences
on the observed efficacy of drugs. As an enzymes reaction is inhibited by a competitive
inhibitor, there is an increase in the local concentration of substrate. Without a mechanism to
clear the substrate, a competitive inhibitor will lose potency. This is not the case for a
noncompetitive inhibitor.

Noncompetitive Inhibition
A noncompetitive inhibitor binds equally well to both free enzyme and the enzyme-substrate
complex. These binding events occur exclusively at a site distinct from the precise active site
occupied by substrate. Figure below provides some illustrations of the more common
noncompetitive binding events.

Examples of Noncompetitive Inhibition where Inhibitor (I) binding occurs at a site distinct
from the Substrate (S) binding site and the Catalytic center (c) of the active site. (a) In this
model, the binding of S induces a conformational change to align the catalytic center near S
for catalysis. However, when I binds at a separate site, the conformational change does not
occur and enzyme activity is inhibited. (b) In this model, I can sterically hinder S binding and
release. However, unlike Figure 1-B, I and S can occupy the enzyme at the same time. In
contrast to a competitive inhibitor, a noncompetitive inhibitor will lower the apparent Vmax
value, yet there is no effect on the apparent Km value for its substrate. Essentially, the Ki of
the inhibitor does not change as a function of the substrate concentration. In some
circumstances, a compound may have unequal affinity for both free enzyme and the enzymesubstrate complex. This mixture of competitive and noncompetitive phenotypes is called
mixed inhibition.

Part 5 Example of Lineweaver Burk Plot

Set up four tubes with different concentrations of catechol (the substrate). (Catechol is
arelative of the active ingredients in the poison ivy plant and, like them, can cause serious
contact sensitivity if it gets on one's skin.)
o A = 4.8 mM; B = 1.2 mM; C = 0.6 mM; D = 0.3 mM

Add a fixed amount of enzyme preparation to Tube A and measure the change in
absorbance (Optical Density) at 540 nm) at 1 minute intervals for several minutes.

Record the average change in OD540 per minute ( OD540).

Because the OD is directly proportional to the concentration of the products, we can

use it as a measure of the rate or velocity of the reaction (Vi).

Repeat with the other three tubes.

Tube A

Tube B

Tube C

Tube D

[S]

4.8 mM

1.2 mM

0.6 mM

0.3 mM

1/[S]

0.21

0.83

1.67

3.33

0.081

0.048

0.035

0.020

OD540
(Vi)
1/Vi

12

21

The table above summarizes the results.

29

50

Making a Lineweaver-Burk plot of these results shows

(red)

that

1/Vmax = 10, so Vmax = 0.10

1/Km = 0.8, so Km = 1.25 mM

(In other words, when [S] is 1.25 mM, 1/Vi =

and Vi = 0.05 or one-half of Vmax.)

20,