Biochemical Engineering

7RCHE31

Dr. Sudhir Ranganath

Contact Hours/Week: 3 (Lecture) Credits: 3.0

CIE Marks: 50
Total Lecture Hours: 39 SEE Marks: 50
UNIT III
ENZYME CATALYZED REACTIONS
Mechanism & Michealis-Menten kinetics model
& estimation of kinetic parameters

Batch & continuous enzyme reactor kinetics

Multi-substrate & Allosteric enzyme reaction kinetics

Kinetics of enzyme inhibition & estimation of kinetic

parameters

Effects of temperature, pH & shear

Immobilized enzymes
Multi-Substrate Enzyme Kinetics
Many enzymes utilize more than a single substrate

Eg: NAD+ in oxido-reductases

Eg: Hydrolytic depolymerases acting on many identical bonds regardless

of the polymer segment

Eg: Aspartate aminotransferase catalyzes oxaloacetate and glutamate

In such reactions, combination of S and E occurs as:

Orderly Sequential Mechanism

Random Sequential Mechanism

Theorell-Chance Mechanism

Ping-Pong Mechanism
Multi-Substrate Enzyme Kinetics
Orderly Sequential Mechanism

A and then B binds to E

Ternary complex (EAB)

Product conversion P & Q via

intermediate EPQ

Product released in a specific order

Eg: Conversion of pyruvate into

lactate by lactate dehydrogenase with
NADH being the second substrate
Multi-Substrate Enzyme Kinetics
Random Sequential Mechanism
No definite sequence of substrate
binding
Ternary complex (EAB)
Product conversion P & Q via
intermediate EPQ
Product released in any order
Eg: Phosphorylation of Glucose by
ATP using hexokinase into
glucose-6-phosphate

Theorell-Chance Mechanism A and then B binds to E

A B Q No ternary complex formation or
S.S. concentrations of ternary
P complexes are zero
P released upon binding of B to EA
Eg: NADH and acetaldehyde
E EA EQ E
conversion to ethanol using
alcohol dehydrogenase
Multi-Substrate Enzyme Kinetics
Ping-Pong Mechanism

Double displacement mechanism

One substrate is bound and product

released before the second
substrate binds to the enzyme

The second product is released later

Eg: Phosphate-transferring enzyme

phosphoglycerate mutase
Multi-Substrate Enzyme Kinetics
Effect of substrate on kinetic parameters
Multiple substrates compete with each other for active site

Substrates forming dimer, oligomer or trimers are essentially different

substrates

Polymers with monomer-monomer branches: viewed as multiple substrates

Amyloglucosidase is
used to hydrolyze
starches of different
MW
Allosteric Enzyme Kinetics
Allosteric Enzymes: Enzymes having more than one substrate-binding
sites.

The binding of one substrate or activator to the enzyme helps binding or

non-binding of other substrate molecules…. process is known as allostery
or cooperative binding

Egs., are regulatory enzymes.

Allosteric Enzyme Kinetics
The rate expression for allosteric enzyme kinetics is given as:

$% '( [%]+
!= − = ” .[%]+ ……… (1)
$& ,(

Where, / is the cooperativity coefficient and / > 1 indicates positive

cooperativity.

Eq (1) results in a sigmoidal curve

when ! versus 2 is plotted.

A comparison of allosteric enzyme

kinetics and Michaelis-Menten
kinetics is shown here.
Allosteric Enzyme Kinetics
Equation (1) is rearranged as follows:

# ”
!" = "!" ( − !"*+ ……. (2)
$% &#

#
The cooperativity coefficient " is determined by plotting !" versus !" ( ,
$% &#
and by calculating the slope which is equal to ".

Depending on the value of " ,

substrate binding may be
cooperative when " > 1 , or non-
cooperative (prohibitive) when " <
1, or not affected from the other
bound substrates when " = 1 ,
which is a major assumption for M-
M kinetics.
Allosteric Enzyme - Video
Allosteric Regulation - Video
Thank you