TEKNIK REAKSI

OLEH :
AHMADI FACHRY
M.ADLAN ADAM
HAFIZH MAULANA

to the binding energy of the substrate to the enzyme through multiple bonds with the specific
functional groups on the enzyme (amino side chains, metal ions). The interactions that stabilize
the enzyme-substrate complex are hydrogen bonding and hydrophobic, ionic, and London van
der Waals forces. If the enzyme is exposed to extreme temperatures of pH environment (i.e..
both high and low pH values), it may unfold losing its active sites. When this occurs, the enzyme
is said to be denatured. See Problems P7-15B.
There are two models for substrate-enzyme interactions: the lock anda key model and the
induced fit model, both of which are shown in Figure 7-6. For many years the lock and the lock
and key model was preferred because of the stereospecific effects of one enzyme acting on one
substrate. Howefer, the induced fit model is the more useful model. In the induced fit model both
the anzyme molecule a d substrate molecules are distorted. These changes in conformation
distort one or more of substrate bonds, thereby stressing and weakening the bond to make the
molecule more susceptible to rearrangement or attachment.

GAMBAR (a-b)
GAMBAR (substrate (o-glucose))

There are six classes of enzymes and only six:

1. Oxidoreductases

AH2 + B + E

2. Transferases

AB + C + E

3. Hydrolases

AB + H2O + E

4. Isomerases

A+E

5. Lyases

AB + E

6. Ligases

A + B +E

A + BH2 +E
AC + B + E
AH + BOH + E
isoA + E
A+B+E
AB + E

More information about enzyme can be found on the following two web sites:
http://us.expasy.org/enzyme/and www.chem.qmw:ac:uk/iubmb/enzyme
These sites also give information about enzymatic reactions in general.

7.2.2 Mechanisms
In developing some of the elementary principles of the kinetics of enzyme reactions, we shall
discuss an enzymatic reactions that has been suggested by Levine and LaCourse as part of a
system that would reduce the size of an artificial kidney. The desired result is the production of
an artificial kidney that could be worn by the patient and would incorporate a replaceable unit for
the elimination of the nitrogenous waste produts such as uric acid and creatinine. In the
microencapsulation scheme proposed by Levine and LaCourse, the enzyme urease would be
used in the removal of urea from the bloodstream. Here, the catalytic action of urease would
cause urea to decompose into ammonia and carbon dioxide. The mechanism of the reaction is
believed to procced by the following sequence of elementary reactions :

1.

The enzyme urease (E) reacts with the substrate urea (S) to form an enzymesubstrate complex (ES).
NH2CONH2 + Urease

2.

[NH2CONH2 Urease]

(7-13)

This complex (ES) can decompese back to urea (S) and urease (E):
[NH2CONH2 Urease]

Urease + NH2CONH2

(7-14)

3.

Or it can react with water (W) to give the products (P) ammonia and carbon
dioxide, and recover the enzyme urease (E)
[NH2CONH2 Urease] + H2O

2NH3 + CO2 + Urease

(7-15)

We see that some of the enzyme added to the solution binds to the urea, and some remains
unbound. Although we can easily measure that total concentration of enzyme, (E), it is difficult
to measure the conversation of free enzyme, (E).
Letting E, S, W, ES, and P represent the enzyme, substrate, water, the enzyme-substrate
complex, and the reaction products, respectively, we can write reactions (7-13). (7-14), and (715) symbolically in the forms
S+E

ES

(7-16)

ES

E+S

(7-17)

ES + W

P+E

(7-18)

Here P = 2NH3 + CO2

The corresponding rate laws for Reaction (7-16), (7-17), and (7-18) are
r1s= -k1 (E)(S)

(7-16A)

r2s= -k2 (ES)

(7-17A)

r3P= -k3 (ES)(W)

(7-18A)

The net rate of disappearance of substrate, -rs. is

r1s= -k1 (E)(S)- k2 (ES)

