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Lecture 14-Estimation of MM Kinetic Parameters

This document provides information about the Biochemical Engineering course 7RCHE31 taught by Dr. Sudhir Ranganath. It includes 3 units on enzyme-catalyzed reactions, mechanisms, kinetics modeling using Michaelis-Menten and other approaches. It discusses batch and continuous reactor kinetics, multi-substrate reactions, and effects of temperature, pH and shear. It also covers immobilized enzymes and their industrial applications. Examples are provided on deriving rate equations using Briggs-Haldane approach, determining kinetic parameters from experimental data using Lineweaver-Burk, Eadie-Hofstee and Hanes-Woolf plots. Sample problems are given to derive rate equations and calculate kinetic parameters.

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Amogha G C 1SI19CH002
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65 views17 pages

Lecture 14-Estimation of MM Kinetic Parameters

This document provides information about the Biochemical Engineering course 7RCHE31 taught by Dr. Sudhir Ranganath. It includes 3 units on enzyme-catalyzed reactions, mechanisms, kinetics modeling using Michaelis-Menten and other approaches. It discusses batch and continuous reactor kinetics, multi-substrate reactions, and effects of temperature, pH and shear. It also covers immobilized enzymes and their industrial applications. Examples are provided on deriving rate equations using Briggs-Haldane approach, determining kinetic parameters from experimental data using Lineweaver-Burk, Eadie-Hofstee and Hanes-Woolf plots. Sample problems are given to derive rate equations and calculate kinetic parameters.

Uploaded by

Amogha G C 1SI19CH002
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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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

Mechanisms, Michealis-Menten kinetics model and


estimation of kinetic parameters

Batch and continuous enzyme reactor kinetics

Multi-substrate enzyme reaction kinetics and estimation


of kinetic parameters

Effects of temperature, pH and shear

Immobilized enzymes and their industrial applications


2. Quasi-Steady-State Approach: Briggs-Haldane Equation
Quasi-Steady-State Approach: Briggs-Haldane Equation
In many cases the assumption or rapid equilibrium is not valid, although the
enzyme-substrate reaction still shows saturation-type kinetics.

By applying the quasi-steady-state assumption i.e., "[$%]⁄"' = ), to the


following equation:

"[$%] 12 3 [4]
= *+ $ % − *-+ $% − *. [$%], we get: /0 = ………. (1)
"' 152 617

We know that: $ = $) − $% …………. (2)

12 38 - 34 [4]
Substituting (2) in (1), we get: /0 = ……….. (3)
152 617

[3 ][4]
Solving (3) for [ES], we get: /0 = 952:98 7 ………… (4)
92
6[4]

"[<] "[<] * $ [%]


Substituting (4) in ; = = *. [$%], we get: ; = = 952. :9)7
"' "' 6[4]
92
Quasi-Steady-State Approach: Briggs-Haldane Equation
#[%] () *+ [,]
!= =
#' -./ + -1
+ [2]
-/

34 [,]
Which can be written as: ! =5 ………. (5)
4 6[,]

Where, 78 = (-./ + -1 )⁄-/ and <8 = -1 [=> ].

(5) is known as the Briggs-Haldane equation.


Experimental Determination of Kinetic Parameters
Determination of !" and #" with high precision is difficult.

Experimental data are obtained from initial-rate experiments.

A batch reactor is charged with a known amount of substrate [%& ] and


enzyme [(& ].

The product or substrate concentration is plotted against time.

The initial slope of this curve is estimated as: ) = , - ⁄,. = , % ⁄,. = 0.

The value of ) depends on the values of [(& ] and [%& ] in the feed to the
reactor.

Many such experiments can be used to generate many pairs of ) and [S]
data.

These data could be plotted as the figure in the next slide, but the
accurate determination of !" and #" from such a plot is very difficult.
Experimental Determination of Kinetic Parameters
Double-reciprocal Plot (Lineweaver-Burk plot)
Equation (5) can be linearized in a double-reciprocal form as:

! ! '% !
= + ………. (6)
" $% $% [)]

A plot of 1/- versus 1/[.] yields a linear line with a slope of 01 ⁄21 and y-axis
intercept of 1⁄21 as depicted in the figure. A double-reciprocal plot gives good
estimates on 21 .
Eadie-Hofstee Plot
Equation (5) can be
rearranged as:

'
! = #$ − &$ [)] …. (7)

A plot of ! versus !/[,]


results in a line of slope −&$
and y-axis intercept of #$ as
depicted in the figure.

Eadie-Hofstee plots are


subject to large errors since
both coordinates contain !,
but there is less bias on
points at low [S].
Hanes-Woolf Plot
Equation (5) can also be
rearranged as:

["] &' *
$
= ('
+ ( [+] …. (8)
'

A plot of [+] versus [S]/.


results in a line of slope 1/01
and y-axis intercept of 31 ⁄01 ,
as depicted in the figure.

This plot is used to determine


01 more accurately.
Problem 1
When glucose is converted to fructose by glucose isomerase, the slow
product formation step is also reversible as:

k1 k2
S+E ES P+E
k-1 k-2

a. Derive the rate equation by employing the Briggs-Haldane approach.


b. At what condition, this equation can be simplified to Michaelis-Menten
equation?
Problem 1: Solution to (a)
Briggs-Haldane approach assumes quasi steady state ie., change of [ES] with
time is negligible.

(1)

Rate of product formation is given by

(2)

Enzyme is conserved, hence (3)

From (1), we get (4)

Combining (3) & (4) &


(5)
rearranging, we get
Problem 1: Solution to (a)
Substituting (3) & (5) into (2), we get

Substituting [ES] in terms of [E0] and rearranging, we get

Dividing numerator & denominator by k1 and rearranging, we get


Problem 1: Solution to (b)

From Briggs-Haldane approach,


we have

When the first step of the reaction, the complex formation step, is much
faster than the product formation step, k1 and k-1 are large compared to k2
and k-2 , therefore,

Michaelis-Menten approach
Problem 2
The enzyme Fumarase, has the following reaction mechanism and kinetic
constants:

k1 k2
S+E ES P+E
k-1
where, k1 = 109 M-1 s-1
k-1 = 4.4 x 104 s-1
k2 = 103 s-1
a. What is the value of Km for this enzyme?

b. At an enzyme concentration of 10-6 M, what will be the initial rate of


product formation at a substrate concentration of 10-3 M?
Problem 2: Solution to (a) and (b)
Thank you

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