104. Chapter 6 Homogeneous Reaction Catalysis:
ITK-329 Kinetika & Katalisis
Chapter 6
Homogeneous Reaction Catalysis:
ENZYMATIC REACTION FUNDAMENTAL
Development of Kinetic Expression in Enzymatic Reactions
Dicky Dermawan

105. General Properties of Enzymes
# accelerate specific reaction # very selective # work on mild condition k1 k2
E + S (ES)*
(ES)* + W P + E k3
W = Solvent, usually water,
Considered constant in concentration
Example: Urease O
NH2CNH2 + Urease NH2CNH2-Urease
NH2CNH2-Urease NH2CNH2 + Urease O O
NH2CNH2-Urease + HOH NH3 + CO2 + U

106. Development of Kinetic Expression
E + S (ES)*
(ES)* + W P + E k3
W = Pelarut (konstan) Et
NH2CNH2 O ES
Enzyme balance:

107. Michaelis – Menten Equation
where
Property at low substrate concentration:
[S] <<<< Km
First order!!!
Property at high substrate concentration:
[S] >>>> Km
Zero order!!!

108. Plot of Michaelis – Menten Equation
-rs = rp Km
[S]
Estimating Vmax and KM
Burke Lineweaver Plot
Alternatively, Eadie plot can be constructed

109. Example : 7-7
Determine the Michaelis – Menten Parameters for the reaction:
The rate of reaction is given as a function if urea concentration in the following table
Use: a. Burke – lineweaver plot b. Eadie plot Compare the results and determine which method fits better

110. P7-11B
Beef liver catalase has been used to accelerate the decomposition of hydrogen peroxide to yield water and oxygen. The concentration of hydrogen peroxide is given as a function of time for a reaction mixture with pH of 6.76 and maintain at 30oC,
t (min) 10 20 50
100
C H2O2 (mol/liter)
0.02
0.0158
0.0106
0.005
Determine the Michaelis-Menten parameters Vmax and Km (b) If the total enzyme concentration is tripled, what will the substrate concentration be after min ?

111. Integration of Rate Equation
Ex. 7-8
Calculate the time needed to convert 80% of the ure xammonia and carbon dioxide in a 0.5 L batch reactor. The initial concentration of urea is 0.1 mol/L, and the urease concentration is g/L. The reaction is to be carried out isothermally. At this temperature, if total enzyme concentration used is 5 g/L:
Ex. 7-8
If the reaction is carried out in 15 minutes using enzyme concentration of g/L, what will be the conversion?

112. P7-10C Integration of Rate Equation

113. Enzymatic Reaction Inhibition
In addition to pH, another factor that greatly influences the rates of enzyme-catalyzed reactions is the presence of an inhibitor.
The most dramatic consequences of enzyme inhibition are found in living organisms, where the inhibition of any particular enzyme involved in a primary metabolic sequence will render the entire sequence inoperative, resulting in either serious damage or death of the organism. For example, the inhibition of a 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 inhibitors such as the ones used in the treatment of leukemia and other neoplastic diseases.

114. Enzymatic Reaction Inhibition
The three most common types of reversible inhibition occurring in enzymatic reactions are competitive, uncompetitive, and noncompetitive.
The enzyme molecule is analogous to the heterogeneous catalytic surface in that it contains active sites.
When competitive inhibition occurs, the substrate and inhibitor are usually similar molecules that compete for the same site on the enzyme.
Uncompetitive inhibition occurs when the inhibitor deactivates the enzyme-substrate complex, usually by attaching itself to both the substrate and enzyme molecules of the complex.
Noncompetitive inhibition occurs with enzymes containing at least two different types of sites. The inhibitor attaches only to one type of site and the substrate only to the other.

115. Enzymatic Reaction Inhibition: Competitive Inhibition
Competitive inhibition is of particular importance in pharmacokinetics (drug therapy). If a patient were administered two or more drugs simultaneously which react within the body with a common enzyme, cofactor, or active species, this could lead to competitive inhibition of the formation of the respective metabolites and produce serious consequences.
In this type of inhibition another substance, I, competes with the substrate for the enzyme molecules to form an inhibitor-enzyme complex, (E I). k1 k2
S + E ES
I + E EI k3 k4 k5
ES P + E
Mechanism:

116. Competitive Inhibitionk1 k2
S + E ES
I + E EI k3 k4 k5
ES P + E
Mechanism:
I = Inhibitor = Penghambat laju reaksi
Enzyme balance:

117. Competitive Inhibition (cont’)k1 k2
S + E SE
I + E EI k3 k4 k5
ES P + E
Mechanism:
Competitive Inhibition (cont’)
; substituting expression for [ES]:
; substituting expression for [E]:

118. Competitive Inhibition (cont’)k1 k2
S + E SE
I + E EI k3 k4 k5
SE P + E
Mechanism:
…… Jika penambahan suatu zat I ke dalam kultur menyebabkan kenaikan harga KM tanpa perubahan harga Vmax, maka I menginhibisi secara kompetitif.