(7-19)

This rate law is of not much use to us in making reaction engineering calculations
because we cannot measure the concentration of enzyme substrate complex (ES) in terms of
measured variables. The net rate of formation of the enzyme-substrate complex is
rES = -k1 (E)(S)- k2 (ES) - k3 (W) (ES)

(7-20)

Using the PPSH. rES=0, we solve equation (7-20) for (ES)

( )( )

(ES) =

(7-21)

( )

and substitute for (ES) into [Equation (7-19)]

( )( )

-rs= k1 (E)(S) - k2

Simplifying
( )( )(

-rs=

(7-22)

We still cannot use this rate law because we cannot measure the unbound enzyme concentration
(E): however, we can measure the total enzymw concentration. Et
In the absence of enzyme denaturization , the total concentration of the enzyme in the
system, (Et), is constant and equal to the sum of the concentrations of the free or unbounded
enzyme, (E), and the enzyme-substrate complex (ES):
(Et) = (E) + (ES)

(7-23)

Substituting for (ES)

( )( )

(Et)= (E) +

Solving for (E)

(E) =

)(

(
( )

))
( )

substituting for (E) in equation (7-22), the rate law for substrate consumption is

-rs =

( )(
( )

)( )
( )

(7-24)

Note : Throughout, Et = (Et) = total concentration of enzyme with typical units (kmol/m3 or
g/dm3).

7.2.3 Michaelis-Menten Equation

Because the reaction of urea is carried out in aquaeous solution. water is, of coarse, in excess,
and the concentration of water is therefore considered constant. Let

kcat = k3 (W) and KM =

Dividing the numerator and denominator of Equation (7-24) by k1, we obtain a form of
the Michaelis-Menten equation :
-rs =

)( )

( )

(7-25)

The parameter kcat is also referred to as theturnover number. It is the number of substrate
molecules converted to product in a given time on a single-enzyme molecule when the enzyme is
saturated with substrate . For example, turnover number for decomposition H2O2 by the enzyme
catalase is 40 x 106 s-i. That is. 40 million molecules of H2O2 are decomposed every second on a
single-enzyme molecule saturated with H2O2. The constant KM (mol/dm3) is called the Michaelis
constant and for simple systems is measure of the attraction of enzyme for its substrate, so it's
also called the affinity constant. The Michaelis constant, KM. For the decomposition of H2O
discussed earlier is 1.1 M while that for chymotrypsin is 0.1 M.
If, in addition, we let Vmax represent the maximum rate of reaction for a given total
enzyme concentration.

Vmax= Kcat (Et)

The michaelis-Menten equation takes the familiar form

-rs =

( )
( )

(7-26)

For a given enzyme concentration, a sketch of the rate of disappearance of the substrate is shown
as a function of the substrate concentration in Figure 7-7.

Grafik Figure 7-6 Michaelis-Menten plot identifying the parameters Vmax and Km

A plot of this type is sometimes called a Michaelis-Menten plot. At low substrate concentration.
KM > (S)
( )

-rs =

and the reaction is apparent first order in the substrate concentration, at high substrate
concentrations,
(S) > Vmax
Consider the case when the substrate concentration is such that the reaction rate is equal to onehalf the maximum rate.
rs=
then

( )
( )

(7-27)

Solving equation (7-27) for the Michaelis constant yields

KM = (S1/2)

(7-28)

The Michaelis constant is equal to the substrate concentration at which the rate of reaction is
equal to one half the maximum rate.

The parameters Vmax and Km characterize the enzymatic reactions tht are described by
Michaelis-Menten kinetics. Vmax is dependent on total enzyme concentration, whereas KM is not.
Two enzymes may have the same values for kcat but have different reaction rates because
of different values of KM. One way to compare the ratio kcat/KM. When this ratio approaches 108
to 109 (dm3/mol/s) the reaction rate approaches becoming diffusion-limited. That is, it takes a
long time for the enzyme and substrate to find each other. But once they do they reach
immediately. We will discuss diffusion-limited reactions in Chapters 11 and 12.
Example 7-3

(E7-3.2)

A plot of the reciprocal reaction rate versus the reciprocal urea concentration should be a straight
line with an intercept 1/Vmax and slope KM/Vmax. This type of plot is called a Lineweaver-Bark
plot. The data in Table E7-3.1 are presented in Figure E7-3.1 in the form of a Lineweaver-Bark
plot. The Intercept is 0.75, s0

= 0.75 m3.s/kmol

TABLE E7-3.1 RAW AND PROCESSED DATA

Curea
(kmol/m3)

-rurea
(kmol/m3..s)

1/Curea
(m3/kmol)

1/-rurea
(m3..s/ kmol)

0.20

1.08

5.0

0.93

0.02

0.55

50.0

1.82

0.01

0.38

100.0

2.63

0.005

0.20

200.0

5.00

0.002

0.09

500.0

11.11

Figure E7-3.1 (a)Michaelis-Menten plot; (b)lineweaver-Burk plot

Therefore, the maximum rate of reaction is

Vmax= 1.33 kmol/m3.s = 1.33 mol/dm3.s
From the slope, which is 0.02 s we can calculate the Michaelis constant. KM:
= slope = 0.02 s
KM = 0.0266 kmol/m3
Subtituting Km and Vmax into Equation (7-26) give us

-r =

(E7-3.3)

where Curea has units of kmol/m3 and -rs has units of kmol/m3.s. Levine and LaCourse suggest
that the total concentration of urease, (Et), corresponding to the value of Vmax above is
approximately 5 g/dm3.
In addition to the Lineweaver-Burk plot, one can also use a Hanes-Woolf plot or and
Eadie-Hofstee plot. Here S Curea and -rs -rurea equation (7-26)
-rs =

( )

(7-26)

( )

Can be rearranged in the following form. For the Eadie-Hofstee form,

-rs = Vmax - KM (

( )

(E7-3.4)

For the Hanes-Wolf form. we have

( )

( )

(E7-3.5)

For the Eadie-Hofstee model we plot -rs as a function of (-rs/S) and for the Hanes-Woolf model,
we plot [(S)/-rs] as a function of (S). The Eadie-Hofstee plot does not bias the points at low
substrate concentrations, while the Hanes-Woolf plot gives a more accurate evaluation of Vmax in
Table E7-3.2, we add two columns to Table E7-3.1 to generate these plots (Curea
)
Table E7-3.2 RAW AND PROCESSED DATA
S
(kmol/m3)
0.20
0.02
0.01
0.005
0.002

-rs
(kmol/m3s)
1.08
0.55
0.38
0.20
0.09

1/S
(m /kmol)
5.0
50.0
100.0
200.0
500.0
3

1/-rs
(m s/kmol)
0.93
1.82
2.63
5.00
11.11
3

Sl-rs
(s)
0.185
0.0364
0.0263
0.0250
0.0222

Plotting the data in Table E7-3.2, we arrive at figures E7-3.2 and E7-3.3.
Figure E7-3.2 Hanes-Woolf plot

Figure E7-3.3 Eadie-Holfstee plot

-rs/S
(1/s)
5.4
27.5
38
40
45

Regression
Equation (7-26) was used in the regression program of Polymath with the following results for
Vmax and Km
Nonlinear regression (L-M)
Model : rate = Vmax*Curea/(Km+Curea)
Variable

Ini guess

Vmax
Km

1
0.02

Value
1.2057502
0.0233322

Nonlinear regression settings

Max # iterations = 64
Precision
R^2
R^2adj
Rmsd
Variance

95% confidence
0.0598303
0.003295
Vmax=1.2 mol/dm3s
KM =0.003295 mol/dm3

= 0.9990611
= 0.9987481
= 0.0047604
= 1.888E-04

The product-Enzyme Complex

In many reaction the enzyme and product complex (EP) is formed directly from the enzyme
substrate complex (EP) according to the sequence
E+S