119. Enzymatic Reaction Inhibition: Uncompetitive Inhibition
Here, the inhibitor does not compete with the substrate for the enzyme; instead, it ties up the enzyme-substrate complex by forming an inhibitor-enzyme-substrate complex, thereby restricting the breakdown of the (E S) complex to produce the desired product. k1 k2
S + E ES
I + ES EIS k3 k4 k5
ES P + E
Mechanism:

120. Uncompetitive Inhibitionk1 k2
S + E ES
I + ES EIS k3 k4 k5
ES P + E
Mechanism:

121. Uncompetitive Inhibition (cont’)k1 k2
S + E ES
I + ES EIS k3 k4 k5
ES P + E
Mechanism:
Enzyme balance:

122. Uncompetitive Inhibition (cont’)k1 k2
S + E ES
I + ES EIS k3 k4 k5
ES P + E
Mechanism:

123. Uncompetitive Inhibition (cont’)k1 k2
S + E ES
I + ES EIS k3 k4 k5
ES P + E
Mechanism:
Finally:
Inhibisi uncompetitive mengakibatkan penurunan harga Vmax dan KM secara bersama-sama

124. Non Competitive Inhibition
In noncompetitive inhibition, the substrate and inhibitor molecules react with different types of sites on the enzyme molecule, and consequently, the deactivating complex, IES, 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 an inhibitor molecule attaches to the enzyme molecule at the inhibitor site, the substrate molecule attaches to the enzyme at the substrate site.
EI + S EIS k1 k2
S + E ES
I + E EI k3 k4 k7 k8
ES + I EIS k9
ES P + E k5 k6
Mechanism:

125. Non Competitive Inhibition (cont’)
Use “rate limiting” concept assuming reaction (5) as rate limiting step:
Other reactions are in equilibrium with equilibrium constant of KS, KI, KS’ and KI’ for reaction (1) to (4), respectively.
EI + S EIS k1 k2
S + E ES
I + E EI k3 k4 k7 k8
ES + I EIS k9
ES P + E k5 k6
Mechanism:
Further, assume KS= KS’ and KI = KI’ to obtain:

126. Non Competitive Inhibition (cont’)
Further, assume KS= KS’ and KI = KI’ to obtain:
Enzyme balance:
EI + S EIS k1 k2
S + E ES
I + E EI k3 k4 k7 k8
ES + I EIS k9
ES P + E k5 k6
Mechanism:
From
use
to obtain:

127. Enzymatic Reaction Inhibition: Comparison & Summary
a. Competitive Inhibition
c. Non Competitive Inhibition k1 k2
S + E ES
I + E EI k3 k4 k7 k8
ES + I EIS k9
ES P + E k5 k6 k1 k2
S + E ES
I + E EI k3 k4 k5
ES P + E
EI + S EIS
b. Uncompetitive Inhibition k1 k2
S + E ES
I + ES EIS k3 k4 k5
ES P + E

128. Enzymatic Reaction Inhibition: Comparison & Summary
No Inhibition
Competitive Inhibition
Uncompetitive Inhibition
Noncompetitive Inhibition

129. Enzymatic Reaction Inhibition: Comparison in Plot
Burke – Lineweaver
Eadie
(a) ? (b) ? (c) ?

130. P7-14B

131. Inhibisi oleh Substrat
Special Case:
Inhibisi oleh Substrat k1 k2
S + E SE
S + ES SES k3 k4 k5
SE P + E
Mechanism:

132. Inhibisi oleh Substrat (cont’)
Enzyme balance:

133. Inhibisi oleh Substrat (cont’)

134. Curve Fittings: Substrate-inhibited Enzymatic Reaction
Perkirakan harga Vmax, KM, dan Ki berdasarkan plot hasil percobaan dari reaksi enzimatik yang diinhibisi substrat di bawah ini.