ES

P+E

Applying the PPSH, we obtain

(

(7-29)

which is often referred to as the Briggs-Haldane Equation (see Problem P7-10) and the
application of the PPSH to enzyme kinetics often called the Briggs-Haldane approximations
7.2.4 Batch Reactor Calculations for Enzyme Reactions
A mole balance on urea in the bach reactor gives

Because this reactions is liquid phase, the mole balance can be put in the following form:

(7-30)

The rate low for urea decomposition is

(7-31)

Substituting Equation (7-31) into Equation (7-30) and then rearranging and integrating. We get

(7-32)

We can write Equation (7-32) in terms of conversion as

(

(7-32)

The parameters KM and Vmax can readily be determined from batch reactor data by using the
integral method of analysis. Dividing both sides of Equation (7-32) by tKm/Vmax and rearranging
yields

We see than KM and Vmax be determined from the slope and intercept of a plot of 1/t ln [1/t(1-x)]
versus X/t. We could also express the Michaelis Menten equation in terms of the substrate
concentration S :

(7-33)

Where S0 is the initial concentration of substrate. In cases similar to Equation (7-33) where there
is no possibility of confusion. We shall not bother to enclose the substrate or other species in
parentheses to represent concentration [i.e..Cs (S) S ]. The corresponding plot in terms of
substrate concentration is shown in Figure 7-8.

Figure 7-7 Evaluating Vmax and KM.

Example 7-4 Batch Encymatic Reactors

Calculate the time needed to convert 99% of the urea to ammonia and carbon dioxide in a 0.5dm3
bacth reactor. The initial concentration of urea is 0.1 mol/dm3, and the urease concentration is
0.001 g/dm3. The reaction is to be carried out isothermally at the same temperature at which the
data in Table E7-3.2 were obtained.
Solution
We can use Equation (7-32).
(7-32)
Where KM = 0.0266 mol/dm3. X = 0.99 and Curea 0 = 0.1 mol/dm3. Vmax was 1.33 mol/dm3-s.
However, for the conditions in the batch reactor, the enzyme concentration is only 0.001 g/dm3
compared with 5 g in Example 7-3. Because Vmax = E1.k3. , Vmax for the second enzyme
concentration is

Km = 0.0266 mol/dm3 and X = 0.99

Subtituting into Equation (7-32)

)(

= 460s + 380s
= 840s (14 minutes)

Effect of Temperature
The effect of temeprature on enzymatic reactions is very complex. If the enzyme structure would
remain unchanged as the temperature is increased, the rate would probably follow the Arrhenius
temperature dependence. However, as the temperature increases, the enzyme can unfold and/or
become denatured and lose it catalytic activity. Consequently, as the temperature increases, the
reaction rate, -rs, increases up to a maximum with increasing temperature and then decreases as
the temperature is increased further. The descending part of this curve is called temeprature
inactivation or thermal denaturizing.10 Figure 7-8 shows an example of this optimum in enzyme
activity.11

Figure 7-8 Catalytic breakdown rate of H2O2 depending on temperature. Courtesy of S. Aiba,
A.E.. Humphrey and N.F Mills. Biochemical Engineering.Academic Press (1973)

10

M.L..Shuller and F.Kargi. Bioprocess Engineering Basic Concepts. 2nded. (Upper Saddle
River. N.J.: Prentice Hall. 2002). P. 77
11

S. Aiba. A.E. Humphrey. and N.F. Mills. Biochemical Engineering. (New York: Academic
Press. 1973). P. 47

Side note : Lab-on-a-chip. Enzyme-catalyzed polymerization of nucleotides is a key step in

DNA identification. The microfluidic device shown in Fig-ure SN7.1 is used to identify DNA
strands. It was developed by Professor Mark Burnss group at the University of Michigan.

Figure SN7.1 Microfluidic device to identify DNA. Courtesy of Science, 282, 484 (1998)

In order to identify DNA, its concentration must be raised to a level that can be easily quantified.
This increase is typically accomplished by replicating the DNA in the following manner. After a
biological sanple (e.g., purified saliva, blood) is injected ito the micro device. It is heated and the
hydrogen bonds connecting the DNA strands are broken. After breaking, a primer attaches to the
DNA to form a DNA primer complex. DNA*. An enzyme E then attaches to this pair forming the
DNA* enzyme complex, DNA* E. Once this complex is formed a polymerization reaction
occurs as nucleotides (dNTPs-dATP, dGTP, dCTP, and dTTP-N) attach to the primer one
molecule at a time as shown in Figure SN7.2. the enzyme interacts with the DNA strand to add
the proper nucleotide in the proper order. The addition continues as the enzyme moves down the
strand attaching the nucleotides until the order end of DNA strand is reached. At this point the
enzyme drops off the strand and duplicate. Double-stranded DNA molecule is formed. The
reaction sequence is

Figure SN7.2 Replication Sequence

The schematic in Figure SN7.2 can be written in terms of singles-step reaction where N is one of
the four nucleotides.
Complex Formation :
DNA + Primer DNA*
DNA* + E

DNA * E

Nucleotide addition/polymerization
DNA * E + N DNA * N1 E
DNA * N1 E+ N DNA * N2 E
The process then continues much like a zipper as the enzyme moves along the strand to add more
nucleotides to extend the primer. The addition of the last nucleotide is
DNA * Ni-1 E+ N DNA * Ni E
Where I is the number of nucleotide molecules on the originalDNA minus the nucleotides in
primer. Once a complete double-stranded DA is formed, the polymerization stops, the enzme
drops off, and separation occurs,
DNA * N1 E 2DNA + E
Here 2DNA strands really represents one double-stranded DNA helix. Once replicated in the
device, the length of DNA molecules can be analyzed by electrophoresis to indicate relevant
genetic information.

7-3 Inhibition of Enzyme Reactions

In addition to temperature and solution pH, another factor that greatly influences the rates of
enzyme-catalyzed reactions is presenceof an inhibitor. Inhibitors are species that interact with
enzymes and render the enzymes ineffective to catalyze its specific reaction. The most dramatic
consequence of enzyme inhibition are found in living organisms where the inhibition of any
particular enzyme involved in a primary metabolic pathway will render the entire pathway
inoperative, resulting in either serious damage or deathof the organisms. For example, the
inhibition of single enzyme, cytochrome oxidase by cyanide will cause the aerobic oxidation
process to stop : death occurs in a very few minutes. There are also beneficial inhibitor such as
the one used in the treatment of leukimia and other neoplastic diseases.aspirin inhibits the
enzyme that catalyzes the synthesis of prostaglandin involved in the pain-producing process.
The three most common types of reversible inhibition occuring in enzymatic reaction are
competitive, uncompetitive, and noncompetitive. The enzyme molecule is analogous to a
heterogeneous catalytic surface in that in contains active sites. When competitive inhibition
occurs, the substrate and inhibitor are usually similar molecules that compete for the samesite on
the enzyme. Uncompetitive inhibition occurs when the inhibitor deactivates the enzyme-substrate
complex, sometimes by attaching it self to both the substrate and enzyme molecules of the
complex. Noncompetitive inhibition occurs with the enzymes containing at least two different
types f sites. The substrate attaches only to one type of site, and the inhibitor attaches only to the
other to render the enzyme inactive.

7.3.1 Competitive Inhibition

Competitive inhibition is of particular importance in pharmacokinetics (drug therapy). If a
patient were administered two or more drugs that react simultaneosly within the body with a
common enzyme, cofactor, or active species, this interaction could lead to competitive inhibition
in the formation of the respective metabolites and produce serious consequence.
In competitive inhibition another substance, I. competes with the substrate for the enzyme
molecules to form an inhibitor-enzyme complex, as shown here.
Reaction steps

Competitive Inhibition Pathway

(1)

E+S

k1

E S

(2)

ES

k2

E+S

(3)

ES

k3

P+E

(4)

1+E

k4

E 1 (inactive)

(5) E 1

k5

E+1

In addition to three Michaelis-Menten reaction steps. There are two additional steps as the
inhibitor reversely ties up the enzyme as shown in reaction steps 4 and 5.
The rate law for the formation of product is the same [ef. Equation (7-18A)] as it was
before in the absence of inhibitor.
r1 = k3 ( E S)

(7-34)

Reaction Steps

Uncompetitive Pathway

(1)

E+S

k1

E S

(2)

ES

k2

E+S

(3)

ES

k3

P+E

(4)

1+ES

k4

1 E S (inactive)

k5

1ES

(5) 1 E + S

Starting with equation for rate of formation of product. Equation (7-34), and then applying
the pseudo-steady-state hypothesis to the intermediate (1 E S) . we arrive at the rate law for
uncompetitive inhibition
( )
( )(

( )

(7-40)

Rearranging
( )

( )

(7-41)

The Lineweaver-Burk plot is shown in Figure 7-11 for different inhibitor concentrations.
The slope (KM/Vmax) remains the same as the inhibition (I) concentration is increased, whilethe
intercept (1+ (I)/ K1) increases.

Figure 7-11 Lineweaver Burk plot for uncompetitive inhibition

In noncompetitive inhibition, also called mixed inhibition, the substrate and inhibitor molecules
react with different types of sites on the enzyme molecule. Whenever the inhibitor is attached to
the enzyme it is inactive and cannot form products. Consequently, the deactivating complex (1
E S) can be formed by two reversible reaction paths.
1. After a substrate molecule attaches to the enzyme molecule at the substrate site, the
inhibitor molecule attaches to the enzyme at the inhibitor site.
2. After the inhibitor molecule attaches to the enzyme molecule at the inhibitor site, the
substrate molecule attaches to the enzyme at the substrate site.
These paths, along with the formation of the product. P, are shown here. In noncompetitive
inhibition, the enzyme can be be tied up in its inactive form either, before or after forming the
enzyme substrate complex as shown in steps 2,3 and 4.

Reaction Steps

Noncompetitive Pathway

(1)

E+S

k1

E S

(2)

E+1

k2

1 E (inactive)

(3) 1 + E S

k3

1 E S (inactive)

(4) S + 1 E

k4

1 E S (inactive)

k5

(5) E S

Again starting with the rate law for the rate of formation of product and then applying the
PSSH to the complexes (1 E) and (1 + E S) we arrive at the rate of the law for the
noncompetitive inhibition.
( )
( )(

( )

(7-42)

The derivation of the rate law is given in the Summary Notes on the web and CD-ROM.
Equation (7-42) is in the form of the rate law that is given for an enzymatic reaction exhibiting
noncompetitive inhibition. Heavy metal ions such as Pb2+, Ag+, and Hg2+ as well as inhibitors
that react with the enzyme to form chemical derivatives, are typical examples of noncompetitive
inhibitors.
Rearranging

( )

( )

( )

(7-43)

Figure 7-12 Lineweaver-Burk plot for noncompetitive enzyme inhibition

For noncompetitive inhibition, we see in Figure 7-12 that both the slope *
and intercept *

( )

( )

)+

)+ increase with increasing inhibitor concentration. In

practice, uncompetitive inhibition and mixed inhibition are observed anly for enzymes with two
ormore substrate. S1 and S2.
The three types of inhibition are compared with a reaction in which no inhibitors are
present on the Lineweaver-Burk plot shown in Figure 7-13

Figure 7-13 Lineweaver-Burk plots are three types of enzyme inhibition